Methcathinone exposure alters host behavior and gut microbiota community in zebrafish

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Methcathinone (MCAT) is a novel addictive drug, and its effects on the gut microbiota and brain-gut axis are still unclear. This study aimed to determine the alteration of host behavior and gut microbiota community in zebrafish under methcathinone exposure. Methods: In this study, MCAT was administeredto zebrafish, and their behavioral characteristics were evaluated. Changes in the gut microbiota and metabolic pathways in zebrafish were also analyzed. Results: We found that after the administration of MCAT, the zebrafish was anxious and aggressive, with hyperlocomotion, and the abundance and diversity of the gut microbiota significantly decreased. Additionally, the composition of the gut microbiota shifted. These alterations in the composition of the gut microbiota further led to changes in their metabolic pathways, some of which might be related to the behavioral response induced by MCAT. Conclusion: Our findings provide insights into alteration of behavior and gut microbiota composition of zebrafish induced by MCAT which may further contribute to its neurobehavioral responses. Drug addiction gut microbiota behavioral response metabolic pathways zebrafish model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Substance use disorders (SUDs) are characterized by chronic dependence on special substances (including alcohol, cocaine, amphetamine, opioids, nicotine etc.), and they represent mental and physical disorders. Increasing evidence indicates that persistent intake of addictive substances alters the gut microbiota, contributing to the development of SUDs. In alcohol-abusing individuals, the abundance and diversity of the gut microbiota decrease, while the proportion of pro-inflammatory taxa increases (Bajaj et al., 2019). Repeated cocaine intake altered the gut microbiota profile in a rat model (Scorza et al., 2019 ), and gut microbiota depletion elevated conditioned place preference in mice (Kiraly et al., 2016 ). The gut-brain axis represents a bidirectional relationship between the gut microbiota and brain. The gut microbiota regulates the metabolism of neurotransmitters; in turn, their synthesis, release, and transfer are modified after alteration of the gut microbiota due to SUDs. Dopamine is a crucial neurotransmitter for drug reward and reinforcement. Moreover, any drug with addictive potential can increase dopamine levels. More than half of the dopamine produced in the body is synthesized by enteric neurons and intestinal epithelial cells, and its levels are regulated by the gut microbiota (Hamamah et al., 2022 ). Several neurotransmitters, including gamma-aminobutyric acid (GABA), serotonin, and dopamine, are produced directly by bacteria colonization of the gut, contributing to drug dependence (Yano et al., 2015 ; Strandwitz et al., 2018). Additionally, fecal microbiota transplantation (FMT) has the potential to rescue gut microbiota dysbiosis, relieve alcohol-related liver disease, and reduce alcohol craving and consumption (Bajaj et al., 2021 ). In a morphine-dependent mice, FMT attenuates naloxone-induced opioid withdrawal (Thomaz et al., 2021 ). These results indicate the possibility of applying FMT as a compulsory treatment. Owing to its simple manufacturing process and lack of proper regulation, synthetic cathinone has recently emerged as a popular drug of abuse through sensationalized media attention (Baumann et al., 2016). It can often be trafficked through the internet and even retail shops, partially resulting in the widespread availability of personal mood elevation and entertainment experimentation. Methcathinone (MCAT), as a parent β-ketone analog of methamphetamine and representative compound to a series of designer drugs, is re-used as a notably popular novel psychoactive substance. Considering the structural similarity between MCAT and amphetamine, we focused on enhancing monoaminergic neurotransmission, including the inhibition of monoamine reuptake, release of neurotransmitters stored inside synaptic vesicles, and direct interaction with receptors (Blough et al., 2019 ; Davies et al., 2020 ). Chronic MCAT and methamphetamine users showed significantly reduced dopamine transporter density by positron emission tomography, suggesting that functional regulation of the dopaminergic system is an expected consequence of drug abuse. Similar to several psychostimulants like methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA), MCAT can induce euphoric and stimulant feelings and desired effects for hours, including improved mood, euphoria, excitement, increased energy and motivation, and enhanced alertness and awareness (Papaseit et al., 2016 ; Papaseit et al., 2021 ). Uncontrolled craving for the aforementioned desired pleasures drives such users to escalate consumption. As overdose use of MCAT induces a wide range of toxic neurological and psychopathological effects, such as hallucinations, delusions, hyperthermia, and even death (Baumann et al., 2014 ; Spiller et al., 2011 ), it has become a widely used recreational drug, with increasingly reported overdose incidents. Although the behavioral and physiological effects of MCAT and its analogs have been studied in rats and mice (Gatch et al., 2015 ), the addictive behavior, potential psychomotor effects, and gut microbiota functions of MCAT have never been reported in other experimental animals. Despite numerous clinical cases of MCAT abuse, little is known about the effects of neurotransmitters on fecal microbiota. The goal of this study was to systematically evaluate the effects of MCAT on fecal microbiota function in a zebrafish model. We mapped the gut microbiota community and predicted alterations in metabolic pathways following MCAT administration. By combining behavioral testing and change in gut microbiota composition in MCAT-consuming animals, our findings highlighted the interaction between gut microbiota and the psychostimulant effects of MCAT. Materials and Methods Zebrafish husbandry Wild-type adult zebrafish (AB strain) were obtained from the China Zebrafish Resource Center (Wuhan, China). Adult zebrafish were raised in a recirculation system with a 14 h-light/10 h-dark circadian cycle at 28.5°C, according to standard zebrafish breeding protocols (Ding et al., 2023 ). The culture water supplied for the system was filtered by reverse osmosis (pH = 7.0–7.5). The adult zebrafish were fed twice daily at 08:30 and 17:30 with newly hatched Artemia (Jiahong Feed Co., Tianjin, China); however, they were not fed the day before the MCAT exposure experiments. The use of zebrafish in this study protocol was performed following the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Chemicals and exposure Methcathinone was purchased from the Wuhan Zhongchang National Research Standard Technology Co., Ltd., China (CAS number: 5650-44-2, #C-019). Stock solutions of 1.0 mg/mL were prepared by dissolving MCAT in the purified water and stored at − 4 ℃. Working solutions were prepared by diluting the stock solution to the final concentrations (1.0, 5.0, and 10.0 µg/mL) immediately before experiments. Zebrafish were exposed to culture water (control) or three doses of MCAT for 10 min in 2 weeks before the experiments. All exposed solutions were replaced every time to ensure the exact MCAT concentration. Behavioral tests Behavioral tests were recorded using a behavioral analysis system with camera equipment, and movements were extracted and analyzed using Ethovision XT 11.5 software (Noldus IT, Wageningen, Netherlands). All the zebrafish were 12 months old and tested in a clear acrylic tank at the same time each day. As the MCAT condition leads to hyperlocomotion (Zhou et al., 2023 ), we decided to apply 1.0, 5.0, and 10.0 µg/mL treatments for our initial behavioral experiments to explore the effect of different levels. Novel tank test Each zebrafish was placed in a tank with measuring 24 cm × 8 cm × 20 cm. The back side of the tank was covered with a light plate to aid in video recording. Tester fishes were dropped from the top of the tank. Videos were recorded for 10 min from a lateral viewpoint of