This mouse study 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.
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.
Papers cited by this study that are also in Blossom
Bouso, J. C., González, D., Fondevila, S. et al. · PLOS ONE (2012)
Santos, R. G., Landeira-Fernandez, J., Strassman, R. J. et al. · Journal of Ethnopharmacology (2007)
Argento, E., Capler, R., Thomas, G. et al. · Drug and Alcohol Review (2019)
Palhano-Fontes, F., Barreto, D., Onias, H. et al. · Psychological Medicine (2018)
Alcohol use disorder (AUD) is a major public‑health problem with high morbidity and mortality and limited effective treatments; relapse rates exceed 50% and are higher when anxiety disorders co‑occur. Previous clinical and preclinical work has suggested that psychedelic substances, including ayahuasca (AYA), may have therapeutic effects in depression, anxiety and substance use disorders. Ayahuasca is a traditional Amazonian decoction combining Psychotria viridis leaves, which contain N,N‑dimethyltryptamine (DMT), and Banisteriopsis caapi vines, which provide β‑carbolines (harmine, harmaline, tetrahydroharmine) that inhibit monoamine oxidase A and permit oral DMT activity. Ethanol affects dopaminergic, serotonergic and opioid systems and produces neuroplastic adaptations; dynorphin (and its precursor prodynorphin) and κ‑opioid receptors have been implicated in anxiety, depression and addiction, and ethanol‑induced behavioural sensitization (EIBS) is a model of ethanol‑related neuroadaptation measured as progressively enhanced locomotor activation following repeated exposure. Almeida and colleagues set out to evaluate whether oral ayahuasca administration modifies ethanol‑induced behavioural sensitization and withdrawal‑related anxiety in mice, and whether AYA alters molecular markers in striatum and hippocampus related to dopamine (D1, D2), serotonin (5‑HT1a, 5‑HT2a) and the dynorphin/prodynorphin system. The study therefore combined behavioural testing (open field locomotion for sensitization and elevated plus maze for anxiety) with biochemical assays (UPLC‑ESI‑MS/MS quantification of AYA alkaloids and Western blotting of receptor and peptide levels) to probe potential neuroplastic effects of AYA on ethanol responses.
Seventy‑two adult male Swiss mice (6–7 weeks old) were used; they were group‑housed under controlled temperature and a 12:12 light:dark cycle. One animal died during the protocol (in the SAL/AYA/ET group). Experimental procedures were approved by the institutional ethics committee. Ethanol was prepared as 15% v/v in saline and administered intraperitoneally at 2.2 g/kg. Lyophilised ayahuasca tea was reconstituted and dosed orally by gavage to deliver 1.76 mg DMT/kg per day. Alkaloid content of the lyophilised AYA was quantified by UPLC‑ESI‑MS/MS using a Waters UPLC system with tandem MS in positive ion mode and multiple reaction monitoring; samples were diluted in ammonium formate/formic acid buffer and injected (5 μL) with DMT‑d6 as internal standard. Sample preparation followed a validated multi‑step dilution procedure. Behavioral apparatus included an open field (40 cm diameter) with automated tracking (EthoVision) for locomotion and an elevated plus maze (two open and two closed arms) for anxiety‑related measures. Grooming and head‑dipping were scored manually from recorded videos by a trained, blinded observer. The behavioural protocol comprised three phases. During acquisition mice received IP injections of ethanol (2.2 g/kg) or saline every other day over nine days (D1, D3, D5, D7, D9) and locomotion was recorded for 10 min after a 5‑minute post‑injection home‑cage interval. During an eight‑day withdrawal/treatment phase mice were given AYA (1.76 mg DMT/kg) or water by gavage daily; on day 7 of this phase animals were assessed on the elevated plus maze (five minutes, six hours after AYA). In the expression phase animals were challenged with a single injection of ethanol or saline and placed in the open field for 10 min to test sensitization expression. Groups followed a factorial design (examples include SAL/SAL/ET and SAL/SAL/SAL) with nominal group sizes around n = 10 reported for some groups. Blood ethanol concentrations were measured by headspace extraction and GC‑FID. For Western blotting, striatum and hippocampus were dissected after euthanasia, homogenised, and protein quantified; specified protein loads (e.g. 75–200 μg depending on target) were separated by SDS‑PAGE, transferred to PVDF membranes and probed with antibodies to D1, D2, 5‑HT1a, 5‑HT2a, dynorphin A and prodynorphin, with β‑actin normalisation. Statistical analysis used repeated‑measures ANOVA for acquisition, factorial ANOVA for expression and elevated plus maze data (with Bonferroni post‑hoc tests), and one‑way ANOVA with Tukey post‑hoc for Western blots. Significance was set at p < 0.05 and data are reported as mean ± SEM.
