Repeated use of 3,4-methylenedioxymethamphetamine is associated with the resilience in mice after chronic social defeat stress: A role of gut–microbiota–brain axis

3,4-Methylenedioxymethamphetamine (MDMA), the most widely used illicit compound worldwide, is the most attractive therapeutic drug for post-traumatic stress disorder (PTSD). Recent observational studies of US adults demonstrated that lifetime MDMA use was associated with lower risk of depression. Here, we examined whether repeated administration of MDMA can affect resilience versus susceptibility in mice exposed to chronic social defeat stress (CSDS). CSDS produced splenomegaly, anhedonia-like phenotype, and higher plasma levels of interleukin-6 (IL-6) in the saline-treated mice. In contrast, CSDS did not cause these changes in the MDMA-treated mice. Analysis of gut microbiome found several microbes altered between saline + CSDS group and MDMA + CSDS group. Untargeted metabolomics analysis showed that plasma levels of N-epsilon-methyl-L-lysine in the saline + CSDS group were significantly higher than those in the control and MDMA + CSDS groups. Interestingly, there were positive correlations between plasma IL-6 levels and the abundance of several microbes (or plasma N-epsilon-methyl-L-lysine) in the three groups. Furthermore, there were also positive correlations between the abundance of several microbes and N-epsilon-methyl-L-lysine in the three groups. In conclusion, these data suggest that repeated administration of MDMA might contribute to stress resilience in mice subjected to CSDS through gut-microbiota-brain axis.


Introduction
3,4-Methylenedioxymetahmphetamine (MDMA; ecstasy), a synthetic ring-substituted methamphetamine, is one of the most widely used recreational drugs in the world since it can produce the subjective prosocial feeling, the enhancement of empathy and sociability (Boote, 2018;Dunlap et al., 2018;Nutt, 2019;Parrott et al., 2017). A recent observational study using a large nationally representative sample of US adults (n = 213,437) demonstrated that lifetime MDMA use was associated with lower risk of depression (Jones and Nock, 2022b). Furthermore, a new study using US adults (n = 484,732) demonstrated that lifetime MDMA use was associated with lowered odds of psychological distress and suicidal thoughts (Jones and Nock, 2022a).
Moreover, another study using US representative sample of noninstitutionalized adults (n = 241,675) from 2015 -2020 National Survey on Drug Use and Health showed that MDMA use was associated with a decreased likelihood of serious psychological distress, major depressive episode, and suicidal thinking whereas the use of the hallucinogen LSD (lysergic acid diethylamide) was associated with an increased likelihood of major depressive episode and suicidal thinking (Yang et al., 2022). Taken together, it is likely that lifetime use of MDMA in adults is associated with lower risk of depression, although the reasons underlying the relationship between lifetime MDMA use and resilience remain unclear.
Therapeutic potential of MDMA for post-traumatic stress disorder (PTSD) has attracted a lot of interest from the scientific community Hoskins et al., 2021;Reiff et al., 2020; Abbreviations: CSDS, chronic social defeat stress; FDR, false discovery rate; IL-6, interleukin-6; MDMA, 3,4-methylenedioxymethamphetamine; PTSD, posttraumatic stress disorder. Tedesco et al., 2021). Mithoefer et al. (2011;2018) demonstrated a significant efficacy of MDMA-assisted psychotherapy for chronic PTSD patients. A recent randomized, double-blind, placebo-controlled phase 3 trial showed that MDMA could produce significant and robust reduction in PTSD symptoms in severe PTSD patients compared with placebo (Mitchell et al., 2021). However, the precise mechanisms underlying MDMA's beneficial effects for PTSD symptoms remain to be elucidated.
Gut-microbiota-brain axis consists of bidirectional communication between the gastrointestinal (GI) tract and the brain (Chang et al., 2022;Cryan et al., 2019;Góralczyk-Bińkowska et al., 2022;Wei et al., 2022b). Multiple lines of evidence suggest that gut-microbiota-brain axis may contribute to resilience versus susceptibility in rodents after repeated stress (Bailey et al., 2011;Cathomas et al., 2019;Szyszkowicz et al., 2017;Wang et al., 2020aWang et al., , 2021;Wei et al., 2022aWei et al., , 2022bYang et al., 2017Yang et al., , 2019Zhang et al., 2017Zhang et al., , 2019. We previously reported higher levels of Bifidobacterium in the resilient mice compared with susceptible mice after chronic social defeat stress (CSDS), and oral supplementation with Bifidobacterium produced resilience in mice exposed to CSDS . Furthermore, antibiotic-induced microbiome depletion confers stress resilience in mice after CSDS, suggesting a role of microbiome in stress resilience (Wang et al., 2020a). Moreover, repeated oral administration of betaine produced stress resilience in mice after CSDS through gut-microbiota-brain axis . In addition, Zhang et al. (2019) reported that abnormal composition of gut microbiota is associated with resilience versus susceptibility to inescapable electric stress in a learned helplessness model. Taken all together, it is likely that gut-microbiota-brain axis might play a crucial role in resilience versus susceptibility in rodents exposed to stress. At present, there are no articles reporting a role of gut-microbiota-brain axis in beneficial effects of MDMA.
The serotonin (5-hydroxytryptamine: 5-HT) system in the body plays an important role in the mechanisms of action of MDMA (Green et al., 2003;Schenk and Highgate, 2021). In addition of inhibition for 5-HT transporter, MDMA causes excessive release of 5-HT, and it has less potent effects of other neurotransmitters such as dopamine and norepinephrine. Importantly, approximately 95% of 5-HT in the body is synthesized and secreted by enterochromaffin cells in the GI tract (Liu et al., 2021;Mawe and Hoffman, 2013;Shine et al., 2022). From the current findings, we have a hypothesis that 5-HT in the GI tract may play a role in beneficial actions of MDMA.
The present study was undertaken to examine whether repeated administration of MDMA contributes to resilience in mice after subsequent CSDS. First, we examined anhedonia-like phenotype using sucrose preference test since anhedonia is a core symptom of stress-related disorders such as depression (Pizzagalli, 2014). As the auxiliary analysis, we examined the role of gut-microbiota-brain axis including microbes-derived metabolites in the stress resilience after MDMA administration since gut microbiota might play a role in stress resilience.