the tank. For data analysis, the tank was artificially divided evenly into top, middle, and bottom zones from top to bottom. The time spent in the bottom and top zones, freezing time, and the latency to reach the middle and top zones during the tests were quantified. Mirror biting test A rectangular tank (18 cm × 5 cm × 15 cm) with a mirror positioned vertically on the left side was used. The back side of the tank was covered with a light plate to aid in data recording. Experimental fishes (one at a time) were placed inside the tank and monitored continuously for 6 min from a lateral viewpoint. The region in which the tester fish could touch the mirror was designated as the mirror zone (3 cm wide, the same as the average body length of an adult zebrafish). Endpoints used to assess mirror biting response and reflect aggression-related behaviors included time and entry frequency into the mirror zone. Social preference test Social preference tests were performed in a rectangular tank (40 cm × 15 cm × 15 cm) separated into three compartments (10 cm × 15 cm × 15 cm, 20 cm × 15 cm × 15 cm, and 10 cm × 15 cm × 15 cm) from left to right using two transparent dividers. The left and right end tanks were used as either a conspecific tank (CT) or empty tank (ET), alternating between trials to avoid lateral bias. The tank was then placed on a light plate to facilitate video tracking. Before each test trial, four companion fishes were placed in the CT, and water with no fish was placed in the ET. The test fishes were placed in the central tank and allowed to acclimate to the new environment for 5 min before starting a 15 min video recording for behavioral tracking. The center tank was identified as the social contact, center, and empty zones, which were isometric based on their distance from the CT. Social preference was assessed by calculating the time spent in the social contact and empty zones, as well as the average speed in the social contact zone. Open field test Individual fish was placed in the center of a tank (40 cm × 40 cm × 20 cm) and allowed to swim freely for 5 min. Videos were recorded from the top of the tank for 15 min. A square area of > 10 cm 2 away from the nearest edge of the tank was defined as the center zone and the rest as the peripheral zone. Total distance, time spent in the center and peripheral zones, latency to the center zone, and frequency of high-speed movement (60% above the average speed) were calculated. Fecal sample collection and 16S rRNA sequencing At the time of sampling, the whole intestine of each fish was aseptically removed and collected as a single sample, as previously described (Xiao et al., 2022 ). Bacterial 16S rRNA was amplified, extracted, pooled in equimolar ratios, and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA), as previously reported (Zhu et al., 2019 ). Briefly, bacterial DNA was extracted using an E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). Sequencing of 16S rRNA gene was performed as described previously (Guo et al., 2021 ; Wang et al., 2022 ), using primers to amplify the V3-V4 hypervariable regions of the bacterial 16S rRNA gene (338F, 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′). Amplicons were purified and paired-end sequenced (2 × 300) using an Illumina MiSeq System (Illumina, San Diego, CA, USA). Statistical analysis Data were analyzed using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA). In all the experiments, comparisons were performed using a two-tailed unpaired Student’s t-test or one-way analysis of variance with the Dunnett’s multiple comparison test. Results are presented as mean ± standard error of the mean (SEM), and p < 0.05 was considered statistically significant. In these figures, * indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Results Behavior test 1. Novel tank test A novel tank diving test was used to evaluate the effects of MCAT on anxiety and locomotion in zebrafish (Fig. 1 A). After MCAT exposure for 10 min (Supplementary Fig. 1A), the time spent in the bottom zone by 1.0 and 5.0 µg/mL MCAT-treated fish was significantly lower than that in the control fish, but the 10.0 µg/mL MCAT-treated fish was much higher (Fig. 1 B); although opposite in the top zone (Fig. 1 C). The fishes treated with 1.0 and 5.0 µg/mL of MCAT also showed lower latency to the middle and top zones of the novel tank diving test (Fig. 1 D, E). To investigate whether the decrease in travel distance was due to a reduction in fish motivation, we examined the latency to the middle and top zones and freezing time. Compared with the control fishes, the fishes treated with 1.0 and 5.0 µg/mL of MCAT had more freezing time and less latency to the middle and top zones, although 10.0 µg/mL of the MCAT-treated fish showed opposite again (Fig. 1 F), indicating lower and medium doses of MCAT-treated fish had a lower endurance of anxious behavior, but higher dose of MCAT led to a high level of anxiety. 2. Mirror biting test A mirror biting test was used to evaluate the aggressiveness of the adult zebrafish (Fig. 1 G). The 1.0 and 5.0 µg/mL MCAT-treated fishes showed a higher time and frequency in the mirror zone than the control fishes, and 10.0 µg/mL of the MCAT-treated fish was lower than that of the control, with a significant level (n = 20, p = 0.05; binomial test) (Fig. 1 H, I, J), which indicated that MCAT enhanced the aggressiveness of the zebrafish. 3. Social preference test Zebrafish are highly social and naturally form groups (shoals) with structured social relationships. A social preference test was performed to investigate the effects of MCAT on social interactions in zebrafish (Fig. 2 A). Both the 1.0 and 5.0 µg/mL MCAT-treated fish showed a preference for social partners, and they spent more time in the social area of the tank with quicker pace, while less time in the empty zone (Fig. 2 B, C, D). The average time spent and speed in the social zones by 1.0 and 5.0 µg/mL MCAT-treated fish was higher than that of the control fishes with statistical significance, while 10.0 µg/ml MCAT-treated fish was almost the same, indicating the higher dose might suppress the social preference (n = 10, p = 0.05; unpaired t-test). Open field test Based on the aforementioned results of the behavioral tests, the open field test was performed with 5.0 µg/mL MCAT-treated zebrafish. As shown in Fig. 2 E, the total distance moved and mobility time increased during the test (Fig. 2 F, G), and the time spent in the center zone increased at a higher speed in the zebrafish treated with MCAT (Fig. 2 H, I). This was likely due to the decreased time spent swimming in the center of the test box rather than reducing free exploration along the walls. MCAT treatment altered the gut microbiota community To confirm whether MCAT treatment altered the gut microbial community in zebrafish, we established a chronic MCAT exposure model. Based on the aforementioned results of the behavioral tests, the zebrafish were treated with 5.0 ug/mL of MCAT 10 min twice daily for 15 days, and fecal samples were collected and 16S rRNA sequencing was performed (Supplementary Fig. 1B). The operational taxonomic unit (OTU) count index decreased in the MCAT group compared to that in the control group (Fig. 3 A). The Chao community richness at the OTU and specie levels decreased significantly in the MCAT group (Fig. 3 B), although the Simpson index at the OTU level and Shannon index at the species level were comparable (Supplementary Fig. 2A). It indicated that α diversity decreased after MCAT treatment. Compared with the control group, principal coordinates analysis (PCoA) showed that the cluster of the MCAT group shifted on the OTU, genus, and specie levels (Fig. 3 C), but no significant differences were observed at the phylum, class, order, and family levels (Supplementary Fig. 2B). These results indicated that MCAT treatment altered the gut microbiota community, with a significant decrease in both abundance and diversity of the gut microbiota. Gut microbiome profiles shift after MCAT treatment The gut microbiota composition was assessed at each taxonomic level to evaluate the effect of MCAT treatment on the gut microbiota profile. Five domain phyla comprised > 95% of the gut