Alkaloid quantification confirmed the presence of DMT and β‑carbolines in the lyophilised ayahuasca (table values reported in the paper). Habituation locomotor data showed no group differences. During the acquisition phase repeated‑measures ANOVA identified a significant main effect of time (F4,236 = 5.60, p < 0.001) and a treatment × time interaction (F4,236 = 3.69, p < 0.001). Mice repeatedly given ethanol showed significantly greater locomotor activity than saline controls on all acquisition days (p < 0.05) and activity in ethanol‑treated animals increased from Day 1 to Day 9 (p < 0.001), indicating development of behavioural sensitization. Following the withdrawal/treatment period, challenge‑day locomotor activity (expression phase) yielded a significant treatment effect by factorial ANOVA (F1,57 = 6.33, p < 0.001). Specifically, mice that had received ethanol in acquisition and ayahuasca during withdrawal/treatment (ET‑AYA‑ET group) showed significantly lower locomotor activity on challenge than ethanol‑sensitised mice that received vehicle during withdrawal (ET‑SAL‑ET; p < 0.05), indicating attenuation of EIBS by AYA. Plasma ethanol concentrations measured after the final test did not differ among ethanol‑treated groups. Elevated plus maze data showed an acquisition phase × withdrawal interaction for number of open‑arm entries (F1,57 = 5.38, p < 0.01); the ET‑AYA‑ET group had increased entries into open arms compared with SAL‑SAL‑ET, SAL‑AYA‑ET and ET‑SAL‑ET groups (p < 0.01), interpreted by the authors as reduced anxiety‑like behaviour during withdrawal after AYA. Grooming frequency exhibited significant treatment effects during acquisition and withdrawal (both F1,57 = 10.11, p < 0.01) and an acquisition × withdrawal interaction (F1,57 = 9.05, p < 0.01); the ET‑SAL‑ET group showed increased grooming relative to the other groups (p < 0.001). No group differences were observed for head‑dipping. Western blot analyses revealed region‑ and marker‑specific changes. In striatum, D1 receptor levels were reduced in the SAL‑SAL‑ET, SAL‑AYA‑ET, ET‑SAL‑ET and ET‑AYA‑ET groups relative to the SAL‑SAL‑SAL baseline group (p < 0.05), whereas D2 levels showed no differences. Dynorphin and prodynorphin absolute levels did not differ across groups in the striatum, but the dynorphin/prodynorphin (DYN/pDYN) ratio was decreased in SAL‑SAL‑ET, SAL‑AYA‑ET, ET‑SAL‑ET and ET‑AYA‑ET compared with SAL‑SAL‑SAL (p < 0.001). Notably, ET‑AYA‑ET animals showed an increased DYN/pDYN ratio relative to ET‑SAL‑ET (p < 0.001), suggesting partial reversal of ethanol‑induced change by AYA. In the hippocampus, the ET‑SAL‑ET group had elevated 5‑HT1a receptor levels compared with SAL‑SAL‑SAL, SAL‑AYA‑ET and ET‑AYA‑ET groups (p < 0.01). Prodynorphin levels were lower in ET‑SAL‑ET versus SAL‑SAL‑SAL (p < 0.01). The hippocampal DYN/pDYN ratio decreased in SAL‑AYA‑ET (p < 0.01) and ET‑SAL‑ET (p < 0.001) groups compared with SAL‑SAL‑ET. Overall, AYA treatment during withdrawal prevented the ethanol‑associated increase in hippocampal 5‑HT1a and the prodynorphin change, and modified dynorphin/prodynorphin balance in striatum and hippocampus in ways the authors highlight as relevant to anxiety and sensitization.