Animals
Both male adult CD1 (ICR) mice, aged 13 weeks (body weight >40 g), and male adult C57BL/6 mice, aged 7 weeks (body weight 20-25 g), were purchased from Japan SLC, Inc. (Hamamatsu, Japan). The animals were kept in controlled environments with water and food available at all times, as well as 12-hour light/dark cycles (lights on between 07:00 and 19:00 h). The Institutional Animal Care and Use Committee at Chiba University approved the experimental protocol of the study . The guidelines of the National Institutes of Health's Guide for the Care and Use of Laboratory Animals were strictly followed when conducting this investigation. Every attempt was made to reduce pain.
2.2. MDMA, CSDS, collection of fecal samples, behavioral test, and collection of samples MDMA hydrochloride was synthesized from 3,4-methylenedioxyphenyl-2-propanone and methylamine by the one (K.H.) of the authors. Three groups such as saline + control (no CSDS) group (n = 10), saline + CSDS group (n = 9), and MDMA + CSDS group (n = 7) were used in the experiment. Saline (10 ml/kg/day) or MDMA (10 mg/kg/day as hydrochloride salt) was administered intraperitoneally (i.p.) to mice for 14 days (day 1-day 14) (Fig. 1A). The dose (10 mg/kg) of MDMA was used as previous reported (Zhang et al., 2006). Subsequently, CSDS was carried out for 10 days (day 15-day 24) (Fig. 1A), as previously described (Qu et al., 2017Wang et al., 2020a). The C57BL/6 mice were exposed to a different CD1 aggressor mouse for 10 min each day for a total of 10 days (day 15-day 24). The resident CD1 mouse and the invader mouse were kept in one half of the cage after the social defeat session concluded, separated by a perforated Plexiglas divider to allow visual, olfactory, and auditory interaction for the remaining 24 h.
At 24 h after the last session, all mice were housed individually. To minimize circadian impacts on the microbiota, we collected fecal samples from each mouse at around 10:00 on day 25 (Fig. 1A). When each mouse was put in a fresh and clean cage, we collected fecal samples. Immediately following defecation, the fecal samples were placed in a sterile screw-cap microtube, where they were kept until use at -80 • C.
On day 27, sucrose preference test (SPT: one % sucrose and water) was performed from 17:00 to 18:00, to examine anhedonia-like phenotype. Mice were given access to the sucrose solution 48 h before to the test in order to acclimate them to the 1% sucrose solution before verifying the change in sucrose solution consumption. Then, after going without food and water for 4 h, the subjects were exposed for an hour to either water or a 1% sucrose solution in one of two identical bottles. The bottles were weighed both before and just after the test. The ratio of sucrose solution ingestion to total liquid consumption was used to calculate the sucrose preference.
On day 28, blood was collected from heart under inhaled isoflurane (2-5%) anesthesia and placed into a tube containing ethylenediamine-N,N,N',N'-tetraacetic acid potassium salt dehydrate as an anticoagulant. Then plasma samples were collected after centrifugation, and stored at -80 • C before the use. In addition, mouse spleen was removed and weighted.