microbiota in zebrafish, including Proteobacteria, Actinobacteria, Fusobacteriota, Firmicutes, and Bacteroidetes (Supplementary Fig. 4B). Firmicutes and Bacteroidetes are the two most common phyla in humans and mice. Significant alterations in the ratio of Firmicutes to Bacteroidetes have been observed in patients with various chronic diseases, and they are sensitive markers of overall microbiota alteration in humans (Magne et al., 2020 ). In zebrafish, Proteobacteria and Actinobacteria were the two major components of the gut microbiota, accounting for > 80% of the gut microbiota composition (Supplementary Fig. 4B). Our data showed an increasing trend in the ratio of Proteobacteria to Actinobacteriota and Firmicutes to Bacteroidetes, although no significant change was observed (Supplementary Fig. 3). Compared to the control zebrafish, the composition of the gut microbiota in the MCAT group shifted significantly at each taxon level. A Venn diagram showed the shared and unique bacteria in both groups. After the MCAT treatment, the proportions of three phyla, six classes, twelve orders, and twelve families decreased. In contrast, two orders and three families increased significantly in the MCAT group (Supplementary Fig. 4–7). At the genus level, 289 genera were shared by both groups; 240 and 176 genera were unique to the control and MCAT group, respectively (Fig. 4 A). The proportion of 12 genera decreased and that of three genera (Tardiphaga, Sphingomonas, and Vagnococcus) increased significantly in the MCAT group (Fig. 4 B-C). At the specie level, 334 species were shared; 373 and 293 species were unique to the control and MCAT groups, respectively (Fig. 5 A). Twelve species decreased, and three species ( Rhodococcus erythropolis , uncultured bacterium Tardiphaga, Sphingomonas paucimobilis , and Sphingomonas) increased in the MCAT group (Fig. 5 B-C). Linear discriminant analysis effect size (LEfSe) was performed to identify specific bacteria that exhibited remarkably different proportions in the control and MCAT groups (Fig. 6 ). These data indicate that MCAT treatment shifts the gut microbiota profile and induces dysbiosis. Predicted metabolic pathway analysis after MCAT treatment Based on the 16S rRNA sequencing data, we inferred the abundance of functional genes in the gut microbiota and predicted their metabolic pathways using the software package PICRUSt2 (Jiang et al., 2019 ). After investigating the level 3 KEGG functional classes, 41 metabolic pathways showing statistically different abundance between the two groups were identified. These 41 metabolic pathways belong to 19 superclasses. Interestingly, four pathways were related to antibiotics biosynthesis, including acarbose and validamycin biosynthesis, penicillin and cephalosporin biosynthesis, biosynthesis of vancomycin group antibiotics, and biosynthesis of enediyne antibiotics. This suggests that MCAT treatment influences the levels and composition of antibiotics produced by the gut microbiota (Fig. 7 A). The proportions of facultative anaerobic, gram-positive, and gram-negative bacteria were altered after the MCAT treatment (Supplementary Fig. 8). The network between the gut microbiota and metabolic pathways is shown based on the correlation coefficients (Fig. 7 B). Focal adhesion, apoptosis, protein digestion and absorption, hematopoietic cell lineage, arrhythmogenic right ventricular cardiomyopathy (ARVC), regulation of the actin cytoskeleton, acarbose and validamycin biosynthesis, osteoclast differentiation, ferroptosis, staurosporine biosynthesis, and dilated cardiomyopathy were negatively correlated with Fusobacteriota. Parathyroid hormone synthesis, secretion, and action, biosynthesis of various secondary metabolites part 1, gonadotropin-releasing hormone (GnRH) signaling pathway, pancreatic cancer, AGE RAGE signaling pathway in diabetic complications, indole alkaloid biosynthesis, Fc gamma R-mediated phagocytosis, malaria, relaxin signaling pathway, chronic myeloid leukemia, and biosynthesis of vancomycin group antibiotics were positively correlated with Verrucomicrobiota. However, biosynthesis of the vancomycin group antibiotics was also positively related to Fusobacteriota, and dilated cardiomyopathy was negatively related to Verrucomicrobiota (P < 0.05). 3.6 Effect of MCAT on histopathology of intestinal tissue This study examined the protective effects of MCAT on zebrafish colonic morphology. To investigate this, histological analysis of the zebrafish intestinal segments was performed following H&E and alcian blue-PAS staining. The control group (Fig. 8 A and 8 C) exhibited normal histological features with no morphological changes. However, the MCAT-treated group showed tissue inflammation and severe pathological changes, including high epithelial degradation, goblet cell hyperplasia, and lymphocyte accumulation. The MCAT-exposed group showed a greater number of goblet cells in the intestine than the other group (Fig. 8 B, D). Discussion Substance use disorders are characterized by chronic dependence on special substances (including alcohol, cocaine, amphetamine, opioids, nicotine etc.), and represent mental and physical disorders. Increasing evidence indicates that persistent intake of addictive substances alters the gut microbiota, contributing to the development of SUDs. In this study, we treated zebrafish with a popular novel psychoactive substance, MCAT, and found that at the behavioral level, MCAT induced dose-dependent changes in locomotion. Anxiety, aggressive, and social behaviors correlated with dosages increasing from 1.0 to 10.0 µg/mL. The effects of MCAT on zebrafish mimic several aspects in humans. MCAT induces a remarkable increase in locomotion in zebrafish, which is the most prominent behavioral effect reported in rodents (Gatch et al., 2015 ; Wojcieszak et al., 2019 ; Jones et al., 2014 ), similar to the hyperactive effects and higher psychomotor capacity produced by synthetic cathinones in humans. Characterized lap-running of the periphery in mice has also been reported for psychoactive synthetic cathinones, such as MDMA (van de Wetering et al., 2017; Nguyen et al., 2016 ). In the novel tank and mirror biting tests, the lower and medium doses of MCAT-treated fish had lower endurance of anxious behavior but enhanced aggressiveness; a higher dose of MCAT led to a high level of anxiety and remained motionless. In the social preference test, the high-dose group swam frenetically along the wall at a high speed, completely ignoring the presence of the social partner in the tank (Fig. 2 A, B, C, D). Lastly, enhancement of spontaneous locomotor activity in zebrafish occurred after exposure to MCAT at a medium dose (5.0 µg/mL) in the open field test for sure (Fig. 2 E, F, G, H, I). It appears that MCAT produces hyperactive locomotion in an exaggerated state, and this behavior mimics the manic behavior reported in overdosing human users (Papaseit et al., 2016 ; Papaseit et al., 2021 ). Overall, the behavioral tests revealed that the effects of MCAT on zebrafish were similar to the psychostimulant effects reported by human users, suggesting that zebrafish are good animal models for studying the neuronal mechanisms of such drugs. Similar to other SUDs, MCAT administration can alter the gut microbiota of the host. Both abundance and diversity decreased after two weeks of MCAT exposure in zebrafish. However, the mechanisms underlying gut microbiota dysbiosis following MCAT treatment remain unclear. Both aerobic and anaerobic bacteria colonize the gut, and oxygen consumption affects the growth of commensal bacteria. Our data suggest that MCAT administration altered the proportion of facultative anaerobic bacteria (Supplementary Fig. 8). Hypoxia-inducible factors (HIF) are a family of proteins that can sense oxygen concentration, and their production is increased in hypoxic tissues. Intestinal HIF-2α increases intestinal lactic acid levels by directly activating lactate dehydrogenase A, thereby influencing the intestinal microbiota composition (Wu et al., 2021 ). Methamphetamine (METH), an analog of MCAT, is one of the most widely consumed psychostimulants worldwide. Increasing evidence confirms that METH abuse is associated with hypoxia and