Almeida and colleagues interpret their findings to indicate that eight days of oral ayahuasca (1.76 mg DMT/kg) attenuated the expression of ethanol‑induced behavioural sensitization in mice and reduced anxiety‑like behaviour during ethanol withdrawal. The authors note that AYA prevented ethanol‑induced upregulation of hippocampal 5‑HT1a receptors and opposed ethanol effects on prodynorphin in the hippocampus and on the dynorphin/prodynorphin ratio in the striatum. They therefore propose that modulation of serotonergic and dynorphinergic systems may underlie the behavioural effects observed. Dose selection and route of administration are discussed: the AYA dose was calibrated to DMT content and chosen to approximate doses used in human clinical studies, and gavage was used to match oral ritual use of ayahuasca. The authors acknowledge that AYA composition can vary by plant source and preparation, justifying quantification of alkaloids in the lyophilised material. They also consider confounding effects of stress: repeated saline injections can produce cross‑sensitization to ethanol, and the authors observed enhanced challenge‑day locomotion in some saline‑treated animals, suggesting stress contributions to sensitization; ayahuasca did not affect this cross‑sensitization but did attenuate ethanol‑induced sensitization itself. The possibility that reduced EIBS after AYA reflects loss of context–drug contingency (a context‑dependent component of sensitization) is raised and not excluded. The discussion situates results relative to prior work, noting concordance with other preclinical reports of AYA or DMT‑related anxiolytic and antidepressant effects and with one study that found inhibition of ethanol sensitization after AYA. The authors highlight that, despite ethanol‑related reductions in striatal D1 receptor levels, AYA did not prevent this D1 decrease, suggesting AYA’s effects on EIBS are not mediated via restoring D1 expression. They emphasise the relevance of dynorphin/κ‑opioid signalling and serotonergic changes to addiction‑related neuroplasticity and withdrawal‑related anxiety, referencing evidence that dynorphin/κ‑opioid upregulation influences anxiety, sensitization and abstinence. Finally, the authors propose that AYA’s capacity to prevent specific ethanol‑induced molecular alterations in hippocampus and to modulate dynorphin/prodynorphin balance in striatum may underlie its behavioural effects. Key limitations acknowledged in the text include variability in AYA composition, potential stress‑related confounds, and the need for further preclinical studies to clarify mechanisms and translational relevance.
Although ethanol is a licit drug, it is associated with high morbidity and mortality. Ethanol abuse is responsible for about 3 million deaths per year worldwide, being associated with more than 200 diseases, such as cancer, cardiovascular and liver diseases, and traffic injuries, aggression, and homicides. The prevalence of current drinkers differs among regions, reaching 54.1 % in the Americas and 59.9 % in Europe. Therefore, alcohol use disorder (AUD) is most prevalent in these regions (Americas -4.1 %; Europe -3.7 %). AUD is the uncontrolled use of ethanol despite the absence or reduction of pleasant effects and the awareness of the harm caused by its repeated administration. Ethanol withdrawal may lead to symptoms of anxiety, depression, and irritability that act as a negative reinforcement for the continued use of the drug, whose mechanism involves "drinking-to-cope", a process related to negative effect. Treatments available to AUD are based on psychosocial and pharmacological therapies. The psychosocial tools include individual, group and family therapies, and the pharmacological therapies include treatment of the alcohol withdrawal symptoms (anxiolytics and antidepressants) and specific therapies that cause aversion to ethanol (disulfiram) or craving reduction (naltrexone). Thus, treatments are scarce, and relapse occurs in more than half of the patients with AUD, being even higher for patients with co-occurring anxiety disorder. In this context, psychedelic substances have been widely studied as possible therapeutic strategies for addiction treatment. Ayahuasca (AYA) is a psychoactive beverage originally from the Amazon Indians used in religious rituals such as Santo Daime and Barquinha. AYA is prepared by the decoction of the leaves of Psychotria viridis, which contain the hallucinogenic substance N,N-dimethyltryptamine (DMT), and Banisteriopsis caapi vines, whose main constituents are β-carbolines alkaloids, such as harmine (HRM), harmaline (HRL), and tetrahydroharmine (THH). The synergistic effect of both plants is explained by the monoamine oxidase type A inhibition of the β-carbolines alkaloids, which allows DMT