16S rRNA analysis
NucleoSpin® DNA Stool (Macherey-Nagel. catalog number: U0472C) was used to extract DNA from fecal samples.16S rRNA analysis of fecal samples was performed at BGI Japan (Kobe, Japan). The FastPrep-24 5G homogenizer was used to dissolve the fecal samples while they were suspended in a solution containing 4M guanidium thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA (MP Biomedicals, Irvine, CA). Following that, GENE PREP STAR PI-480 was used to extract the DNA from the solution after bead treatment (KURABO, Tokyo, Japan). The DNA sample's final concentration was changed to 10 ng/L. One sample of control (saline + no CSDS) group was not used for subsequent 16S rRNA analysis since the DNA purity was not good. In a nutshell, PCR was used to amplify the V3-V4 hypervariable regions of the 16S rRNA from microbial genomic DNA using the dual-index approach and bacterial universal primers (341F/R806). Following an approach that has already been disclosed, bioinformatics analysis was carried out. The fastq-join application was used to integrate the overlapping paired-end readings using its default parameters. The readings underwent the following quality and chimera screening processes. Usearch6.1 was used to eliminate chimeric sequences and only extract reads with quality value scores of 20 for >99% of the sequence. For 16S rDNA-based taxonomy analysis, non-chimeric data were submitted using the Ribosomal Database Project (RDP) Multiclassifier program. With an 80% confidence criterion, reads acquired in the multi-FASTA format were categorized at the genus or phylum level. α-diversity analyses, including Observed_OTU, Chao, Ace and Shannon, were used to reflect the abundance and diversity of the microbial community. The similarity or dissimilarity of three groups was assessed by β-diversity analysis including principal components analysis (PCA).
Linear discriminant analysis (LDA) effect size (LEfSe) was used for identifying certain bacteria as potential microbial biomarkers discovery. Microbiota-based potential biomarker discoveries were performed with LEfSe using the online galaxy platform (Segata et al., 2011). The LDA scores (LDA > 3.0 and P < 0.05) derived from LEfSe analysis were considered significantly to be enriched or deficient bacterial taxa in the intestinal microbiota among the three groups.