ischemia. METH exposure induces chronic hypoxia and increases placental growth in pregnant women, increasing the risk of neurodevelopmental deficits (Carter et al., 2016 ). Transient and persistent METH consumption can induce vascular changes and promote the development of hypoxia and hypoperfusion in the striatum, leading to dopamine reduction and an increased risk of Parkinson’s disease (Kousik et al., 2011 ). METH reward behaviors are regulated by iron homeostasis and HIF-1α (Yan et al., 2022 ). METH treatment enhances the production of monoamine neurotransmitters, including dopamine, serotonin, and norepinephrine. All of these act as vasoconstrictors involved in the regulation of blood pressure and blood flow. Monoamine neurotransmitters promote platelet aggregation and occlusion. As a result, METH treatment induces retinal hypoxia and increased HIF-1α expression (Lee et al., 2021 ). Additionally, METH treatment enhances the expression of antimicrobial peptides in rat liver, which are transported to the intestine and influence the gut microbiota community (Qu et al., 2020 ). Based on previous studies, it is reasonable to hypothesize that MCAT treatment may induce hypoxia in the gut and enhance the production of antimicrobial peptides in the liver, leading to alterations in the gut microbiota of zebrafish. The potential mechanisms underlying gut microbiota dysbiosis after MCAT treatment require further investigation. Increasing evidence indicates bidirectional communication between the gut microbiota and brain. The alteration of gut microbiota and its metabolites may not only be a consequence of SUD, but may in fact play a role in mediating behavioral responses to drugs of abuse (Salavrakos et al., 2021 ). The expression of central GABA receptors, which play a critical role in emotional regulation capacities, is partially dependent on gut microbiota colonization, and their production decreases in germ-free (GF) mice. The GF mice exhibit weak social cognition and fewer anxiety-like behaviors. Fecal microbiota transplantation from patients with major depressive disorders to GF mice leads to depression-like behavior, whereas FMT from healthy donors has no effect on mouse behavior (Zheng et al., 2016 ). Recently, Cuesta et al. showed that cocaine exposure increases gut norepinephrine levels and facilitates Proteobacteria colonization in mice. Proteobacteria consume glycine from the host and facilitate cocaine-induced addition-like behaviors (Cuesta et al., 2022 ). These studies demonstrate that altered microbiota communities can affect host behavior by modulating host metabolism. In this study, it was predicted that the parathyroid hormone and relaxin signaling pathways were altered after MCAT treatment (Fig. 7 A). Hypoparathyroidism, a rare endocrine disorder where there is deficiency of parathyroid hormone, can cause emotional complaints, including anxiety and depression (Vokes et al., 2019). The relaxin signaling pathway can modulate social recognition in rats via effects within the amygdala, likely through interactions with GABA and oxytocin signaling (Albert-Gasco et al., 2019 ). It is reasonable to hypothesize that MCAT exposure modulates the behavior of zebrafish via the parathyroid hormone and relaxin signaling pathways, which requires further confirmation. In conclusion, we established an MCAT exposure and reward behavioral response model in zebrafish. We also demonstrated that MCAT treatment alters the behavioral response and gut microbiota composition in zebrafish. It has been predicted that a series of metabolic pathway changes are associated with shifts in the gut microbiota profile, which may contribute to neurobehavioral responses to MCAT exposure. Abbreviations MCAT SUDs GABA FMT MDMA CT ET OTU PcoA LEfSe ARVC GnRH HIF METH GF Methcathinone Substance use disorders Gamma-aminobutyric acid Fecal microbiota transplantation 3,4-methylenedioxymethamphetamine Conspecific tank Empty tank Operational taxonomic unit Principal coordinates analysis Linear discriminant analysis effect size Arrhythmogenic right ventricular cardiomyopathy Gonadotropin-releasing hormone Hypoxia-inducible factors Methamphetamine Germ-free Declarations Ethics approval and consent to participate This study was in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and the protocols were approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology (Permit Number: S814, Wuhan, China). Consent for publication Not applicable. Availability of data and materials The datasets generated and analysed during the current study are available in the NCBI repository, [BioProject: PRJNA1253566]. Competing Interests The authors declare no competing interests. Funding This work was supported by the National Key Research and Development Program of China (2022YFC2305100 and 2024YFC3306604). Authors' contributions Aijia Zhang: methodology, validation, formal analysis, investigation, data curation, writing original draft, software. Shuang Ye: methodology, validation, formal analysis, investigation, data curation. Liqi Wang: methodology, validation, formal analysis, investigation, data curation, software. Balibuli Bahetibieke: methodology, validation, formal analysis, investigation, data curation, software. Junzhong Wang: conceptualization, project administration, visualization, supervision, writing, review and editing. Man Liang: conceptualization, project administration, visualization, supervision, writing, review and editing. All authors read and approved the final manuscript. Acknowledgements We would like to acknowledge the participants in Majorbio Company (Shanghai, China) for their contribution, and assistance with this work. And we also would like to thank Editage (www.editage.cn) for English language editing. References Albert-Gasco H, Sanchez-Sarasua S, Ma S, García-Díaz C, Gundlach AL, Sanchez-Perez AM, et al. Central relaxin-3 receptor (RXFP3) activation impairs social recognition and modulates ERK-phosphorylation in specific GABAergic amygdala neurons. Brain Struct Funct. 2019;224(1):453–69. 10.1007/s00429-018-1763-5 . Bajaj JS. Alcohol, liver disease and the gut microbiota. Nat Rev Gastroenterol Hepatol. 2019;16(4):235–46. 10.1038/s41575-018-0099-1 . Bajaj JS, Gavis EA, Fagan A, Wade JB, Thacker LR, Fuchs M, et al. A Randomized Clinical Trial of Fecal Microbiota Transplant for Alcohol Use Disorder. Hepatology. 2021;73(5):1688–700. 10.1002/hep.31496 . Baumann MH, Volkow ND. Abuse of New Psychoactive Substances: Threats and Solutions. Neuropsychopharmacology. 2016;41(3):663–5. 10.1038/npp.2015.260 . Blough BE, Decker AM, Landavazo A, Namjoshi OA, Partilla JS, Baumann MH, et al. The dopamine, serotonin and norepinephrine releasing activities of a series of methcathinone analogs in male rat brain synaptosomes. Psychopharmacology. 2019;236(3):915–24. 10.1007/s00213-018-5063-9 . Baumann MH, Solis E Jr, Watterson LR, Marusich JA, Fantegrossi WE, Wiley JL. Baths salts, spice, and related designer drugs: the science behind the headlines. J Neurosci. 2014;34(46):15150–8. 10.1523/JNEUROSCI.3223-14.2014 . Carter RC, Wainwright H, Molteno CD, Georgieff MK, Dodge NC, Warton F, et al. Alcohol, Methamphetamine, and Marijuana Exposure Have Distinct Effects on the Human Placenta. Alcohol Clin Exp Res. 2016;40(4):753–64. 10.1111/acer.13022 . Cuesta S, Burdisso P, Segev A, Kourrich S, Sperandio V. Gut colonization by Proteobacteria alters host metabolism and modulates cocaine neurobehavioral responses. Cell Host Microbe. 2022;30(11):1615–e16295. 10.1016/j.chom.2022.09.014 . Davies RA, Baird TR, Nguyen VT, Ruiz B, Sakloth F, Eltit JM, et al. Investigation of the Optical Isomers of Methcathinone, and Two Achiral Analogs, at Monoamine Transporters and in Intracranial Self-Stimulation Studies in Rats. ACS Chem Neurosci. 2020;11(12):1762–9. 10.1021/acschemneuro.9b00617 . Ding Z, Huang G, Wang T, Duan W, Li H, Wang Y, et al. Genetic Ablation of GIGYF1, Associated With Autism, Causes Behavioral and Neurodevelopmental Defects in Zebrafish and Mice. Biol Psychiatry. 2023;94(10):769–79. 10.1016/j.biopsych.2023.02.993 . Gatch MB, Rutledge MA, Forster MJ. Discriminative and locomotor effects of five synthetic cathinones in rats and mice. Psychopharmacology. 