absorption by oral administration. Clinical studies have shown the beneficial effects of AYA in the treatment of depression, anxiety, and addiction. Preclinical studies also showed that AYA compounds, such as harmine and others β-carbolines, induced anxiolytic and antidepressant-like effects; however, chronic exposure to AYA tea did not affect anxiety and memory in mice. DMT has a similar chemical structure to serotonin and is a partial agonist at 5-HT1a, 5-HT1b, 5-HT2a, and 5-HT2c receptors, whereas THH blocks serotonin reuptake, increasing serotonin levels in the brain. Ethanol interferes in the dopaminergic, serotonergic, and opioid systemsleading to high dopamine levels in the nucleus accumbens, mainly mediated by D1 receptor, and increasing serotonin receptor expression. Dynorphin, whose precursor is prodynorphin, is a potent endogenous opioid that acts preferentially on κ-opioid receptors. Studies indicate that dynorphin and κ receptors are involved in several psychiatric disorders, including anxiety, depression and addiction. Both chronic and acute exposure to ethanol produce neuroplasticity in the opioid system that reflect changes in the levels, expression and activity of dynorphin and κ receptors. The ethanol-induced behavioral sensitization (EIBS) is a phenomenon associated with neuroadaptation induced by ethanol. It can be measured by the enhancement of locomotor activity following chronic or intermittent ethanol exposure and is divided in two phases: acquisition or development and expression. Acquisition phase is characterized by immediate molecular and cellular changes mainly in the prefrontal cortex and in the ventral tegmental area. The expression phase is related to the long-lasting consequences of these effects and the main substrate is the nucleus accumbens In general, the sensitization expression is revealed after a period of drug withdrawal upon a challenge administration. Thus, this study aimed to evaluate the effects of AYA on EIBS and in anxiety, one of the main symptoms of the ethanol withdrawal, and to investigate if AYA modulates dopamine (D1, D2) and serotonin (5-HT1a, 5-HT2a) receptors, and dynorphin and prodynorphin levels.
Seventy-two adult male Swiss mice (6-7 weeks-old) were housed in polypropylene cages (26 × 36 × 19 cm) with free access to food and water. They were housed in a room with constant temperature (22 ± 1 • C) on a 12:12 h light/dark cycle (lights on at 7:00 am). All experiments were performed during the light period and the behavioral experiments were conducted between 7:30 am and 12 pm. One animal died during the experiments (SAL/AYA/ET group). The experimental procedures were approved by the Institutional Ethical Committee of Federal University of Alfenas under the protocol 34/2017 and were in compliance with the Principles of Laboratory Animal Care of the National Institutes of Health.
Ethanol (Merck S. A., Brazil) was diluted with 0.9 % saline to obtain a 15 % v/v solution, and it was administered at a dose of 2.2 g/kg by intraperitoneal injection. The dose was based on previous studies regarding ethanol sensitization. About 1.5 L of AYA was lyophilized in small portions of 400 mL in LioTop 101 (500 μHg and -53 • C), generating 22.9 g of AYA powder, which had its alkaloids quantified for the experiments. AYA was diluted in filtered water and was administrated by gavage at a dose of 1.76 mg of DMT/kg.
Analyses were performed using a Waters UPLC (Ultra Performance Liquid Chromatography) Acquity System coupled to a Quattro Premier tandem MS (Mass Spectrometer) with electrospray ionization (ESI) operated in positive ion mode (Waters Corporation, Milford, MA). Chromatographic separation was conducted on UPLC BEH C18 2.1 mm × 100 mm, ID 1.7 μm Acquity column by using the following gradient elution of solution A (2 mM ammonium formate buffer with 0.1 % formic acid) and solution B (0.1 % formic acid in methanol). Mobile phase composition: solution A:B 90:10 (0 min), 90:10 (0.1 min), 50:50 (7 min), 50:50 (7.1 min), 90:10 (8 min) with a constant flow rate of 0.3 mL/min. Samples were analyzed using a 5 μL injection volume. The MS/MS analysis was performed using multiple reaction monitoring (MRM). The m/z transitions were 188.9 > 57.8, 116.7, 143.8* for DMT and 195.1 > 63.9, 114.9, 143.8* for the internal standard DMT-d 6 . Transitions used for quantification are indicated with an asterisk.
Sample preparation consisted of the fully validated dilution procedure described elsewhere. Briefly, 2 mM ammonium formate buffer with 0.1 % formic acid (mobile phase A) was used to dilute the samples to a final ratio of 1:5000 in three steps (1:10 × 1:10 × 1:50). After dilution, 5 μL of the diluted sample was injected in the UPLC-ESI-MS/MS system. DMT-d 6 (100 ng/mL) was added in all samples as the internal standard.