Untargeted metabolomics analysis
Untargeted metabolomics profiles from plasm samples were analyzed by using ultra-performance liquid chromatography-tandem quadruple time-of-flight mass spectrometry (UPLC-QTOF/MS) technique, as previously reported (Wan et al., 2022a;Yang et al., 2023). With the help of R statistical environment Ver 4.0.5. and Mass Spectrometry-Data Independent AnaLysis (MS-DIAL) software version 4.60 (Tsugawa et al., 2015), metabolomics data were analyzed. Metabolites were detected at least 50% from the analyzed samples and the coefficient of variation (CV) values of 30% of metabolites in pooled quality control (QC) samples, and annotation level 2 proposed by Schymanski et al. (2014) was used for data analysis.

Statistical analysis
The data are shown as the mean ± standard error of the mean (S.E. M.). Analysis was performed by using PASW Statistics 20 (formerly SPSS statistics; SPSS, Tokyo, Japan). Body weight data were analyzed using repeated measure two-way analysis of variance (ANOVA). Comparisons among the three groups were performed by one-way ANOVA, followed by post-hoc Fisher's Least Significant Difference (LSD) test. For the gut microbiota data, differences among the three groups were determined with a Kruskal-Wallis test. For plasma metabolite analysis, we used orthogonal partial least squares discriminant analysis (OPLS-DA) as a multivariate analysis model implemented in SIMCA-P (V.14.0). Significant peaks of metabolites were determined by combination of variable importance in projection (VIP) value > 1, Wilcoxon rank test P values < 0.05, false discovery rate (FDR) < 0.15. The P < 0.05 was considered statistically significant.

Effects of CSDS on body weight, anhedonia-like phenotype, and plasma IL-6
There were no changes of body weight among the three groups (Fig. 1B). CSDS significantly increased the spleen weight in the salinetreated mice. In contrast, MDMA (10 mg/kg/day for 14 days) did not produce splenomegaly in mice after CSDS (Fig. 1C). In SPT, CSDS caused significant reduction in sucrose preference of the saline-treated group, but not the MDMA-treated group, indicating anhedonia-like phenotype in saline-treated mice (Fig. 1D). Furthermore, CSDS significantly increased plasma levels of IL-6 in the saline-treated group, whereas CSDS did not alter plasma IL-6 levels in the MDMA-treated group (Fig. 1E). There was a positive correlation (r = 0.9337, P < 0.001) between the spleen weight and plasma IL-6 levels in the three groups (Fig. 1F), suggesting that systemic inflammation is associated with splenomegaly in mice. The data suggest that MDMA-treated mice show stress resilience in the mice exposed to CSDS.

Composition of gut microbiota
As the auxiliary analysis, we examined the composition of gut microbiota among the three groups. For α-diversity, four indices such as Observed_OUT, Chao, ACE, and Shannon were not different among the three groups ( Fig. 2A-2D). The bacterial population composition of fecal microbiota was analyzed by PCA β-diversity. At the OUT level, PCA analysis showed a significant separation (R = 0.4301, P = 0.001) in the bacterial population composition among the three groups (Fig. 2E).
At the genus level (Fig. 4A), CSDS significantly increased the relative abundance of Oscillibacter in the saline-treated group, but not the MDMA-treated group (Fig. 4B). Furthermore, CSDS significantly increased the relative abundance of Flavonifractor in the saline-treated group (not MDMA-treated group), although CSDS significantly decreased the relative abundance of Lactobacillus in the saline-treated group (not MDMA-treated group) ( Fig. 4C and 4D).
At the species level (Fig. 4E), CSDS significantly increased the relative abundance of Oscillibacter_valericigenes in the saline-treated group, but not the MDMA-treated group (Fig. 4F). Furthermore, CSDS significantly increased the relative abundance of Flavonifractor_plautii, and Alistipes_onderdonkii in the saline-treated group (not MDMA-treated group), although CSDS significantly decreased the relative abundance of Barnesiella_intestinihominis in the saline-treated group (not MDMAtreated group) (Fig. 5G-5I).