2015;232(7):1197–205. 10.1007/s00213-014-3755-3 . Guo W, Zhou X, Li X, Zhu Q, Peng J, Zhu B, et al. Depletion of Gut Microbiota Impairs Gut Barrier Function and Antiviral Immune Defense in the Liver. Front Immunol. 2021;12:636803. 10.3389/fimmu.2021.636803 . Hamamah S, Aghazarian A, Nazaryan A, Hajnal A, Covasa M. Role of Microbiota-Gut-Brain Axis in Regulating Dopaminergic Signaling. Biomedicines. 2022;10(2):436. 10.3390/biomedicines10020436 . Jiang P, Green SJ, Chlipala GE, Turek FW, Vitaterna MH. Reproducible changes in the gut microbiome suggest a shift in microbial and host metabolism during spaceflight. Microbiome. 2019;7(1):113. 10.1186/s40168-019-0724-4 . Jones S, Fileccia EL, Murphy M, Fowler MJ, King MV, Shortall SE, et al. Cathinone increases body temperature, enhances locomotor activity, and induces striatal c-fos expression in the Siberian hamster. Neurosci Lett. 2014;559:34–8. 10.1016/j.neulet.2013.11.032 . Kiraly DD, Walker DM, Calipari ES, Labonte B, Issler O, Pena CJ, et al. Alterations of the Host Microbiome Affect Behavioral Responses to Cocaine. Sci Rep. 2016;6:35455. 10.1038/srep35455 . Kousik SM, Graves SM, Napier TC, Zhao C, Carvey PM. Methamphetamine-induced vascular changes lead to striatal hypoxia and dopamine reduction. NeuroReport. 2011;22(17):923–8. 10.1097/WNR.0b013e32834d0bc8 . Lee M, Leskova W, Eshaq RS, Harris NR. Retinal hypoxia and angiogenesis with methamphetamine. Exp Eye Res. 2021;206:108540. 10.1016/j.exer.2021.108540 . Magne F, Gotteland M, Gauthier L, Zazueta A, Pesoa S, Navarrete P, et al. The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese Patients? Nutrients. 2020;12(5):1474. 10.3390/nu12051474 . Nguyen JD, Aarde SM, Cole M, Vandewater SA, Grant Y, Taffe MA. Locomotor Stimulant and Rewarding Effects of Inhaling Methamphetamine, MDPV, and Mephedrone via Electronic Cigarette-Type Technology. Neuropsychopharmacology. 2016;41(11):2759–71. 10.1038/npp.2016.88 . Papaseit E, Pérez-Mañá C, Mateus JA, Pujadas M, Fonseca F, Torrens M, et al. Human Pharmacology of Mephedrone in Comparison with MDMA. Neuropsychopharmacology. 2016;41(11):2704–13. 10.1038/npp.2016.75 . Papaseit E, Olesti E, Pérez-Mañá C, Torrens M, Fonseca F, Grifell M, et al. Acute Pharmacological Effects of Oral and Intranasal Mephedrone: An Observational Study in Humans. Pharmaceuticals (Basel). 2021;14(2):100. 10.3390/ph14020100 . Qu D, Zhang K, Chen L, Wang Q, Wang H. RNA-sequencing analysis of the effect of luteolin on methamphetamine-induced hepatotoxicity in rats: a preliminary study. PeerJ. 2020;8:e8529. 10.7717/peerj.8529 . Scorza C, Piccini C, Martínez Busi M, Abin Carriquiry JA, Zunino P. Alterations in the Gut Microbiota of Rats Chronically Exposed to Volatilized Cocaine and Its Active Adulterants Caffeine and Phenacetin. Neurotox Res. 2019;35(1):111–21. 10.1007/s12640-018-9936-9 . Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain Res 1693(Pt B). 2018;128–33. 10.1016/j.brainres.2018.03.015 . Spiller HA, Ryan ML, Weston RG, Jansen J. Clinical experience with and analytical confirmation of bath salts and legal highs (synthetic cathinones) in the United States. Clin Toxicol (Phila). 2011;49(6):499–505. 10.3109/15563650.2011.590812 . Salavrakos M, Leclercq S, De Timary P, Dom G. Microbiome and substances of abuse. Prog Neuropsychopharmacol Biol Psychiatry. 2021;105:110113. 10.1016/j.pnpbp.2020.110113 . Thomaz AC, Iyer V, Woodward TJ, Hohmann AG. Fecal microbiota transplantation and antibiotic treatment attenuate naloxone-precipitated opioid withdrawal in morphine-dependent mice. Exp Neurol. 2021;343:113787. 10.1016/j.expneurol.2021.113787 . van de Wetering R, Schenk S. Repeated MDMA administration increases MDMA-produced locomotor activity and facilitates the acquisition of MDMA self-administration: role of dopamine D2 receptor mechanisms. Psychopharmacology. 2017;234(7):1155–64. 10.1007/s00213-017-4554-4 . Vokes T. Quality of life in hypoparathyroidism. Bone. 2019;120:542–7. 10.1016/j.bone.2018.09.017 . Wang J, Zhou X, Li X, Guo W, Zhu Q, Zhu B, et al. Fecal Microbiota Transplantation Alters the Outcome of Hepatitis B Virus Infection in Mice. Front Cell Infect Microbiol. 2022;12:844132. 10.3389/fcimb.2022.844132 . Wojcieszak J, Andrzejczak D, Wojtas A, Gołembiowska K, Zawilska JB. Methcathinone and 3-Fluoromethcathinone Stimulate Spontaneous Horizontal Locomotor Activity in Mice and Elevate Extracellular Dopamine and Serotonin Levels in the Mouse Striatum. Neurotox Res. 2019;35(3):594–605. 10.1007/s12640-018-9973-4 . Wu Q, Liang X, Wang K, Lin J, Wang X, Wang P, et al. Intestinal hypoxia-inducible factor 2α regulates lactate levels to shape the gut microbiome and alter thermogenesis. Cell Metab. 2021;33(10):1988–e20037. 10.1016/j.cmet.2021.07.007 . Xiao F, Zhu W, Yu Y, Huang J, Li J, He Z, et al. Interactions and Stability of Gut Microbiota in Zebrafish Increase with Host Development. Microbiol Spectr. 2022;10(2):e0169621. 10.1128/spectrum.01696-21 . Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015;161(2):264–76. 10.1016/j.cell.2015.02.047 . Yan PJ, Ren ZX, Shi ZF, Wan CL, Han CJ, Zhu LS, et al. Dysregulation of iron homeostasis and methamphetamine reward behaviors in Clk1-deficient mice. Acta Pharmacol Sin. 2022;43(7):1686–98. 10.1038/s41401-021-00806-1 . Zhou J, Deng W, Chen C, Kang J, Yang X, Dou Z, et al. Methcathinone Increases Visually-evoked Neuronal Activity and Enhances Sensory Processing Efficiency in Mice. Neurosci Bull. 2023;39(4):602–16. 10.1007/s12264-022-00965-z . Zhu Q, Xia P, Zhou X, Li X, Guo W, Zhu B, et al. Hepatitis B Virus Infection Alters Gut Microbiota Composition in Mice. Front Cell Infect Microbiol. 2019;9:377. 10.3389/fcimb.2019.00377 . Zheng P, Zeng B, Zhou C, Liu M, Fang Z, Xu X, et al. Gut microbiome remodeling induces depressive-like behaviors through a pathway mediated by the host's metabolism. Mol Psychiatry. 2016;21(6):786–96. 10.1038/mp.2016.44 . Additional Declarations No competing interests reported. Supplementary Files SupplementaryFig1.jpg SupplementaryFig2.jpg SupplementaryFig3.jpg SupplementaryFig4.jpg SupplementaryFig5.jpg SupplementaryFig6.jpg SupplementaryFig7.jpg SupplementaryFig8.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6395322","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":458794635,"identity":"70b849c7-7e96-4e05-a127-d799e4daee5a","order_by":0,"name":"Aijia Zhang","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Aijia","middleName":"","lastName":"Zhang","suffix":""},{"id":458794636,"identity":"7b097bd6-8083-4c67-a48f-04f067a7089a","order_by":1,"name":"Shuang Ye","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Ye","suffix":""},{"id":458794637,"identity":"b5acc03f-968b-4555-a748-43807d790b83","order_by":2,"name":"Liqi Wang","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Liqi","middleName":"","lastName":"Wang","suffix":""},{"id":458794638,"identity":"1d31262e-be60-4a03-b1ab-07e174e9d8a3","order_by":3,"name":"Balibuli Bahetibieke","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Balibuli","middleName":"","lastName":"Bahetibieke","suffix":""},{"id":458794639,"identity":"4717ad04-2e49-4b79-b17e-b4b96c58961c","order_by":4,"name":"Junzhong Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIie3RPUvDQBjA8ScErstJ14R+iYOD0xDsfRVDoC5pF0E6FoRzudJZ6AfpWHmGLrGuAR0UQQQzpAgFNy814HQho9D7w71wPL/pAFyu/1pgFqsvu9nfQzfi3RlCOxFoiH/ShbDNA35E6lme9ub4eb5CKcG/f6IwnFhJPhmdherdj/R2FI9zTDSQNKaQXtmIWGeChQoJKzLBxwovKFAxoLBOZjbyWB4IPZBIoaTQ37eTIuMvhgSG8DdPoaeBklYii1JAsEXG8lx4c3WZaCQ8WrLUSsJFxr+Ca5Rso3n1rWLZu715Lcrp0EpMZNB8A/k9/Xpj9vl6ZFc1l6p1zuVyuY62H4bTVUnGYCv1AAAAAElFTkSuQmCC","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Junzhong","middleName":"","lastName":"Wang","suffix":""},{"id":458794640,"identity":"64c99539-8e0b-47de-9f0c-06cc5ea3d247","order_by":5,"name":"Man Liang","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Man","middleName":"","lastName":"Liang","suffix":""}],"badges":[],"createdAt":"2025-04-07 14:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6395322/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6395322/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83140837,"identity":"db02dc96-5477-4502-8321-3061a285f606","added_by":"auto","created_at":"2025-05-20 