The locomotor activity of each animal was measured in the openfield apparatus (40 cm in diameter surrounded by a 50 cm-high wall). A video camera installed 3 m above the open field was connected to a computer and recorded the videos. The total locomotion was quantified by EthoVision software (Noldus Information Technology, the Netherlands). The elevated plus maze consisted of two open arms (30 × 5 × 0.25 cm) and two closed arms of the same size and height (20 cm). The maze was elevated 60 cm above the floor. Percentage of time spent in the open arms, percentage of time spent in the distal portions of the open arms (the last half of the open arms), frequency of entries into the open arms, frequency of grooming, and frequency of head dipping were analyzed in the elevated plus maze by the same trained researcher all over the study. The investigator blindly watched the videos randomly. The grooming and head dipping analyses were performed manually and were based on visual monitoring and manual scoring of each episode. The investigator considered grooming as self-cleaning (licking of forepaws, hind paws and legs; nose, face and head wash; oral friction directed to anogenital region or tail) and head dipping as exploring out the open arms (when mice lower their head far enough for the eyes to disappear).
Mice were habituated in the open field for 10 min for three consecutive days before the experiments (data not shown). Then, the effect of AYA on behavioral sensitization to ethanol was evaluated by the following protocol (3 phases): Acquisition phasemice received ethanol (2.2 g/kg) or saline intraperitoneal (IP) every other day across nine days (days 1, 3, 5, 7, and 9) and they were placed in the open field for 10 min. Withdrawal phase and treatment (8 consecutive days) -mice received AYA (1.76 mg of DMT/kg) or filtered water by gavage, and after 30 min they were placed in the open field for 10 min (data not shown). Expression phaseon the last day of the protocol, mice were challenged with ethanol (2.2 g/kg) or saline and placed in the open field for 10 min. As the peak in locomotor activation induced by ethanol occurs between 5 and 15 min after injection, in all phases, mice were kept in their home cages for the first 5 min before being placed in the open field to measure total locomotor activity. On the 7th day of withdrawal phase and treatment, mice were placed in the elevated plus maze for five minutes, six hours after treatment with AYA. This test was performed one day before the mice were tested for ethanol-induced sensitization expression. It In the behavioral experiments, the groups were divided according to a factorial design, as follow: • SAL/SAL/ET group (n = 10): animals received saline during the acquisition, water during withdrawal phase and treatment, and challenged with ethanol (expression). Immediately after the last open field test, the mice were euthanized by cervical dislocation, and their blood was collected to measure blood ethanol concentration. For the Western blot procedure, the brains were removed, and the striatum and hippocampus were manually and rapidly dissected on top of a petri dish containing ice to preserve the samples, immediately frozen in liquid nitrogen and stored at -80 • C. Anatomical atlas Paxinos and Franklin (2012)was used as guide. The same researcher performed all euthanasia and dissections of the brain structures and received appropriate training. Western blot analysis included the SAL/SAL/SAL group (animals that received saline during the acquisition, water during withdrawal phase and treatment, and challenged with saline; n = 10) as baseline reference. A summary of the experimental schedule is shown in Fig..
Blood samples were collected in heparinized microtubules (1 UI/mL) and centrifuged (10,000 rpm, 5 min) for plasma separation and then stored at -20 • C. The headspace extraction procedure was based on. Briefly, an aliquot of 100 μL of plasma was introduced into a glass vial, followed by the addition of 900 μL of distilled water and 1000 μL of n-butanol (internal standard). After heating this sealed system for 30 min at 70 • C, 0.5 mL of a vapor aliquot (headspace) was drawn using an air-tight syringe prior to the gas chromatograph analysis (gas chromatography with a flame ionization detector, GC-FID, 6890, Agilent, Palo Alto, CA, USA). The oven temperature was isothermal at 130 • C, and the injector port and detector were set to 250 • C. Separations were performed on a Poraplot Q fused-silica capillary column (10 m × 0.32 mm; Varia, Midelburg, Netherlands). The retention times for ethanol and n-butanol were 0.85 and 3.1 min, respectively.