Correlations among plasma IL-6 levels and the relative abundance of bacteria (or plasma metabolites)
There was a positive correlation between plasma N-epsilon-methyl-L-lysine and plasma IL-6 ( Fig. 6A). There were also positive correlations between the genus Oscillibacter (or the species Flavonifractor_plautii) and plasma IL-6 ( Fig. 6B and 6C). Furthermore, there were positive correlations between N-epsilon-methyl-L-lysine and the relative abundance of the genus Oscillibacter, the genus Flavonifractor, the species Flavoni-fractor_plautii, or the species level Alistipes_onderdonkii (Fig. 6D-6G).

Discussion
The major findings of this study are as follows. First, repeated treatment with MDMA did not cause splenomegaly, anhedonia-like phenotype and increases of plasma IL-6 levels in mice after subsequent CSDS although CSDS caused splenomegaly, anhedonia-like phenotype and increase of plasma IL-6 levels in the saline-treated group. There was a positive correlation between spleen weight and plasma IL-6 levels. Second, repeated treatment with MDMA restored the marked alterations in the beta-diversity of microbiota in the host gut after CSDS. At the genus level, CSDS showed marked alterations in the several bacteria in host gut. Interestingly, MDMA significantly ameliorated higher abundance of Oscillibacter in the CSDS-exposed mice. At the species levels, MDMA significantly ameliorated higher abundance of Oscillibacter_valericigenes in the CSDS-exposed mice. Third, we found 4 plasma metabolites altered among the three groups. Among the four metabolites, N-epsilon-methyl-L-lysine was the most attractive metabolite. Fourth, there was a positive correlation between plasma IL-6 levels and N-epsilon-methyl-L-lysine. Furthermore, there were positive correlations between plasma Il-6 levels and the relative abundance of the genus Oscillibacter (or the species Flavonifractor_plautii). Moreover, there were also positive correlations between plasma levels of N-epsilonmethyl-L-lysine and the relative abundance of the genus Oscillibacter, the genus Flavonifractor, the species Flavonifractor_plautii, or the species level Alistipes_onderdonkii. Collectively, these findings suggest that repeated treatment with MDMA might be associated with the resilience in mice after subsequent CSDS through anti-inflammatory action via gut-microbiota-brain axis.
We previously reported that CSDS causes splenomegaly in the susceptible mice with depression-like behaviors compared to CSDS resilient mice without depression-like behaviors and control (no CSDS) mice (Zhang et al., 2021b). Furthermore, a single dose of lipopolysaccharide (LPS) causes splenomegaly in mice, and spleen weight was positively correlated with blood levels of IL-6 (Ma et al., , 2022b2022c;Zhang et al., 2020Zhang et al., , 2021a. In this study, we found a positive correlation between spleen weight and plasma Il-6 levels, suggesting that systemic inflammation was associated with splenomegaly in mice, consistent with previous reports (Ma et al., , 2022b(Ma et al., , 2022cZhang et al., 2020Zhang et al., , 2021aZhang et al., , 2021b. It has been reported that CSDS resilient mice had lower blood levels of IL-6 than CSDS susceptible mice in response to acute stress (Hodes et al., 2014). Furthermore, blockade of IL-6 receptor in the periphery promotes rapid and sustained antidepressant-like effects by normalizing the altered composition of gut microbiota in CSDS susceptible mice (Zhang et al., 2017). Moreover, antibiotic-induced microbiome depletion is associated with resilience and lower IL-6 levels in mice after subsequent CSDS (Wang et al., 2020a). Collectively, it seems that systemic inflammation such as higher IL-6 levels in the blood is associated with increases in spleen weight of mice with anhedonia-like phenotype after CSDS. It is noteworthy that repeated treatment with MDMA could attenuate systemic inflammation in mice exposed to CSDS despite MDMA was not administered to mice during CSDS (10 days).
In this study, we found that MDMA significantly ameliorated the higher abundance of Oscillibacter_valericigenes in the CSDS-exposed mice. Although the precise functions of Oscillibacter_valericigenes are unclear, a recent study showed that Oscillibacter_valericigenes might cause inflammation through TLR2 (Toll-like receptor 2) ligands (Li et al., 2022). Thus, it is possible that reduction of the abundance of Oscil-libacter_valericigenes by MDMA may play a role in long-lasting prophylactic effects of MDMA in mice exposed to CSDS although further study is needed.
