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/lp/pubmed-central/ayahuasca-and-its-dmt-and-carbolines-containing-ingredients-block-the-r6n3d1ObRx
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- Pubmed Central
- Copyright
- Copyright © 2018 Cata-Preta, Serra, Moreira-Junior, Reis, Kisaki, Libarino-Santos, Silva, Barros-Santos, Santos, Barbosa, Costa, Oliveira-Lima, Berro and Marinho.
- ISSN
- 1663-9812
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- 10.3389/fphar.2018.00561
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Abstract
fphar-09-00561 May 25, 2018 Time: 17:50 # 1 ORIGINAL RESEARCH published: 29 May 2018 doi: 10.3389/fphar.2018.00561 Ayahuasca and Its DMT- and b-carbolines – Containing Ingredients Block the Expression of Ethanol-Induced Conditioned Place Preference in Mice: Role of the Treatment Environment 1 1 1 Edited by: Elisangela G. Cata-Preta , Yasmim A. Serra , Eliseu da C. Moreira-Junior , Andrew Robert Gallimore, 1 1 1 1 Henrique S. Reis , Natali D. Kisaki , Matheus Libarino-Santos , Raiany R. R. Silva , Okinawa Institute of Science 1 1 2 3 Thaísa Barros-Santos , Lucas C. Santos , Paulo C. R. Barbosa , José L. Costa , and Technology, Japan 1 4 1 Alexandre J. Oliveira-Lima , Lais F. Berro and Eduardo A. V. Marinho * * Reviewed by: 1 2 Department of Health Sciences, Universidade Estadual de Santa Cruz, Ilhéus, Brazil, Department of Philosophy and Regina A. Mangieri, Human Sciences, Universidade Estadual de Santa Cruz, Ilhéus, Brazil, Faculty of Pharmaceutical Sciences, University of University of Texas at Austin, Campinas, Campinas, Brazil, Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, United States Jackson, MS, United States Andrey E. Ryabinin, Oregon Health & Science University, United States Ayahuasca is a hallucinogenic beverage produced from the decoction of Banisteriopsis *Correspondence: caapi (Bc) and Psychotria viridis (Pv), b-carboline- and N,N-dimethyltryptamine(DMT)- Lais F. Berro berro.lf@gmail.com containing plants, respectively. Accumulating evidence suggests that ayahuasca may Eduardo A. V. Marinho have therapeutic effects on ethanol abuse. It is not known, however, whether its edumarinho@hotmail.com; effects are dependent on the presence of DMT or if non-DMT-containing components eavmarinho@uesc.br would have therapeutic effects. The aim of the present study was to investigate the Specialty section: rewarding properties of ayahuasca (30, 100, and 300 mg/kg, orally), Bc (132, 440, and This article was submitted to 1320 mg/kg, orally) and Pv (3.75, 12.5 and 37.5 mg/kg, i.p.) extracts and their effects on Neuropharmacology, a section of the journal ethanol (1.8 g/kg, i.p.) reward using the conditioned place preference (CPP) paradigm Frontiers in Pharmacology in male mice. Animals were conditioned with ayahuasca, Bc or Pv extracts during 8 Received: 28 November 2017 sessions. An intermediate, but not a high, dose of ayahuasca induced CPP in mice. Accepted: 10 May 2018 Published: 29 May 2018 Bc and Pv did not induce CPP. Subsequently, the effects of those extracts were tested Citation: on the development of ethanol-induced CPP. Ayahuasca, Bc or Pv were administered Cata-Preta EG, Serra YA, before ethanol injections during conditioning sessions. While Bc and Pv exerted no Moreira-Junior EC, Reis HS, effects on ethanol-induced CPP, pretreatment with ayahuasca blocked the development Kisaki ND, Libarino-Santos M, Silva RRR, Barros-Santos T, of CPP to ethanol. Finally, the effects of a post-ethanol-conditioning treatment with Santos LC, Barbosa PCR, Costa JL, ayahuasca, Bc or Pv on the expression of ethanol-induced CPP were tested. Animals Oliveira-Lima AJ, Berro LF and Marinho EAV (2018) Ayahuasca were conditioned with ethanol, and subsequently treated with either ayahuasca, Bc and Its DMT- and -carbolines – or Pv in the CPP environment previously associated with saline or ethanol for 6 days. Containing Ingredients Block Animals were then reexposed to ethanol and ethanol-induced CPP was quantified on the Expression of Ethanol-Induced Conditioned Place Preference in Mice: the following day. Treatment with all compounds in the ethanol-paired environment Role of the Treatment Environment. blocked the expression of ethanol-induced CPP. Administration of an intermediate, but Front. Pharmacol. 9:561. doi: 10.3389/fphar.2018.00561 not a high, dose of ayahuasca and Bc, as well as Pv administration, in the saline-paired Frontiers in Pharmacology | www.frontiersin.org 1 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 2 Cata-Preta et al. Ayahuasca and Ethanol Reward compartment blocked the expression of ethanol-induced CPP. The present study sheds light into the components underlying the therapeutic effects of ayahuasca on ethanol abuse, indicating that ayahuasca and its plant components can decrease ethanol reward at doses that do not exert abuse liability. Importantly, the treatment environment seems to influence the therapeutic effects of ayahuasca and Bc, providing important insights into clinical practice. Keywords: ayahuasca, N,N-dimethyltryptamine, b-carboline alkaloids, ethanol, reward, conditioned place preference, mice drug abuse, a previous study from our group investigated the INTRODUCTION effects of Aya on behaviors induces by acute and chronic Eth Alcohol (ethanol, Eth) use disorder (AUD) and its health administration in mice (Oliveira-Lima et al., 2015). Aya blocked consequences are a major public health problem. The World the development and expression of acute and chronic Eth- Health Organization estimated that the global prevalence of AUD induced hyperlocomotion and behavioral sensitization without in 2010 was 4.1%, including Eth dependence (2.3%) and harmful affecting baseline locomotor activity (Oliveira-Lima et al., 2015). use of Eth (1.8%) (World Health Organization [WHO], 2014). Those findings suggest that Aya may have therapeutic effects per Eth use is the third leading risk factor for poor health globally, se that are not completely dependent on sociocultural religious and it is estimated that nearly 2.5 million deaths every year variables. are attributable to Eth use (World Health Organization [WHO], Importantly, it is well known that DMT has no significant 2010). Thus, despite the substantial amount of resources that psychoactive effect when ingested orally due to its breakdown governments and international organizations invest in programs by monoamine oxidase A (MAO-A) in the gastrointestinal tract to prevent substance use disorders, AUD is still high across the (Lanaro et al., 2015). According to the proposed pharmacokinetic globe. Currently available treatments are only partially effective model of Aya, b-carboline alkaloids present in B. caapi inhibit (Mason, 2017) and further research on new treatment approaches MAO-A, thereby allowing the entry of DMT from P. viridis is needed. to systemic circulation and central nervous system, and its Throughout the last two decades, accumulating evidence subsequent psychoactive effects (Holmstedt and Lindgren, 1967; has suggested that ayahuasca (Aya) may have therapeutic McKenna, 2004). Thus, it was believed that the main