The protocol was based on the one described by Torres et al.. Briefly, the striatum and hippocampus were dissected and stored at -• C until the homogenates were prepared in ice-cold buffer containing mM Tris-HCl (pH 7.4), 1.0 mM PMFS, and 10 μg/mL protease inhibitor cocktail (Sigma-Aldrich, St Louis, USA). The protein content in the extracts was measured with the Bradford dye method (Bradford, 1976). The whole extract was treated with Laemmli sample buffer and boiled for 5 min. Equal quantities of protein (D1, D2, and 5-HT1a: 75 μg; 5-HT2a: 100 μg; dynorphin and prodynorphin: 200 μg) from each sample were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 15 % polyacrylamide) and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking the non-specific sites with 5% (w/v) casein, the membranes were incubated overnight at 4-8 • C with the following primary antibodies: rabbit polyclonal anti-D1, 1:500 (Abcam, Cambridge, UK); rabbit polyclonal anti-D2, 1:500 (Abcam, Cambridge, UK); rabbit polyclonal anti-Dynorphin A, 1:500 (Abcam, Cambridge, UK); rabbit polyclonal anti-Prodynorphin, 1:500 (Abcam, Cambridge, UK); rabbit polyclonal anti-5-HT1a, 1:1000 (Abcam, Cambridge, UK); rabbit polyclonal anti-5HT2a, 1:500 (Abcam, Cambridge, UK). All membranes were washed with Tris-buffered saline containing 0.1 % Tween 20 and then incubated with a peroxidase-conjugated secondary antibody for 2 h at room temperature. After three washes, the immunoreactive bands were visualized using the ECL detection system (Thermo Scientific, Rockford, USA), and the images were captured with ImageQuant LAS 500 (GE Healthcare, Delaware, USA). The band intensities were quantified with IQuantCapture 500, v.1.0.0 (GE Healthcare, Delaware, USA) and the results were normalized to the intensity of β-actin (Sigma-Aldrich, St. Louis, USA).
The acquisition phase of the behavioral sensitization protocol (D1-D9) was analyzed by repeated measures of analysis of variance (ANOVA) Fig.. Experimental design. The development of behavioral sensitization was evaluated by the open field test. For habituation in the open field, the animals received IP saline for three consecutive days (H1 to H3). After 5 min, they were evaluated in the open field for 10 min. On D1, D3, D5, D7, and D9, the animals received an injection of saline or 2.2 g/kg ethanol according to the experimental group. During the eight days of withdrawal/treatment, the animals received water or ayahuasca (AYA) [1.76 mg of N,N-dimethyltryptamine (DMT)/ kg] via gavage according to the experimental group. On the 7th day of withdrawal phase and treatment, mice were placed in the elevated plus maze for 5 min. The animals then received an injection of saline or ethanol according to the experimental group to evaluate the expression of ethanol-induced behavioral sensitization (EIBS). The locomotor activity was measured in the open field for 10 min. The animals were euthanized (#) immediately after the test. followed by Bonferroni post-hoc test. The locomotor activity in the expression phase of EIBS and data from elevated plus maze test were analyzed by factorial ANOVA followed by Bonferroni post-hoc test. Western blot data were analyzed by one-way ANOVA followed by Tukey post-hoc test. Statistic 7.0 software (Statsoft, Tulsa, OK, USA) was used to analyze behavioral data and GraphPad Prism software version 6.0 was used to analyze Western blot data. p < 0.05 was considered statistically significant, and data was reported as mean ± SEM. All data were plotted by GraphPad Prism software version 6.0.
Tableshows values of HRL, HRM, THH, and DMT concentration in lyophilized AYA.
Locomotor activity during habituation showed no significant differences among the groups (data not shown). ANOVA repeated measures revealed a significant time effect (F 4,236 = 5.60, p < 0.001) and treatment × time interaction (F 4,236 = 3.69; p < 0.001; Fig.) during the acquisition phase. Animals from the ethanol group showed significantly increased locomotor activity when compared with saline group on all days during the acquisition of sensitization (p < 0.05, Fig.). In addition, animals of the ethanol group showed increased locomotor activity on Day 9 when compared with Day 1 (p < 0.001; Fig.). On the challenged day, a factorial ANOVA revealed a significant treatment effect (F 1,57 = 6.33, p < 0.001). Mice of ET-AYA-ET group had significantly lower locomotor activity when compared with the ET-SAL-ET group (p < 0.05, Fig.).). Fig.shows number of entries into the open arms. A factorial ANOVA revealed a significant interaction between acquisition phase × withdrawal (F 1,57 = 5.38, p < 0.01). Animals from ET-AYA-ET group showed increased number of entries into the open arms when compared with other groups (SAL-SAL-ET, SAL-AYA-ET, ET-SAL-ET; p < 0.01). For grooming analysis, there was a significant treatment effect during acquisition phase of EIBS and during withdrawal phase (F 1,57 = 10.11, p < 0.01; for both) and significant acquisition phase × withdrawal interaction (F 1,57 = 9.05, p < 0.01). There was an increase in the frequency of grooming for the ET-SAL-ET group when compared with the other groups (SAL-SAL-ET, SAL-AYA-ET, ET-AYA-ET; p < 0.001). There was no significant difference on head dipping measures among groups (Fig.).