N-epsilon-methyl-L-lysine is generated by metabolic transmethylation of the endogenous amino acid L-lysine, and methylation of L-lysine is implicated in transcriptional regulation (Levy, 2019). In this study, we found a positive correlation between plasma IL-6 levels and plasma levels of N-epsilon-methyl-L-lysine. Although the precise functions of N-epsilon-methyl-L-lysine are unknown, the current data suggest that it may play a role in inflammation. In addition, we found positive correlations between plasma levels of N-epsilon-methyl-L-lysine and the relative abundance of the genus Oscillibacter, the genus Flavonifractor, the species Flavonifractor_plautii, or the species Alistipe-s_onderdonkii. The data suggest that these microbes may contribute to production of N-epsilon-methyl-L-lysine. In addition, Wang et al. (2020) reported that the abundance of the genus Oscillibacter was associated with plasma N-epsilon-methyl-L-lysine in the Mongolian sheep. Thus, it seems that the genus Oscillibacter may play a role in the production of N-epsilon-methyl-L-lysine. Interestingly, we also found positive correlations between plasma IL-6 levels and the relative abundance of two microbes (the genus Oscillibacter and the species Flavonifractor_plautii), suggesting that these two microbes may play a role in systemic inflammation. Interestingly, N-epsilon-methyl-L-lysine was detected in human and rat urine (Kalász et al., 2005;Löwer et al., 1975), indicating production of this metabolite in the body. Collectively, it is likely that these microbes may contribute to production of N-epsilon-methyl-L-lysine in  the GI tract, resulting in systemic inflammation. Nonetheless, further detailed study is needed to investigate the role of gut microbiome in the production of N-epsilon-methyl-L-lysine.
As mentioned in the introduction, the 5-HT system plays an important role in the mechanisms of action of MDMA in the body (Green et al., 2003;Schenk and Highgate, 2021). Since approximately 95% of 5-HT in the body is synthesized in the GI tract (Liu et al., 2021;Mawe and Hoffman, 2013;Shine et al., 2022), the data of this study suggest that gut-microbiota may play a role in long-lasting prophylactic effects of MDMA in CSDS model. Considering a major role of MDMA in the 5-HT system, it is likely that 5-HT system in the GI tract could contribute to resilience-enhancing effects of MDMA through gut-microbiota-brain axis. Further study is needed to ascertain the role of 5-HT system in the GI tract as well as gut microbiota and microbes-derived metabolites for resilience-enhancing effects of MDMA.
Accumulating evidence suggests that vagus nerve plays a key role in the gut-microbiota-brain axis (Chang et al., 2022;Wei et al., 2022b). It is reported that vagotomy blocked the beneficial effects of Lactobacillus rhamnosus on anxiety-and depression-like behaviors in mice exposed to stress (Bravo et al., 2011), suggesting the vagus nerve in the communication of brain and body. Furthermore, we reported a role of subdiaphragmatic vagus nerve in depression-like behaviors in rodents after fecal microbiota transplantation of mice with depression-like behaviors Wang et al., 2020b. Given the role of vagus nerve in communication of brain and body (Chang et al., 2022;Pavlov and Tracey, 2022;Shine et al., 2022;Wei et al., 2022b), it is of great interest to investigate whether vagotomy could block MDMA-induced resilience in rodents exposed to stress.
As aforementioned in the introduction, combination of MDMA with psychotherapy for PTSD has attracted a lot of interest from the scientific community (Smith et al., 2022;Tedesco et al., 2021). A new US population-based survey study demonstrated that lifetime use of MDMA was associated with significantly lower risk of self-reported overweightness and obesity, and lower odds of self-reported physical diseases such as heart condition and/or cancer, hypertension, and diabetes (Jones et al., 2022). Importantly, Price et al. (2022) reported that chronic SSRI (selective serotonin reuptake inhibitor) use dampens the response to MDMA-assisted therapy in the treatment of PTSD, suggesting a role of 5-HT system in the beneficial effects of MDMA for PTSD. Collectively, it is possible that lifetime MDMA use could have long-lasting beneficial effects in later life, although the precise mechanisms underlying MDMA's beneficial effects in PTSD remain to be elucidated. Further study on brain-body crosstalk is needed to ascertain the role of gut-microbiota-brain axis in the mechanisms of actions of MDMA on stress resilience. Collectively, it is of great interest to examine the role of gut-microbiota-brain axis on resilience and vulnerability in [4-imidazoleacetic acid (Kruskal-Wallis test, FDR-corrected P = 0.11), N-epsilon-methyl-L-lysine (Kruskal-Wallis test, FDR-corrected P = 0.11), 3-hydroxymethylglutaric acid (Kruskal-Wallis test, FDR-corrected P = 0.12), 3-methylhistamine (Kruskal-Wallis test, FDR-corrected P = 0.13)] among the three groups. The number of mice (n = 7 -10). *P < 0.05, **P < 0.01, ***P < 0.001.