psychoactive properties on substance use disorders (for review, see effects of Aya were attributable to DMT, with b-carboline Nunes et al., 2016). Aya is a brew frequently prepared by alkaloids such as harmine and harmaline simply facilitating its the decoction of Banisteriopsis caapi and Psychotria viridis, effects. However, more recent evidence suggests that b-carboline b-carboline- and N,N -dimethyltryptamine(DMT)-containing alkaloids also have psychoactive properties and could exert plants, respectively. Originally used for religious purposes by therapeutic effects in drug abuse (for review, see Brierley and Amerindian populations of the Amazon Basin, the use of Aya Davidson, 2012). Studies have shown a decrease in morphine and has spread throughout the world (Tupper, 2008), being currently cocaine intake after harmaline administration (Glick et al., 1994), used in syncretic religions such as Santo Daime and União do as well as harmine-induced attenuation of morphine withdrawal Vegetal and other contexts, such as Aya retreats (Labate et al., (Aricioglu-Kartal et al., 2003). Therefore, it remains unknown 2009; Goulart, 2011; Luna, 2011). Case-control studies have whether the effects of Aya are dependent on the presence of found a lower prevalence of substance use and substance-related DMT or if non-DMT containing components, such as the plant problems in Aya users from União do Vegetal and Santo Daime Banisteriopsis caapi, would have therapeutic effects per se. religions relative to control groups (Grob et al., 1996; Da Silveira The aim of the present study was to investigate the et al., 2005; Doering-Silveira et al., 2005; Fabregas et al., 2010; rewarding properties of Aya (prepared by the decoction of Barbosa et al., 2016), and decreased substance use after joining the stems of the Banisteriopsis caapi vine combined with the those churches (Halpern et al., 2008; Labate et al., 2014). In leaves of the Psychotria viridis bush), Banisteriopsis caapi and addition, studies have shown that Aya-assisted therapy resulted Psychotria viridis extracts. We also investigated the effects of in decreased drug use and craving in drug dependent individuals those compounds on the rewarding properties of Eth using (Thomas et al., 2013; Loizaga-Velder and Verres, 2014). the conditioned place preference (CPP) paradigm (Liu et al., Although increasing evidence supports the effectiveness of 2008). We evaluated the effects of Aya, Banisteriopsis caapi Aya for the treatment of drug abuse, it remains unknown whether or Psychotria viridis extracts alone on CPP as well as on the Aya has anti-addictive properties alone or if other factors, such development of CPP to Eth. Additionally, we also investigated if as contextual and religious influences, play a major role in the the treatment environment could influence the effects of those results described above. In what seems to be the only study extracts on the subsequent expression of Eth-induced CPP. Thus, to date investigating the effects of Aya on an animal model of we also tested the effects of treatments with Aya, Banisteriopsis caapi or Psychotria viridis extracts in the CPP environment previously associated with saline (unpaired) or Eth (paired) on Abbreviations: Aya, ayahuasca; EBc, extract of Banisteriopsis caapi; EPv, extract of the subsequent expression of CPP to Eth. Psychotria viridis; Eth, ethanol. Frontiers in Pharmacology | www.frontiersin.org 2 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 3 Cata-Preta et al. Ayahuasca and Ethanol Reward 2015), oral administration of DMT would not allow us to MATERIALS AND METHODS evaluate the effects of this compound in the behavioral tests. Thus, EPv and its Veh were administered i.p. at 10 ml/kg of Animals body weight. The selected dose range of Aya and Eth was Three-month-old Swiss male mice from our own colony were based on previous studies from our group (Marinho et al., used. Animals weighing 35–40 g were group housed (5–7 per 2015; Oliveira-Lima et al., 2015; Silva et al., 2017). Doses of cage) in polypropylene cages (32 42 18 cm) under controlled Aya were chosen based on their ability to prevent acute Eth- temperature (22–23 C) and light (12 h light, 12 h dark; lights induced hyperlocomotion and locomotor sensitization in mice on at 6 h 45) conditions. Rodent chow (Nuvilab, Quimtia (Oliveira-Lima et al., 2015). For comparative purposes, the doses SA, Colombo, PR, Brazil) and water were available ad libitum of EBc and EPv were calculated based on the concentration throughout the experiments. Animals were maintained according of harmine and DMT, respectively, in the Aya extract used in to the National Institutes of Health Guide for the Care and the present study, as previously described (Oliveira-Lima et al., Use of Laboratory Animals (8th Edition, revised 2011) and 2015). in accordance with the Brazilian Law for Procedures for Animal Scientific Use (#11794/2008). The Institutional Animal Conditioned Place Preference Care and Use Committee of UESC approved the experimental The CPP apparatus consisted of 2 conditioning compartments procedures. of equal size (40 20 20 cm): 1 black with white vertical bands in the walls and a black wooden floor and Preparation of Extracts and Compounds 1 white with black horizontal bands in the walls and a Analysis green (red) smooth floor, both connected by a central choice A batch of Aya had been previously obtained from a member compartment (40 10 15 cm) that was accessible by sliding of the Santo Daime church, lyophilized and analyzed for doors. The CPP procedure consisted of the following phases: the quantification of the amount DMT, tetrahydroharmine, Experiments 1, 2, 3, and 4 – habituation, pre-conditioning harmine and harmaline, as previously described (Oliveira-Lima test, conditioning, post-conditioning test; Experiments 5, 6, 7, et al., 2015). Samples of B. caapi and P. viridis were obtained and 8 – habituation, pre-conditioning test, conditioning, post- from members of the Santo Daime church and submitted to conditioning test, treatment, Eth reexposure and post-treatment extraction. The extracts were, then, lyophilized, rendering a test. freeze dried material that was analyzed as described below. The ratio of dry extract/volume of liquid (extract) was calculated to Habituation and Pre-conditioning Test establish the doses to be administered in the experiments for all In order to avoid a novelty effect and establish if animals showed extracts. a preference for either of the compartments, a habituation In order to quantify the amount of the main compounds session and a pre-conditioning test were conducted (Days 1 of B. caapi (tetrahydroharmine, harmine, and harmaline) and and 2, respectively) in which animals were placed in the center P. viridis (DMT) in our preparation, the extracts were analyzed of the apparatus with the door open with free access to each by liquid chromatography mass spectrometry (LC-MS/MS) compartment for 15 min. No injection was administered on the conducted on a high performance liquid chromatography habituation day or on the day of the pre-conditioning test. equipment Prominence system (Shimadzu, Kyoto, Japan). Harmine hydrochloride and harmaline hydrochloride were Conditioning purchased from Sigma . The synthesis of tetrahydroharmine was An unbiased design was used because mice showed no preference performed as previously described (Callaway et al., 1996). DMT for either of the compartments in the pre-conditioning test. was synthesized according to a modified procedure based on Therefore, animals were randomly assigned to an experimental the selective dimethylation method (Giumanini and Casalini, group and a ‘drug-paired compartment’ in a counterbalanced 1980; Pires et al., 2009). The stock solutions (1.0 mg/ml) of fashion, with the “black” compartment as the drug-paired one harmine, harmaline, tetrahydroharmine and DMT were prepared for half of the animals and the “white” compartment for the in methanol and stored at 20 C until the performance of the other half. One “drug-paired compartment” and one “Sal-paired LC-MS/MS. compartment” were defined for all animals. The conditioning trials were performed during 8 consecutive days (Days 3–10). Drugs During the conditioning sessions, the doors remained closed Eth (Merck ), Aya, extract of B. caapi (EBc) and extract of so the animals would be confined to one of the conditioning P. viridis (EPv) were dissolved in 0.9% saline (Sal) solution, compartments. Animals received Sal on even days. On odd days, which was used as vehicle (Veh) solution for the Aya, EBc animals received Aya (30, 100, and 300 mg/kg, Experiment 1a), and EPv treatments. Eth and Sal solutions were administered EBc (132, 440, and 1320 mg/kg, Experiment 1b), EPv (3.75, 12.5, intraperitoneally (i.p.) at 10 ml/kg of body weight. Aya, EBc and 37.5 mg/kg, Experiment 1c) or Eth (1.8 g/kg, Experiments and Veh were orally administered by gavage at a volume of 2a,b,c, 3a,b, 4a,b, and 5a,b). Five (Eth), 30 (Aya, EBc) or 20 (EPv) 10 ml/kg. Because DMT has no significant psychoactive effect min after treatments, mice were confined to the assigned drug- when ingested orally due to its breakdown by monoamine or Sal-paired compartment for 10 min. On Experiments 2a, 2b, oxidase A (MAO-A) in the gastrointestinal tract (Lanaro et al., and 2c, animals received Veh, Aya (100 or 300 mg/kg), EBc (440 Frontiers in Pharmacology | www.frontiersin.org 3 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 4 Cata-Preta et al. Ayahuasca and Ethanol Reward or 1320 mg/kg) or EPv (12.5 or 37.5 mg/kg) 25 (Aya, EBc) or 15 and in the non-drug-paired compartments). The CPP protocol (EPv) min before Eth injections. design is illustrated in Figure 1. Different cohorts of mice were used for each experiment (as well as sub-experiment) described Post-conditioning Test below. Twenty four hours after the last conditioning session (Day 11), animals were placed in the center of the apparatus with the Experimental Design door open with free access to each compartment for 15 min. No Experiment 1: Effects of Treatment With Ayahuasca, injection was administered on the day of the post-conditioning Banisteriopsis caapi or Psychotria viridis on the CPP test. Paradigm In order to evaluate if Aya, EBc or EPv would induce CPP, mice Treatment were submitted to the habituation, pre-conditioning test and Aya Treatment for 8 consecutive days (Days 12 to 19), animals (Experiment 1a, n = 8 per group), EBc (Experiment 1b, n = 8 received every other day administrations of Veh, Aya (100 and per group) or EPv (Experiment 1c, n = 8 per group) conditioning 300 mg/kg, Experiments 3a and 3b), EBc (440 and 1320 mg/kg, followed by post-conditioning test as previously described. Experiments 4a and 4b) or EPv (12.5 or 37.5 mg/kg, Experiments 5a and 5b) and, 30 (Aya, EBc) or 20 (EPv) min after treatments, Experiment 2: Effects of Pretreatment With were confined to the assigned Eth- or Sal-paired compartment for Ayahuasca, Banisteriopsis caapi or Psychotria viridis 10 min. on the Development of Ethanol-Induced CPP Mice were submitted to the habituation, pre-conditioning test, Ethanol Reexposure Eth conditioning preceded by Aya (Experiment 2a, n = 8 per Twenty four hours after the last treatment session (Day 20), group), EBc (Experiment 2b, n = 8 per group) or EPv (Experiment animals received an injection of Eth (1.8 g/kg) and, five min 2c, n = 8 per group) pretreatments and post-conditioning test as after injection, were confined to the Eth-paired compartment for previously described. 10 min. Experiment 3: Role of the Treatment Environment in Post-treatment Test the Effects of Ayahuasca on the Expression of Twenty four hours after the Eth reexposure session (Day 21), Ethanol-Induced CPP animals were placed in the center of the apparatus with the Mice were submitted to the pre-conditioning test, Eth door open with free access to each compartment for 15 min. No conditioning and post-conditioning test as previously described. injection was administered on the day of the post-treatment test. Twenty-four hours after the post-conditioning test, the treatment All behavioral sessions started between 8 and 9 am for phase began. For 8 days, animals received an oral administration all experiments in order to minimize the effect of time of of either Veh or Aya every other day on even days and, 30 min the day and circadian rhythms on the behavioral tasks being after injection, were confined to the compartment previously conducted. Because several groups were ran concomitantly paired with Eth (Experiment 3a, n = 8 per group) or Sal within a given experiment, the order of animals being submitted (Experiment 3b, n = 8 per group) for 10 min. On odd days, to the behavioral sessions in the CPP was randomized for each animals received an oral administration of Veh associated with phase described above, so that all groups had animals being the opposite (Eth- or Sal-paired) compartment. The treatment tested at the same time. The CPP apparatus was cleaned with phase was followed by the Eth reexposure and drug-free Eth-water (5%) solution before each behavioral session/test to post-treatment test sessions as previously described. eliminate possible bias due to odors left by previous mice. Experiment 4: Role of the Treatment Environment in During test sessions, the time spent in each compartment was the Effects of Banisteriopsis caapi on the Expression registered. Movement was tracked using the ANY-maze software of Ethanol-Induced CPP (version 5.1, Stoelting) and a webcam suspended overhead. Expression of drug-induced CPP was evidenced by the CPP Mice were submitted to the pre-conditioning test, Eth score (difference between the time spent in the drug-paired conditioning and post-conditioning test as previously described. FIGURE 1 | Conditioned place preference (CPP) protocol design. H, habituation; PreCT, drug-free pre-conditioning test; Conditioning, ayahuasca (30, 100, or 300 mg/kg, Experiment 1), Banisteriopsis caapi (132, 440 or 1320 mg/kg, Experiment 2), ethanol (1.8 g/kg, Experiments 5, 6, 7 and 8) – preceded by vehicle or ayahuasca (100 or 300 mg/kg, Experiment 3) or Banisteriopsis caapi (440 or 1320 mg/kg, Experiment 4) pretreatment – or saline conditioning; PostCT, drug-free post-conditioning test; Treatment, vehicle, ayahuasca (100 or 300 mg/kg, Experiments 5 and 6) or Banisteriopsis caapi (440 or 1320 mg/kg, Experiments 7 and 8) treatment in the compartment previously paired with ethanol (Experiments 5 and 7) or saline (Experiments 6 and 8); ER, ethanol (1.8 g/kg) reexposure in previously ethanol-paired compartment; PTT, drug-free post-treatment test. Frontiers in Pharmacology | www.frontiersin.org 4 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 5 Cata-Preta et al. Ayahuasca and Ethanol Reward Twenty-four hours after the post-conditioning test, the treatment Experiment 1: Effects of Treatment With phase began. For 8 days, animals received an oral administration Ayahuasca, Banisteriopsis caapi or of either Veh or EBc every other day on even days and, Psychotria viridis on the CPP Paradigm 30 min after injection, were confined to the compartment Experiment 1a (Ayahuasca) previously paired with Eth (Experiment 4a, n = 8 per group) Analysis of the pre-conditioning test showed that animals or Sal (Experiment 4b, n = 8 per group) for 10 min. On had no preference for the subsequently Sal- or Aya-paired odd days, animals received an oral administration of Veh compartments [t(23) = 0.89; p > 0.05] (data not shown). associated with the opposite (Eth- or Sal-paired) compartment. One-way ANOVA showed no significant differences between The treatment phase was followed by the Eth reexposure groups (Aya 30, Aya 100, or Aya 300) during the pre- and drug-free post-treatment test sessions as previously conditioning test [F(2,21) = 1.20, p > 0.05] (data not shown). described. Two-way RM ANOVA showed a significant interaction effect between time (pre- vs. post-conditioning) and Aya dose Experiment 5: Role of the Treatment Environment in [F(2,21) = 5.99, p < 0.01] (Figure 2A). Bonferroni post hoc the Effects of Psychotria viridis on the Expression of test showed that animals treated with 100 mg/kg, but not 30 Ethanol-Induced CPP or 300 mg/kg, Aya showed higher levels of CPP score in the Mice were submitted to the pre-conditioning test, Eth post-conditioning test compared to the pre-conditioning test, conditioning and post-conditioning test as previously described. indicating a marked preference for the 100 mg/kg Aya-paired Twenty-four hours after the post-conditioning test, the treatment compartment. phase began. For 8 days, animals received an i.p. administration of either Veh or EPv every other day on even days and, Experiment 1b (Banisteriopsis caapi) 20 min after injection, were confined to the compartment Analysis of the pre-conditioning test showed that animals previously paired with Eth (Experiment 5a, n = 8 per group) or had no preference for the subsequently Sal- or EBc-paired Sal (Experiment 5b, n = 8 per group) for 10 min. On odd days, compartments [T(23) = 0.86; p > 0.05] (data not shown). One- animals received an i.p. administration of Veh associated with the way ANOVA showed no significant differences between groups opposite (Eth- or Sal-paired) compartment. The treatment phase (EBc 132, EBc 440, or EBc 1320) during the pre-conditioning was followed by the Eth reexposure and drug-free post-treatment test [F(2,21) = 1.12, p > 0.05] (data not shown). Two-way RM test sessions as previously described. ANOVA showed no significant effect of time (pre- vs. post- conditioning) [F(1,21) = 0.01, p > 0.05], EBc dose [F(2,21) = 0.55, Statistical Analysis p > 0.05] or interaction between the two factors [F(2,21) = 0.64, All variables were checked for normality (Shapiro–Wilk test) p > 0.05] (Figure 2B). and homogeneity of variances (Levene’s test), which validated Experiment 1c (Psychotria viridis) the use of the parametric test. Within-group comparisons were Analysis of the pre-conditioning test showed that animals performed using the paired samples t-test. CPP score was analyzed using the Student’s t-test. Multiple comparisons were had no preference for the subsequently Sal- or EPv-paired compartments [T(23) = 0.21; p > 0.05] (data not shown). One- performed using one- or two-way analysis of variance (ANOVA), way ANOVA showed no significant differences between groups with repeated measures (RM) or not, and Bonferroni post hoc test when necessary. A probability of p < 0.05 was considered a (EPv 3.75, EPv 12.5, or EPv 37.5) during the pre-conditioning test [F(2,21) = 0.78, p > 0.05] (data not shown). Two-way statistically significant difference. RM ANOVA showed no significant effect of time (pre- vs. post-conditioning) [F(1,21) = 0.74, p > 0.05] or interaction between time and EPv dose [F(2,21) = 0.24, p > 0.05], but RESULTS showed a significant effect of EPv dose alone [F(2,21) = 3.77, p < 0.05] (Figure 2C). Bonferroni post hoc test, however, Banisteriopsis caapi Compounds showed no differences in the CPP scores of groups during Analysis the post-conditioning session compared to the pre-conditioning LC-MS/MS analysis indicated the following active constituents session. in our EBc sample: Experiment 2: Effects of Pretreatment – Tetrahydroharmine: < 0.1 mg/100 mg With Ayahuasca, Banisteriopsis caapi or – Harmine: 0.876 mg/100 mg Psychotria viridis on the Development of – Harmaline: 0.927 mg/100 mg Ethanol-Induced CPP Psychotria viridis Compounds Analysis Experiment 2a (Ayahuasca) LC-MS/MS analysis indicated the following active constituent in Analysis of the pre-conditioning test showed that animals our EPv sample: had no preference for the subsequently Sal- or Eth-paired compartments [T(23) = 0.67; p > 0.05] (data not shown). One- – N,N -dimethyltryptamine (DMT): 3.2 mg/100 mg way ANOVA showed no significant differences between groups Frontiers in Pharmacology | www.frontiersin.org 5 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 6 Cata-Preta et al. Ayahuasca and Ethanol Reward FIGURE 2 | Effects of (A) ayahuasca (Aya), (B) extract of Banisteriopsis caapi (EBc) and (C) extract of Psychotria viridis (EPv) on the conditioned place preference (CPP) paradigm. CPP score (difference between the time spent in the drug-paired and in the saline-paired compartments) during the pre-conditioning and post-conditioning test sessions. Data are reported as means SEM. p < 0.05 compared with the pre-conditioning test. (Vehicle, Aya 100 or Aya 300) during the pre-conditioning test Experiment 2b (Banisteriopsis caapi) [F(2,21) = 2.12, p > 0.05] (data not shown). Two-way RM Analysis of the pre-conditioning test showed that animals ANOVA showed a significant interaction effect between time had no preference for the subsequently Sal- or Eth-paired (pre- vs. post-conditioning) and Aya treatment [F(2,21) = 3.12, compartments [T(23) = 0.1; p > 0.05] (data not shown). One- p < 0.05] (Figure 3A). Bonferroni post hoc test showed way ANOVA showed no significant differences between groups that animals treated with Veh before Eth administrations (Vehicle, EBc 440 or EBc 1320) during the pre-conditioning showed higher levels of CPP score in the post-conditioning test [F(2,21) = 0.92, p > 0.05] (data not shown). Two-way test compared to the pre-conditioning test, indicating a RM ANOVA showed a significant effect of time (pre- vs. post- marked preference for the Eth-paired compartment. This