12:11:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1151831,"visible":true,"origin":"","legend":"\u003cp\u003eNovel tank diving test was used to evaluate the effects of MCAT on anxiety levels and locomotion of zebrafish (A). After MCAT exposure for 10 min, the time spent in the bottom zone by 1.0 and 5.0 µg/mL MCAT-treated fish was significantly lower than that in a control group, but much higher than that in a 10.0 µg/mL MCAT-treated fish group (B); while opposite in the top zone (C). Fishes treated with 1.0 and 5.0 µg/mL of MCAT also showed lower latency to the middle and top zones of the novel tank diving test (D, E). The fishes treated with 1.0 and 5.0 µg/mL of MCAT had more freezing time and less latency to the middle and top zones, while the 10.0 µg/mL MCAT-treated fish showed opposite (F). A mirror-biting test was used to evaluate the aggressiveness of adult zebrafish (G). The 1.0 and 5.0 µg/mL MCAT-treated fish groups showed a higher time and frequency in the mirror zone than the control group, and it was lower in the 10.0 µg/mL MCAT-treated fish group than that in the control group, with a significant level (H, I, J). n=20, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001. MCAT, methcathinone\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/c269e0acd93fb0b1d1b51099.jpg"},{"id":83140842,"identity":"2f7229cb-25b6-4956-ab4b-9c29af65ded0","added_by":"auto","created_at":"2025-05-20 12:11:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":965854,"visible":true,"origin":"","legend":"\u003cp\u003eSocial preference test was performed to investigate the effects of MCAT on social interactions of zebrafish (A). Both 1.0 and 5.0 µg/mL MCAT-treated fishes showed a preference for social partners, and they spent more time in the social area of the tank with quicker pace, while they spent less time in the empty zone (B, C, D). The open field test was performed with 5.0 μg/mL MCAT-treated zebrafish. As shown in Fig. 2E, the total distance moved and mobility time increased during the test (F, G), and the time spent in the center zone was increased with higher speed in zebrafish treated with MCAT (H, I). n=20, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001. MCAT, methcathinone\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/1c0c648c44daee2d530eac19.jpg"},{"id":83142321,"identity":"6fe3b974-a556-45de-9a12-1ca4540bf0c0","added_by":"auto","created_at":"2025-05-20 12:27:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":250767,"visible":true,"origin":"","legend":"\u003cp\u003e5.0 μg/mL MCAT treatment decreased the abundance and diversity of gut microbiota in zebrafish. After repeated MCAT exposure, the OTU count (A), α diversity (B), and β diversity (C) were compared between control and MCAT group. n=20. MCAT, methcathinone; OTU, operational taxonomic unit\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/43397e739eeffc4bf7061f17.jpg"},{"id":83141161,"identity":"7f4bec58-bde1-4013-bc5f-2449537c663f","added_by":"auto","created_at":"2025-05-20 12:19:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":304326,"visible":true,"origin":"","legend":"\u003cp\u003e5.0 μg/mL MCAT treatment shifted the gut microbiota at the genus level in zebrafish. A Venn diagram (A). After repeated MCAT exposure, genus level composition of the gut microbiota in control and MCAT group (B). The genera with significant differences between the control and MCAT group (C). n=20. MCAT, methcathinone\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/cab8597a5374eb1cb75d51d4.jpg"},{"id":83141163,"identity":"f9b1b61b-f8a8-4422-b5e6-6749c1dbd684","added_by":"auto","created_at":"2025-05-20 12:19:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":373863,"visible":true,"origin":"","legend":"\u003cp\u003e5.0 μg/mL MCAT treatment shifted the gut microbiota at the species level in zebrafish. A Venn diagram (A). After repeated MCAT exposure, species level composition of the gut microbiota in control and MCAT group (B). Species with significant differences between the control and MCAT group (C). n=20. MCAT, methcathinone\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/4f40c842d6e22f0a213a8d40.jpg"},{"id":83140839,"identity":"b787ff8c-8eed-4ae5-b3c7-6f2305dc3efb","added_by":"auto","created_at":"2025-05-20 12:11:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":613257,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in bacterial richness in control and MCAT zebrafish group after repeated 5.0 μg/mL MCAT exposure. Linear discriminant analysis effect size analysis was performed to determine the difference in bacterial richness; the threshold of LDA score was 2.0. The cladogram represents the phylogenetic relationship of significant OTUs associated with each group. MCAT, methcathinone;LDA, logarithmic discriminant analysis; OTUs, operational taxonomic units\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/20b5673d830bbf4fa7045f39.jpg"},{"id":83140834,"identity":"80b0095c-ea9d-4a24-982d-1df86e9f8f43","added_by":"auto","created_at":"2025-05-20 12:11:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":391623,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic pathways were predicted based on the abundance of functional genes in the gut microbiota by PICRUSt2. (A) Heat map showed the predictive metabolic pathways from control and MCAT group (5.0 μg/mL). (B) The correlation network of predictive metabolic pathways and changed microbiome at the phylum level. The line between the microbiome and metabolic pathways indicates correlation: the red line indicates positive correlation, and the blue line indicates negative correlation. MCAT, methcathinone\u003c/p\u003e","description":"","filename":"Fig7A.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/fd223ac9a2829009cfed4f75.jpg"},{"id":83141166,"identity":"0ede656c-4ad9-4fab-8936-b859128241de","added_by":"auto","created_at":"2025-05-20 12:19:00","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":653297,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MCAT-induced intestinal histology of zebrafish via H\u0026amp;E and alcian blue-PAS staining. The intestinal structure was examined post intoxication using H\u0026amp;E staining under an 40× objective lens (scale bar = 100 μm). Yellow arrows indicate vacuolar degeneration, red arrows indicate goblet cells, and the blue arrow indicates inflammatory infiltration (A, B). Compared with a control group (C), an MCAT-exposed group (5.0 μg/mL) displayed a greater presence of goblet cells in the intestines with respect to the other gradual decrease (D) with alcian blue-PAS staining. MCAT, methcathinone;H\u0026amp;E, hematoxylin and eosin; PAS, periodic acid Schiff\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/0d194bc8c5f94da6b7aed496.jpg"},{"id":87462656,"identity":"8c71b71c-1ea7-444e-a247-b0c2dbbcb3c5","added_by":"auto","created_at":"2025-07-24 06:32:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5424846,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/d13b9748-c01f-4dc2-b7af-44c982793c92.pdf"},{"id":83140850,"identity":"67b253b3-7b3c-4204-8bee-c8a6dbd9b506","added_by":"auto","created_at":"2025-05-20 12:11:00","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":332970,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/89a387ca0311499eca7e9df6.jpg"},{"id":83140844,"identity":"0caaf1f9-6f04-4a09-b220-c50de8a5f3d3","added_by":"auto","created_at":"2025-05-20 12:11:00","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":599264,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/2c8d88468311a2496f6cf807.jpg"},{"id":83142839,"identity":"60df59b6-4760-4fca-886e-df995fba8978","added_by":"auto","created_at":"2025-05-20 12:35:00","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":291855,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/385b8b38ac98eb3d03f0c9e7.jpg"},{"id":83144030,"identity":"cc3b0e65-0a89-41ba-b718-c40854b1984a","added_by":"auto","created_at":"2025-05-20 12:43:00","extension":"jpg","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":460176,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/6f8e88858c8a1ec4d2e94543.jpg"},{"id":83141165,"identity":"288bfdd1-be70-4476-b61d-a309eb08e971","added_by":"auto","created_at":"2025-05-20 12:19:00","extension":"jpg","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":524137,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/d870791bb32b5843afb8d0cb.jpg"},{"id":83142322,"identity":"6798d82b-cfbd-4017-8d4e-80656d092833","added_by":"auto","created_at":"2025-05-20 12:27:00","extension":"jpg","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":757512,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/26a7c1d7a144ed5c7cf5f1e0.jpg"},{"id":83140860,"identity":"2e6760c2-b8ba-45a0-bb1e-e4ea67664758","added_by":"auto","created_at":"2025-05-20 12:11:00","extension":"jpg","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":828514,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/e32156f658ea159774f75474.jpg"},{"id":83142326,"identity":"85e0e03c-320c-44b7-9799-2930d5d21b1a","added_by":"auto","created_at":"2025-05-20 12:27:00","extension":"jpg","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":325185,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395322/v1/b46e63dbeeb8f6118fa7c182.