Tableshows the plasma ethanol concentrations. There was no statistical difference among groups that received ethanol.
Figs.andshow the Western blot analysis of the following proteins in the striatum and hippocampus, respectively: serotoninergic receptors (5-HT1a and 5-HT2a), dynorphin, prodynorphin, dynorphin and prodynorphin ratio (DYN/pDYN), and dopaminergic receptors (D1 and D2). In the striatum, D1 levels were decreased in the SAL-SAL-ET, SAL-AYA-ET, ET-SAL-ET, and ET-AYA-ET groups when compared with the SAL-SAL-SAL group (p < 0.05, Fig.), whereas no differences were observed in D2 levels (Fig.). Although dynorphin and prodynorphin levels were equal for all groups, the dynorphin/prodynorphin (DYN/ pDYN) ratio decreased in the SAL-SAL-ET, SAL-AYA-ET, ET-SAL-ET, and ET-AYA-ET groups when compared with the SAL-SAL-SAL group (p < 0.001, Fig.). Additionally, an increase in the DYN/pDYN ratio of the ET-AYA-ET group was observed when compared with the ET-SAL-ET group (p < 0.001, Fig.). In the hippocampus, the ET-SAL-ET group had increased 5-HT1a levels when compared with the SAL-SAL-SAL, SAL-AYA-ET, and ET-AYA-ET groups (p < 0.01, Fig.). Moreover, the prodynorphin levels were lower in the ET-SAL-ET group when compared with the SAL-SAL-SAL group (p < 0.01, Fig.). The DYN/pDYN ratio decreased in the SAL-AYA-ET (p < 0.01) and ET-SAL-ET (p < 0.001) groups when compared with SAL-SAL-ET (Fig.).
A clinical study has indicated that AYA, in a religious context, has been effective as a treatment for drug addiction. However, more preclinical studies are required for a better understanding of the effects of AYA. We found that oral administration of AYA during eight consecutive days at a dose of 1.76 mg DMT/kg was effective to attenuate the expression of ethanol-induced behavioral sensitization in mice. We also observed that AYA caused an anxiolytic effect during ethanol withdrawal, prevented the ethanol-induced changes on 5-HT1a receptor and prodynorphin levels in the hippocampus, and reduced ethanol effects in the dynorphin/prodynorphin in the striatum. This study was conducted with AYA dose based on the DMT levels, since AYA alkaloid levels can vary for several reasons, such as origin of the plant and how the tea is prepared. AYA was administrated by gavage since the oral route is the administration route used during the rituals and the dose used was based on clinical studies that evaluated the effect of AYA on depression, anxiety, and panic syndrome. The dose of DMT used is equivalent to 19.05 mg for a human with 70 kg, 11.75 mL of ayahuasca tea. Galvão-Coelho et al. (2020)showed antidepressant effects of AYA with a dose equivalent to 25.2 mg for a human with 70 kg, which is similar to the dose used in this study. Behavioral sensitization to ethanol can be influenced by several factors, such as the dose of ethanol, the period of treatment, age and animal species, and stress. Repeated stress might induce behavioral sensitization to ethanol, a phenomenon known as cross-sensitization, which has already been described between the stress from repeated saline injections and drugs of abuse, either ethanol or cocaine. Consistent with these studies, we observed that the stress of Notes: HRLharmaline; HRMharmine; THHtetrahydroharmine; DMT -N, N-dimethyltryptamine. repeated injections of saline contributed to enhanced locomotor activity when these mice were challenged with ethanol, since the locomotor activity in the challenge day was higher when compared with the acquisition phase in the animals that received saline. Ayahuasca did not affect cross-sensitization of stress from repeated saline injections; however, it was able to attenuate EIBS. Since EIBS has a context-dependent component, we cannot discard the possibility that reduced EIBS by AYA was partially due to the loss of contingency between the context and ethanol effects. Our results are in agreement with those of Oliveira-Lima et al., who reported inhibition of the expression of ethanol sensitization in mice after eight days of IP treatment with AYA (100 mg and Anxiety is one of the most common symptoms of the withdrawal phase of addictsand anxiety-like