effect conditioning) [F(1,21) = 26.18, p < 0.0001], but not EBc was not observed for animals treated with 100 mg/kg and treatment [F(2,21) = 0.74, p > 0.05] or interaction between the 300 mg/kg Aya before Eth conditioning sessions, indicating two factors [F(2,21) = 2.00, p > 0.05] (Figure 3B). Bonferroni that the development of Eth-induced CPP was blocked by co- post hoc test showed that animals treated with Veh before administration of Aya. Eth administrations showed higher levels of CPP score in the Frontiers in Pharmacology | www.frontiersin.org 6 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 7 Cata-Preta et al. Ayahuasca and Ethanol Reward FIGURE 3 | Effects of pretreatments with (A) ayahuasca (Aya), (B) extract of Banisteriopsis caapi (EBc) or (C) extract of Psychotria viridis (EPv) before ethanol conditioning sessions on the development of ethanol-induced conditioned place preference (CPP). CPP score (difference between the time spent in the ethanol-paired and in the saline-paired compartments) during the pre-conditioning and post-conditioning test sessions. Data are reported as means SEM. p < 0.05 compared with the pre-conditioning test. post-conditioning test compared to the pre-conditioning test, Bonferroni post hoc test showed that animals treated with indicating a marked preference for the Eth-paired compartment, Veh before Eth administrations showed higher levels of CPP an effect that was not affected by treatment with EBc. score in the post-conditioning test compared to the pre- conditioning test, indicating a marked preference for the Eth- Experiment 2c (Psychotria viridis) paired compartment, an effect that was not affected by treatment Analysis of the pre-conditioning test showed that animals with EPv. had no preference for the subsequently Sal- or Eth-paired compartments [T(23) = 0.43; p > 0.05] (data not shown). One- Experiment 3: Role of the Treatment way ANOVA showed no significant differences between groups Environment in the Effects of Ayahuasca (Vehicle, EPv 12.5 or EPv 37.5) during the pre-conditioning on the Expression of Ethanol-Induced test [F(2,21) = 0.00, p > 0.05] (data not shown). Two-way CPP RM ANOVA showed a significant effect of time (pre- vs. post-conditioning) [F(1,21) = 21.15, p < 0.001], but not EPv Experiment 3a (Ethanol-Paired Compartment) treatment [F(2,21) = 0.86, p > 0.05] or interaction between Analysis of the pre-conditioning test showed that animals had no the two factors [F(2,21) = 0.21, p > 0.05] (Figure 3C). preference for the subsequently Sal- or Eth-paired compartments Frontiers in Pharmacology | www.frontiersin.org 7 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 8 Cata-Preta et al. Ayahuasca and Ethanol Reward FIGURE 4 | Effects of post-conditioning treatments with ayahuasca on the expression ethanol-induced conditioned place preference (CPP). (A,C) CPP score (difference between the time spent in the ethanol-paired and in the saline-paired compartments) during the pre-conditioning test and the post-ethanol conditioning test sessions. (B,D) CPP score during the post-treatment test conducted after treatment with ayahuasca (Aya) in the (B) ethanol- or (D) saline-paired compartments and subsequent ethanol reexposure. Data are reported as means SEM. p < 0.05 compared with the Pre-Conditioning Test; p < 0.05 compared with the Vehicle group. [T(23) = 0.53; p > 0.05] (data not shown). Paired samples t-test followed by Bonferroni post hoc test showed that animals showed that animals displayed a significant increase in CPP treated with 100 mg/kg, but not 300 mg/kg, Aya displayed a score during the post-conditioning test compared to the pre- significant decrease in CPP score during the post-treatment test conditioning test [T(23) = 4.32; p < 0.001] (Figure 4A). In the compared to Veh-treated animals [F(2,21) = 2.3, p < 0.05] post-treatment test, one-way ANOVA followed by Bonferroni (Figure 4D), indicating that the expression of Eth-induced CPP post hoc test showed that animals treated with 100 and 300 mg/kg was blocked by treatment with 100 mg/kg Aya in the Sal-paired Aya displayed a significant decrease in CPP score during the post- compartment. treatment test compared to Veh-treated animals [F(2,21) = 7.31, p < 0.01] (Figure 4B), indicating that the expression of Eth- Environment vs. Treatment Interaction induced CPP was blocked by treatment with Aya in the Eth- When performing a combined analysis of the results for paired compartment. Experiments 3a and 3b, two-way ANOVA showed a significant interaction effect between treatment environment (Eth- vs. Experiment 3b (Saline-Paired Compartment) Sal-paired compartment) and Aya treatment [F(2,42) = 4.15, Analysis of the pre-conditioning test showed that animals had no p < 0.05]. Bonferroni post hoc test showed that animals preference for the subsequently Sal- or Eth-paired compartments treated with 300 mg/kg Aya in the Eth-paired environment [T(23) = 0.63; p > 0.05] (data not shown). Paired samples showed lower levels of CPP score during the post-treatment t-test showed that animals displayed a significant increase test compared to animals treated with 300 mg/kg Aya in CPP score during the post-conditioning test compared in the Sal-paired environment. No statistically significant to the pre-conditioning test [T(23) = 8.38; p < 0.0001] differences were observed for groups treated with Veh and (Figure 4C). In the post-treatment test, one-way ANOVA 100 mg/kg Aya. Frontiers in Pharmacology | www.frontiersin.org 8 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 9 Cata-Preta et al. Ayahuasca and Ethanol Reward Experiment 4b (Saline-Paired Compartment) Experiment 4: Role of the Treatment Analysis of the pre-conditioning test showed that animals had no Environment in the Effects of preference for the subsequently Sal- or Eth-paired compartments Banisteriopsis caapi on the Expression [T(23) = 0.01; p > 0.05] (data not shown). Paired samples t-test of Ethanol-Induced CPP showed that animals displayed a significant increase in CPP Experiment 4a (Ethanol-Paired Compartment) score during the post-conditioning test compared to the pre- Analysis of the pre-conditioning test showed that animals conditioning test [T(23) = 2.69; p < 0.05] (Figure 5C). In the had no preference for the subsequently Sal- or Eth-paired post-treatment test, one-way ANOVA followed by Bonferroni compartments [T(23) = 1.19; p > 0.05] (data not shown). post hoc test showed that showed that animals treated with Paired samples t-test showed that animals displayed a significant 440 mg/kg, but not 1320 mg/kg, EBc displayed a significant increase in CPP score during the post-conditioning test decrease in CPP score during the post-treatment test compared compared to the pre-conditioning test [T(23) = 2.91; p < 0.01] to Veh-treated animals [F(2,29) = 5.86, p < 0.01] (Figure 5D), (Figure 5A). In the post-treatment test, one-way ANOVA indicating that the expression of Eth-induced CPP was blocked followed by Bonferroni post hoc test showed that animals by treatment with 440 mg/kg EBc in the Sal-paired compartment. treated with 1320 mg/kg, but not 440 mg/kg, EBc displayed a significant decrease