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Methcathinone exposure alters host behavior and gut microbiota community in zebrafish","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSubstance use disorders (SUDs) are characterized by chronic dependence on special substances (including alcohol, cocaine, amphetamine, opioids, nicotine etc.), and they represent mental and physical disorders. Increasing evidence indicates that persistent intake of addictive substances alters the gut microbiota, contributing to the development of SUDs. In alcohol-abusing individuals, the abundance and diversity of the gut microbiota decrease, while the proportion of pro-inflammatory taxa increases (Bajaj et al., 2019). Repeated cocaine intake altered the gut microbiota profile in a rat model (Scorza et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and gut microbiota depletion elevated conditioned place preference in mice (Kiraly et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The gut-brain axis represents a bidirectional relationship between the gut microbiota and brain. The gut microbiota regulates the metabolism of neurotransmitters; in turn, their synthesis, release, and transfer are modified after alteration of the gut microbiota due to SUDs. Dopamine is a crucial neurotransmitter for drug reward and reinforcement. Moreover, any drug with addictive potential can increase dopamine levels. More than half of the dopamine produced in the body is synthesized by enteric neurons and intestinal epithelial cells, and its levels are regulated by the gut microbiota (Hamamah et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Several neurotransmitters, including gamma-aminobutyric acid (GABA), serotonin, and dopamine, are produced directly by bacteria colonization of the gut, contributing to drug dependence (Yano et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Strandwitz et al., 2018). Additionally, fecal microbiota transplantation (FMT) has the potential to rescue gut microbiota dysbiosis, relieve alcohol-related liver disease, and reduce alcohol craving and consumption (Bajaj et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In a morphine-dependent mice, FMT attenuates naloxone-induced opioid withdrawal (Thomaz et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These results indicate the possibility of applying FMT as a compulsory treatment.\u003c/p\u003e \u003cp\u003eOwing to its simple manufacturing process and lack of proper regulation, synthetic cathinone has recently emerged as a popular drug of abuse through sensationalized media attention (Baumann et al., 2016). It can often be trafficked through the internet and even retail shops, partially resulting in the widespread availability of personal mood elevation and entertainment experimentation. Methcathinone (MCAT), as a parent β-ketone analog of methamphetamine and representative compound to a series of designer drugs, is re-used as a notably popular novel psychoactive substance. Considering the structural similarity between MCAT and amphetamine, we focused on enhancing monoaminergic neurotransmission, including the inhibition of monoamine reuptake, release of neurotransmitters stored inside synaptic vesicles, and direct interaction with receptors (Blough et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Davies et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Chronic MCAT and methamphetamine users showed significantly reduced dopamine transporter density by positron emission tomography, suggesting that functional regulation of the dopaminergic system is an expected consequence of drug abuse. Similar to several psychostimulants like methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA), MCAT can induce euphoric and stimulant feelings and desired effects for hours, including improved mood, euphoria, excitement, increased energy and motivation, and enhanced alertness and awareness (Papaseit et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Papaseit et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Uncontrolled craving for the aforementioned desired pleasures drives such users to escalate consumption. As overdose use of MCAT induces a wide range of toxic neurological and psychopathological effects, such as hallucinations, delusions, hyperthermia, and even death (Baumann et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Spiller et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), it has become a widely used recreational drug, with increasingly reported overdose incidents.\u003c/p\u003e \u003cp\u003eAlthough the behavioral and physiological effects of MCAT and its analogs have been studied in rats and mice (Gatch et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), the addictive behavior, potential psychomotor effects, and gut microbiota functions of MCAT have never been reported in other experimental animals. Despite numerous clinical cases of MCAT abuse, little is known about the effects of neurotransmitters on fecal microbiota. The goal of this study was to systematically evaluate the effects of MCAT on fecal microbiota function in a zebrafish model. We mapped the gut microbiota community and predicted alterations in metabolic pathways following MCAT administration. By combining behavioral testing and change in gut microbiota composition in MCAT-consuming animals, our findings highlighted the interaction between gut microbiota and the psychostimulant effects of MCAT.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eZebrafish husbandry\u003c/h2\u003e \u003cp\u003eWild-type adult zebrafish (AB strain) were obtained from the China Zebrafish Resource Center (Wuhan, China). Adult zebrafish were raised in a recirculation system with a 14 h-light/10 h-dark circadian cycle at 28.5\u0026deg;C, according to standard zebrafish breeding protocols (Ding et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The culture water supplied for the system was filtered by reverse osmosis (pH\u0026thinsp;=\u0026thinsp;7.0\u0026ndash;7.5). The adult zebrafish were fed twice daily at 08:30 and 17:30 with newly hatched Artemia (Jiahong Feed Co., Tianjin, China); however, they were not fed the day before the MCAT exposure experiments. The use of zebrafish in this study protocol was performed following the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemicals and exposure\u003c/h3\u003e\n\u003cp\u003eMethcathinone was purchased from the Wuhan Zhongchang National Research Standard Technology Co., Ltd., China (CAS number: 5650-44-2, #C-019). Stock solutions of 1.0 mg/mL were prepared by dissolving MCAT in the purified water and stored at \u003cb\u003e\u0026minus;\u0026thinsp;4\u003c/b\u003e℃. Working solutions were prepared by diluting the stock solution to the final concentrations (1.0, 5.0, and 10.0 \u0026micro;g/mL) immediately before experiments. Zebrafish were exposed to culture water (control) or three doses of MCAT for 10 min in 2 weeks before the experiments. All exposed solutions were replaced every time to ensure the exact MCAT concentration.