behavior is well demonstrated in rats and mice undergoing ethanol withdrawal. Several clinical studies involving AYA tea consumers showed that AYA decreased anxiety symptoms. Our results showed that AYA prevented the anxiety-like effect during ethanol withdrawal phase in a way unrelated to risk behavior, since no statistical difference were observed in head dipping data. A preclinical study with DMT corroborates this, showing that DMT leads to an antidepressant and anxiolytic effect in rats. Nevertheless, a study evaluating the effects of long-term AYA administration (30 days, daily) on memory and anxiety in rats did not find any changes. Therefore, although isolated compounds of AYA had an anxiolytic effect in mice; to our knowledge, this is the first preclinical study that shows an anxiolytic effect of the AYA tea. Another study revealed the antidepressant effect of ayahuasca in a model of depression in a non-human primate. In this study, both acute and repeated ethanol administration decreased D1 levels in the striatum, and AYA treatment was unable to prevent this effect, suggesting that AYA does not inhibit EIBS expression by changing D1 receptors. These receptors seem to be important in the ethanol sensitization process; Camarini et al.found sensitization is preferably dependent on D1 receptors rather than D2 receptors, since only selective D1 receptor antagonists were able to block the expression of EIBS. In the hippocampus, we observed that AYA prevented the increase in 5-HT1a receptor levels induced by repeated administration of ethanol. Serotonergic receptors are associated with anxiety, one of the main symptoms observed during ethanol withdrawal. In fact, chronic voluntary ethanol intake induced a supersensitivity of 5-HT1a receptors in male C57BL/6 J mice. A postmortem study showed that levels of dynorphin A and dynorphin B were elevated in the hippocampus of AUD patients. Preclinical studies have demonstrated that acute ethanol administration leads to increased prodynorphin and dynorphin expression in the amygdala and the prefrontal cortex. Other studies demonstrated that chronic ethanol administration increased the levels and expression of dynorphin in the paraventricular nucleus of the hypothalamus and prodynorphin in the nucleus accumbens. The upregulation of the dynorphin/κ-opioids receptor ratio in several brain regions influenced anxiety, ethanol sensitization, and abstinence. In addition, Kuzmin et al.suggest that the dynorphin system mediates ethanol effects on learning and memory impairment. We observed that ethanol decreased the dynorphin/prodynorphin ratio in the striatum, effect that was partially reversed by AYA. In the hippocampus, AYA completely prevented the ethanol-induced increase in prodynorphin levels. Both areas have a role in the circuitry of addiction. The striatum, constituted by the caudate nucleus and putamen, has an essential role in cognitive and limbic functions, whereas hippocampus is related to memory as well as motivation and reward. In summary, our results suggest an eight-day AYA treatment (1.76 mg DMT/Kg animal) decreased the expression of EIBS. We also suggest AYA may have an anxiolytic effect at this dose on top of preventing the
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Galvão-Coelho, N. L., de Menezes Galvão, A. C., de Almeida, R. N. et al. · Journal of Psychopharmacology (2020)
Galvão-Coelho, N. L., de Almeida, R. N., de Menezes Galvão, A. C. et al. · Frontiers in Psychology (2019)
Sanches, R. F., Osório, F. L., Dos Santos, R. G. et al. · Journal of Clinical Psychopharmacology (2016)
Thomas, G., Lucas, P., Rielle Capler, N. et al. · Current Drug Abuse Reviews (2013)
Dos Santos, R. G., Osório, F. L., Crippa, J. A. et al. · brazilian Journal of Psychiatry (2016)
da Silveira, D. X., Doering-Silveira, E., Grob, C. S. et al. · Journal of Psychoactive Drugs (2005)
Cameron, L. P., Benson, C. J., Dunlap, L. E. · ACS Chemical Neuroscience (2018)
Favaro, V. M., Yonamine, M., Soares, J. C. K. et al. · PLOS ONE (2015)
da Silva, F. S., Galvão-Coelho, N. L. · brazilian Journal of Psychiatry (2019)
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