in CPP score during the post-treatment test Environment vs. Treatment Interaction compared to Veh-treated animals [F(2,21) = 5.72, p < 0.05] When performing a combined analysis of the results for (Figure 5B), indicating that the expression of Eth-induced CPP Experiments 4a and 4b, two-way ANOVA showed a significant was blocked by treatment with 1320 mg/kg EBc in the Eth-paired interaction effect between treatment environment (Eth- vs. compartment. Sal-paired compartment) and EBc treatment [F(2,42) = 3.53, FIGURE 5 | Effects of post-conditioning treatments with Banisteriopsis caapi on the expression ethanol-induced conditioned place preference (CPP). (A,C) CPP score (difference between the time spent in the ethanol-paired and in the saline-paired compartments) during the pre-conditioning test and the post-ethanol conditioning test sessions. (B,D) CPP score during the post-treatment test conducted after treatment with extract of Banisteriopsis caapi (EBc) in the (B) ethanol- or (D) saline-paired compartments and subsequent ethanol reexposure. Data are reported as means SEM. p < 0.05 compared with the Pre-Conditioning Test; p < 0.05 compared with the Vehicle group. Frontiers in Pharmacology | www.frontiersin.org 9 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 10 Cata-Preta et al. Ayahuasca and Ethanol Reward p < 0.05]. Bonferroni post hoc test showed no statistically not a high, dose of Aya exerted rewarding effects, inducing CPP significant differences between scores obtained in the Eth- vs. in mice. Pretreatment with Aya also blocked the development Sal-paired compartments for groups treated with Veh, 440 or of Eth-induced CPP at all doses tested. Both EBc and EPv had 1320 mg/kg EBc. no effects on CPP alone or on Eth-induced CPP. Treatment with Aya, EBc and EPv in the environment previously paired Experiment 5: Role of the Treatment to Eth blocked the subsequent expression of CPP induced by an Eth reexposure. Regarding treatments in the Sal-paired Environment in the Effects of Psychotria compartment, administration of an intermediate, but not a high, viridis on the Expression of dose of Aya and EBc, as well as administration of EPv at both Ethanol-Induced CPP doses tested, blocked the subsequent expression of Eth-induced Experiment 5a (Ethanol-Paired Compartment) CPP. Statistical analysis of our findings showed a significant Analysis of the pre-conditioning test showed that animals had no interaction effect between treatment environment (Eth- vs. Sal- preference for the subsequently Sal- or Eth-paired compartments paired compartment) and Aya or EBc, but not EPv, treatments, [T(23) = 0.36; p > 0.05] (data not shown). Paired samples t-test suggesting that the treatment environment seems to play an showed that animals displayed a significant increase in CPP important role in the effects of Aya and EBc on Eth reward. score during the post-conditioning test compared to the pre- Compelling evidence indicates that the effects of Aya are conditioning test [T(23) = 2.86; p < 0.01] (Figure 6A). In the mediated by serotonin 5-HT and 5-HT receptors. Firstly, 2A 2C post-treatment test, one-way ANOVA followed by Bonferroni DMT is a 5-HT receptor agonist, which has been proposed 2A=2C post hoc test showed that animals treated with 12.5 and to mediate its psychoactive properties (Glennon et al., 2000). 37.5 mg/kg EPv displayed a significant decrease in CPP score Secondly, tetrahydroharmine is a serotonin reuptake inhibitor during the post-treatment test compared to Veh-treated animals (Buckholtz and Boggan, 1977), and harmine binds to serotonin 5- [F(2,21) = 4.55, p < 0.05] (Figure 6B), indicating that the HT receptors (Grella et al., 1998; Glennon et al., 2000). The 2A=2C expression of Eth-induced CPP was blocked by treatment with effects of harmine at 5-HT receptors have been proposed 2A=2C EPv in the Eth-paired compartment. to be an agonist mechanism (Brierley and Davidson, 2013); however, a 5-HT receptor agonist or antagonist action is yet to Experiment 5b (Saline-Paired Compartment) be confirmed. Importantly, 5-HT and 5-HT receptors play 2A 2C Analysis of the pre-conditioning test showed that animals had no an important role in mediating the influence of serotonin in preference for the subsequently Sal- or Eth-paired compartments drug abuse and in the rewarding properties of drugs (Bubar and [T(23) = 0.22; p > 0.05] (data not shown). Paired samples t-test Cunningham, 2006, 2008; Craige and Unterwald, 2013; Howell showed that animals displayed a significant increase in CPP and Cunningham, 2015). Because of the regional distribution score during the post-conditioning test compared to the pre- of those receptors in the brain, they exert opposing effects on conditioning test [T(23) = 3.59; p < 0.01] (Figure 6C). In the drug abuse through dopamine-dependent mechanisms (Manvich post-treatment test, one-way ANOVA followed by Bonferroni et al., 2012a,b; Murnane et al., 2013; Berro et al., 2017). post hoc test showed that animals treated with 12.5 and Particularly, activation of 5-HT and blockade of 5-HT 2C 2A 37.5 mg/kg EPv displayed a significant decrease in CPP score receptors have been proposed to decrease dopamine levels in during the post-treatment test compared to Veh-treated animals the nucleus accumbens (NAcc) (Howell and Cunningham, 2015). [F(2,21) = 6.3, p < 0.01] (Figure 6D), indicating that the Of note, the development and induction of CPP are dependent expression of Eth-induced CPP was blocked by treatment with on drug-induced increases in NAcc dopamine levels (Koob and EPv in the Sal-paired compartment. Bloom, 1988). Although DMT is a 5HT receptor agonist, studies have 2A=2C Environment vs. Treatment Interaction shown that it has higher affinity for 5-HT compared to 5- 2A When performing a combined analysis of the results for HT receptors (Glennon et al., 2000). Thus, we propose that 2C Experiments 5a and 5b, two-way ANOVA showed a significant DMT-induced activation of 5-HT receptors would have distinct effect of EPv treatment [F(2,42) = 10.66, p < 0.001], but rewarding properties depending on the dose being administered. not treatment environment (Eth- vs. Sal-paired compartment) At low to intermediate doses, DMT would bind preferentially at [F(1,42) = 0.24, p > 0.05] or interaction between the two factors 5-HT receptors, which would then mediate its psychoactive 2A [F(2,42) = 0.63, p > 0.05]. Bonferroni post hoc test showed no properties. By activating 5-HT receptors, which are highly 2A statistically significant differences between scores obtained in the expressed in dopaminergic neurons in the ventral tegmental area Eth- vs. Sal-paired compartments for groups treated with Veh, (VTA) (Howell and Cunningham, 2015), DMT would increase 12.5 or 37.5 mg/kg EPv. dopamine levels in the NAcc and exert rewarding effects. This mechanism would be responsible for the CPP induced by Aya at an intermediate dose in the present study. However, with DISCUSSION increased dosage, DMT would then bind non-specifically at both In the present study we investigated the rewarding properties of 5-HT and 5-HT receptors. Activation of 5-HT receptors, 2A 2C 2C Aya and its