MDMA users.
This study has some limitations. In this study, we did not identify the specific microbiome and metabolites which contribute to MDMAinduced resilience. Although higher levels of N-epsilon-methyl-L-lysine may play a role in the susceptibility in mice exposed to CSDS, we did not investigate the effects of N-epsilon-methyl-L-lysine in a CSDS model. Furthermore, we did not identify specific microbiome which can produce N-epsilon-methyl-L-lysine in the GI tract. Finally, we do not have strong evidence supporting the role of gut-microbiota-brain axis in stress resilience of MDMA from the current exploratory analysis. Therefore, further study is needed to confirm the role of gut-microbiota-brain axis in MDMA-induced stress resilience.
In conclusion, the present study suggests that repeated use of MDMA might be associated with resilience in mice subjected to CSDS through gut-microbiota-brain axis. It is likely that abnormalities in gut-microbiota-brain axis including microbes-derived metabolites may contribute to susceptibility to stress-related disorders. Finally, MDMA would be a prophylactic and therapeutic drug to prevent the onset of stress-related disorders.

Role of the Funding Source
This study was supported by the grants from Japan Society for the Promotion of Science (to K.H., 21H00184 and 21H05612), JST OPERA Program Japan (to C.M JPMJOP1831), and unrestricted grant of Yamada Bee Company, Japan (to C.M).  Conceptualization, Methodology, Formal analysis, Investigation, Resources, Data curation, Supervision, Project administration, Funding acquisition, Writingoriginal draft, Writingreview & editing.

Declaration of Competing Interest
Dr. Hashimoto is the inventor of filed patent applications on "The use of R-ketamine in the treatment of psychiatric diseases", "(S)-norketamine and salt thereof as pharmaceutical", "R-ketamine and derivative thereof as prophylactic or therapeutic agent for neurodegeneration disease or recognition function disorder", "Preventive or therapeutic agent and pharmaceutical composition for inflammatory diseases or bone diseases", and "R-ketamine and its derivatives as a preventive or therapeutic agent for a neurodevelopmental disorder" by the Chiba University. Dr. Hashimoto has also received speakers' honoraria, consultant fee, or research support from Abbott, Boehringer-Ingelheim, Daiichi-Sankyo, Meiji Seika Pharma, Seikagaku Corporation, Dainippon-Sumitomo, Taisho, Otsuka, Murakami Farm and Perception Neuroscience. Other authors declare no conflict of interest.

Data and code availability
: The 16S rRNA sequencing data has been uploaded and saved in the NCBI Sequence Read Archive and is available at the accession number PRJNA914843.