plant components, Banisteriopsis caapi (EBc) and which are highly expressed in GABAergic interneurons within Psychotria viridis (EPv), and their effects on Eth reward using the VTA (Howell and Cunningham, 2015), would antagonize the CPP paradigm. Our findings show that an intermediate, but the effects of 5-HT receptor activation on NAcc dopamine 2A Frontiers in Pharmacology | www.frontiersin.org 10 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 11 Cata-Preta et al. Ayahuasca and Ethanol Reward FIGURE 6 | Effects of post-conditioning treatments with and Psychotria viridis on the expression ethanol-induced conditioned place preference (CPP). (A,C) CPP score (difference between the time spent in the ethanol-paired and in the saline-paired compartments) during the pre-conditioning test and the post-ethanol conditioning test sessions. (B,D) CPP score during the post-treatment test conducted after treatment with extract of Psychotria viridis (EPv) in the (B) ethanol- or (D) saline-paired compartments and subsequent ethanol reexposure. Data are reported as means SEM. p < 0.05 compared with the Pre-Conditioning Test; p < 0.05 compared with the Vehicle group. neurochemistry and prevent the development of CPP to Aya at being high enough to exert 5-HT agonist action, and thereby 2C higher doses, as observed in the present study. block DMT-induced CPP mediated by 5-HT activation. In fact, 2A Based on the hypothesis that higher doses of DMT would although not statistically significant, increased dosage of EPv exert higher 5-HT receptor activation, EPv would also be seemed to have a trend in inducing conditioned place avoidance 2C expected to induce CPP at intermediate, but not high, doses. under our experimental conditions (p = 0.1), which corroborates In the present study, however, EPv did not induce CPP at any a higher 5-HT -mediated decrease in NAcc dopamine levels 2C of the doses tested. The lack of effects observed for EPv could induced by higher doses of i.p. EPv. be due to the administration route used for EPv in the present Conditioning with EBc was not effective in inducing CPP study. While Aya and EBc were administered orally, EPv was under our experimental conditions, suggesting that its effects at administered i.p. due to expected DMT breakdown by MAO-A 5-HT receptors at the doses tested in the present study were 2A=2C in the gastrointestinal tract. Of note, previous studies in humans not meaningful. EBc also had no effects on the development of have suggested that b-carboline-induced MAO inhibition is Eth-induced CPP. Aya, on the other hand, decreased CPP to Eth mainly peripheral and short-lived, and only allows around 15% of when administered before conditioning sessions. Interestingly, the DMT to reach systemic circulation (Riba, 2003; Domínguez- pretreatment with EPv also had no effects on Eth-induced CPP. Clavé et al., 2016). Thus, with oral administration of Aya, the Thus, our findings suggest that Aya-induced modulation of the proportion of DMT that reaches the brain would be much lower development of Eth reward is dependent on the presence of than that of i.p. EPv, even though the DMT concentration in both both EBc and EPv, and seems to be a 5-HT , rather than 2A=2C extracts was the same across doses. Based on this hypothesis, i.p. 5-HT , receptor-dependent mechanism. Ishiguro et al. (2016) 2C EPv would be expected to induce CPP at lower doses than those have previously shown that the reduction of serotonergic neurons used in the present study, with the doses of EPv used herein induced by chronic prenatal Eth exposure was alleviated by Frontiers in Pharmacology | www.frontiersin.org 11 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 12 Cata-Preta et al. Ayahuasca and Ethanol Reward concomitant administration of a 5-HT receptor agonist. In When given in the Sal-paired compartment, EPv also blocked 2A=2C this scenario, our findings suggest that Aya-mediated activation the expression of Eth-induced CPP at all doses tested, suggesting of 5-HT receptors blocked the development of Eth- that activation of 5-HT receptors mediates those effects of 2A=2C 2A=2C induced CPP by preventing Eth-induced changes in serotonergic Aya. Corroborating our previous hypothesis, high doses of i.p. neurotransmission. An optimum ratio of 5-HT receptor EPv would be expected to induce higher activation of 5-HT 2A=2C 2C activation seems to be necessary in order to exert those effects, receptors, which would ultimately decrease NAcc dopamine because higher DMT doses (EPv) did not prevent Eth-induced levels and block the expression of CPP to Eth (Pina and CPP. Importantly, because all three treatments were capable of Cunningham, 2014). However, similarly to Aya, EBc blocked decreasing the expression of Eth-induced CPP, one cannot rule the expression of Eth-induced CPP when administered at an out the possibility that Aya might also be exerting rewarding intermediate dose in the Sal-paired compartment. Thus, our effects and affecting the development of Eth-induced CPP via findings suggest that increasing the dose of EBc in the Aya other chemicals that are not present in the plant extracts but are mixture seems to counteract some of the effects of EPv. Because present in the Aya concoction. this effect was only observed in an experimental condition Our findings also show that chronic treatment with Aya after associated with low brain dopamine levels (presentation to Eth conditioning blocked the subsequent expression of CPP to a neutral, previously habituated environment), EBc seems Eth. Thus, Aya-mediated activation of 5-HT receptors also to modulate Eth-induced CPP through mechanism that are 2A=2C seems to modulate previously established Eth reward. This effect dependent on dopamine availability. seems to be mediated not only by the presence of DMT, but also Finally, a parameter that could be influencing the effects by the presence of b-carbolines in the Aya preparation. When observed herein is locomotor activity. Treatment with Aya and administered in the previously Eth-associated environment, a its components could have affected locomotor activity in animals high dose of EBc, as well as both doses of EPv, also blocked conditioned to Eth, and therefore decreased time spent in the the expression of CPP to Eth. Therefore, although 5-HT Eth-associated compartment during the post-treatment test by 2A=2C activation seems to be sufficient and more effective at blocking increasing locomotor activity and exploration (Experiments 3–5). the expression of CPP to Eth, b-carboline-mediated mechanisms When looking into locomotor activity data in the present study, also appear to modulate behavioral responses induced by Eth- we observed that during the post-conditioning test, animals environment conditioning. showed significantly higher levels of CPP score and locomotor As previously mentioned, b-carbolines have been proposed activity score (difference between the distance traveled in the Eth- to exert psychoactive effects through 5-HT receptors. and Sal-paired compartments) compared to the pre-conditioning 2A=2C However, those mechanisms do not seem to account for the test (Experiments 3, data not shown). Those data suggest that results obtained in the present study because EBc did not animals spent more time and explored more the Eth-paired induce CPP alone or exert effects on the development of Eth- compartment compared to the Sal-paired compartment. During induced CPP. Instead, we hypothesize that other b-carboline- the post-treatment test, however, no statistically significant mediated mechanisms are responsible for the effects of EBc differences were observed for locomotor activity scores between on the expression of Eth-induced CPP. b-carbolines have been groups (Experiments 3, data not shown). Animals no longer shown to exert several effects in brain neurotransmission, not showed a difference between the distance traveled in the Sal- only through MAO-A inhibition and serotonin receptor binding, vs. Eth-paired compartments, suggesting a habituation factor but also through dual specificity tyrosine-dependent kinase 1A resulting in lower levels of exploratory behavior. Those results (DYRK1A) inhibition, dopamine transporter (DAT) inhibition, corroborate previous findings from our laboratory showing and imidazoline I receptor binding (Brierley and Davidson, that the same Aya extract used in the present study did not 2012). Thus, although the mechanisms underlying EBc-induced affect baseline locomotion or induce locomotor sensitization in blockade of Eth-induced CPP remain unknown, this is the first mice, and instead decreased Eth-induced hyperlocomotion and study to show that EBc alone could exert therapeutic potential locomotor sensitization (Oliveira-Lima et al., 2015). Importantly, for the treatment of ethanol abuse without exerting addictive the results obtained for locomotor activity did not correlate to the properties. results obtained for time score, as animals in the Vehicle control Finally, the effects observed with Aya treatment were context- group, for instance, still expressed CPP do Eth during the post- dependent: when administered in the previously Eth-associated conditioning test, an effect that was blocked by treatment with compartment, its effects were observed for both doses tested, Aya. Thus, analysis of locomotor activity data does not support while only the intermediate dose blocked the expression of Eth- an influence of this parameter on the expression of CPP to Eth. induced CPP when Aya was administered in the Sal-paired In summary, although we have not directly investigated the compartment. Therefore, Aya’s ability to induce rewarding effects molecular mechanisms underlying the present findings, our predicted its ability to block the expression of CPP to Eth when results suggest that the expression of the Aya reward is dependent Aya was administered in a neutral (Sal-paired) environment. By on a specific 5-HT occupancy ratio that can be modulated 2A=2C exerting rewarding properties when administered in a neutral by the quantity of EBc and EPv present in the Aya batch compartment, a rewarding dose of Aya would modify the and by the route of administration. Of clinical importance, we incentive salience of the CPP apparatus (Valyear et al., 2017), propose that a proper 5-HT occupancy could engender 2A=2C and animals would no longer express CPP to Eth during the therapeutic efficacy without exerting abuse liability. By showing post-treatment test. that both EBc and EPv exerted therapeutic effects alone, our Frontiers in Pharmacology | www.frontiersin.org 12 May 2018 | Volume 9 | Article 561 fphar-09-00561 May 25, 2018 Time: 17:50 # 13 Cata-Preta et al. Ayahuasca and Ethanol Reward findings also indicate that the combination of EBc and EPv in the All authors agree to be accountable for all aspects of the work form of Aya beverage seems to have unique therapeutic utility. in ensuring that questions related to the accuracy or integrity Importantly, under our experimental conditions, the treatment of any part of the work are appropriately investigated and environment influenced the therapeutic effects of Aya and EBc, resolved. but not EPv, which could be relevant in the context of clinical and/or religious/ritual uses of Aya. The present study further emphasizes the complexity of the Aya formulation and its effects, FUNDING and highlights the need for further investigations in the area. Funding for this study was provided by grants from Fundação Future studies investigating the effects of varying EBc/EPv ratios de Amparo à Pesquisa do Estado da Bahia (FAPESB), Conselho on the therapeutic properties of Aya are needed in order to ensure Nacional de Desenvolvimento Científico e Tecnológico (CNPq) the safety and best therapeutic strategy in using this beverage for and Coordenação de Aperfeiçoamento de Pessoal de Nível the treatment of ethanol abuse. Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grant number 2015/10650-8, JC). and by an intramural funding from Universidade Estadual de AUTHOR CONTRIBUTIONS Santa Cruz. AO-L, LB, and EM were responsible for the study concept and design. EC-P, YS, EM-J, HR, NK, ML-S, RS, TB-S, LS, and JC contributed to the acquisition of data. EC-P, PB, LB, and ACKNOWLEDGMENTS EM assisted with data analysis and interpretation of findings. EG-P, PB, LB, and EM drafted the manuscript. All authors The authors would like to thank Mr. José Carlos Santos de provided critical revision of the manuscript for important Oliveira for his capable technical support. They also thank intellectual content and approved final version for publication. Adilson R. R. Castro for the donation of ayahuasca. Da Silveira, D. X., Grob, C. S., de Rios, M. D., Lopez, E., Alonso, L. K., Tacla, C., REFERENCES et al. (2005). Ayahuasca in adolescence: a preliminary psychiatric assessment. Aricioglu-Kartal, F., Kayir, H., and Tayfun Uzbay, I. (2003). Effects of J. Psychoactive Drugs 37, 129–133. doi: 10.1080/02791072.2005.10399792 harman and harmine on naloxone-precipitated withdrawal syndrome in Doering-Silveira, E., Grob, C. S., de Rios, M. D., Lopez, E., Alonso, L. K., morphine-dependent rats. Life Sci. 73, 2363–2371. doi: 10.1016/S0024-3205(03) Tacla, C., et al. (2005). Report on psychoactive drug use among adolescents 00647-7 using ayahuasca within a religious context. J. 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Wuo-Silva, R., et al. (2015). Effects of rimonabant on the development of single dose-induced behavioral sensitization to ethanol, morphine and cocaine in Copyright © 2018 Cata-Preta, Serra, Moreira-Junior, Reis, Kisaki, Libarino-Santos, mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 58, 22–31. doi: 10.1016/j. Silva, Barros-Santos, Santos, Barbosa, Costa, Oliveira-Lima, Berro and Marinho. pnpbp.2014.11.010 This is an open-access article distributed under the terms of the Creative Commons Mason, B. J. (2017). Emerging pharmacotherapies for alcohol use disorder. Attribution License (CC BY). The use, distribution or reproduction in other forums Neuropharmacology 122, 244–253. doi: 10.1016/j.neuropharm.2017.04.032 is permitted, provided the original author(s) and the copyright owner are credited McKenna, D. J. (2004). 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