\u003c/p\u003e\n\u003ch3\u003eBehavioral tests\u003c/h3\u003e\n\u003cp\u003eBehavioral tests were recorded using a behavioral analysis system with camera equipment, and movements were extracted and analyzed using Ethovision XT 11.5 software (Noldus IT, Wageningen, Netherlands). All the zebrafish were 12 months old and tested in a clear acrylic tank at the same time each day. As the MCAT condition leads to hyperlocomotion (Zhou et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we decided to apply 1.0, 5.0, and 10.0 \u0026micro;g/mL treatments for our initial behavioral experiments to explore the effect of different levels.\u003c/p\u003e\n\u003ch3\u003eNovel tank test\u003c/h3\u003e\n\u003cp\u003eEach zebrafish was placed in a tank with measuring 24 cm \u0026times; 8 cm \u0026times; 20 cm. The back side of the tank was covered with a light plate to aid in video recording. Tester fishes were dropped from the top of the tank. Videos were recorded for 10 min from a lateral viewpoint of the tank. For data analysis, the tank was artificially divided evenly into top, middle, and bottom zones from top to bottom. The time spent in the bottom and top zones, freezing time, and the latency to reach the middle and top zones during the tests were quantified.\u003c/p\u003e\n\u003ch3\u003eMirror biting test\u003c/h3\u003e\n\u003cp\u003eA rectangular tank (18 cm \u0026times; 5 cm \u0026times; 15 cm) with a mirror positioned vertically on the left side was used. The back side of the tank was covered with a light plate to aid in data recording. Experimental fishes (one at a time) were placed inside the tank and monitored continuously for 6 min from a lateral viewpoint. The region in which the tester fish could touch the mirror was designated as the mirror zone (3 cm wide, the same as the average body length of an adult zebrafish). Endpoints used to assess mirror biting response and reflect aggression-related behaviors included time and entry frequency into the mirror zone.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSocial preference test\u003c/h2\u003e \u003cp\u003eSocial preference tests were performed in a rectangular tank (40 cm \u0026times; 15 cm \u0026times; 15 cm) separated into three compartments (10 cm \u0026times; 15 cm \u0026times; 15 cm, 20 cm \u0026times; 15 cm \u0026times; 15 cm, and 10 cm \u0026times; 15 cm \u0026times; 15 cm) from left to right using two transparent dividers. The left and right end tanks were used as either a conspecific tank (CT) or empty tank (ET), alternating between trials to avoid lateral bias. The tank was then placed on a light plate to facilitate video tracking. Before each test trial, four companion fishes were placed in the CT, and water with no fish was placed in the ET. The test fishes were placed in the central tank and allowed to acclimate to the new environment for 5 min before starting a 15 min video recording for behavioral tracking. The center tank was identified as the social contact, center, and empty zones, which were isometric based on their distance from the CT. Social preference was assessed by calculating the time spent in the social contact and empty zones, as well as the average speed in the social contact zone.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOpen field test\u003c/h3\u003e\n\u003cp\u003eIndividual fish was placed in the center of a tank (40 cm \u0026times; 40 cm \u0026times; 20 cm) and allowed to swim freely for 5 min. Videos were recorded from the top of the tank for 15 min. A square area of \u0026gt;\u0026thinsp;10 cm\u003csup\u003e2\u003c/sup\u003e away from the nearest edge of the tank was defined as the center zone and the rest as the peripheral zone. Total distance, time spent in the center and peripheral zones, latency to the center zone, and frequency of high-speed movement (60% above the average speed) were calculated.\u003c/p\u003e\n\u003ch3\u003eFecal sample collection and 16S rRNA sequencing\u003c/h3\u003e\n\u003cp\u003eAt the time of sampling, the whole intestine of each fish was aseptically removed and collected as a single sample, as previously described (Xiao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Bacterial 16S rRNA was amplified, extracted, pooled in equimolar ratios, and paired-end sequenced (2 \u0026times; 300) on an Illumina MiSeq platform (Illumina, San Diego, USA), as previously reported (Zhu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Briefly, bacterial DNA was extracted using an E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). Sequencing of 16S rRNA gene was performed as described previously (Guo et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), using primers to amplify the V3-V4 hypervariable regions of the bacterial 16S rRNA gene (338F, 5\u0026prime;-ACTCCTACGGGAGGCAGCAG-3\u0026prime; and 806R 5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;). Amplicons were purified and paired-end sequenced (2 \u0026times; 300) using an Illumina MiSeq System (Illumina, San Diego, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA). In all the experiments, comparisons were performed using a two-tailed unpaired Student\u0026rsquo;s t-test or one-way analysis of variance with the Dunnett\u0026rsquo;s multiple comparison test. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. In these figures, * indicates p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eBehavior test\u003c/b\u003e \u003c/p\u003e \u003cp\u003e1. Novel tank test\u003c/p\u003e \u003cp\u003eA novel tank diving test was used to evaluate the effects of MCAT on anxiety and locomotion in zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). After MCAT exposure for 10 min (Supplementary Fig.\u0026nbsp;1A), the time spent in the bottom zone by 1.0 and 5.0 \u0026micro;g/mL MCAT-treated fish was significantly lower than that in the control fish, but the 10.0 \u0026micro;g/mL MCAT-treated fish was much higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB); although opposite in the top zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The fishes treated with 1.0 and 5.0 \u0026micro;g/mL of MCAT also showed lower latency to the middle and top zones of the novel tank diving test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). To investigate whether the decrease in travel distance was due to a reduction in fish motivation, we examined the latency to the middle and top zones and freezing time. Compared with the control fishes, the fishes treated with 1.0 and 5.0 \u0026micro;g/mL of MCAT had more freezing time and less latency to the middle and top zones, although 10.0 \u0026micro;g/mL of the MCAT-treated fish showed opposite again (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), indicating lower and medium doses of MCAT-treated fish had a lower endurance of anxious behavior, but higher dose of MCAT led to a high level of anxiety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e2. Mirror biting test\u003c/p\u003e \u003cp\u003eA mirror biting test was used to evaluate the aggressiveness of the adult zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The 1.0 and 5.0 \u0026micro;g/mL MCAT-treated fishes showed a higher time and frequency in the mirror zone than the control fishes, and 10.0 \u0026micro;g/mL of the MCAT-treated fish was lower than that of the control, with a significant level (n\u0026thinsp;=\u0026thinsp;20, p\u0026thinsp;=\u0026thinsp;0.05; binomial test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I, J), which indicated that MCAT enhanced the aggressiveness of the zebrafish.\u003c/p\u003e \u003cp\u003e3. Social preference test\u003c/p\u003e \u003cp\u003eZebrafish are highly social and naturally form groups (shoals) with structured social relationships. A social preference test was performed to investigate the effects of MCAT on social interactions in zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Both the 1.0 and 5.0 \u0026micro;g/mL MCAT-treated fish showed a preference for social partners, and they spent more time in the social area of the tank with quicker pace, while less time in the empty zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C, D). The average time spent and speed in the social zones by 1.0 and 5.0 \u0026micro;g/mL MCAT-treated fish was higher than that of the control fishes with statistical significance, while 10.0 \u0026micro;g/ml MCAT-treated fish was almost the same, indicating the higher dose might suppress the social preference (n\u0026thinsp;=\u0026thinsp;10, p\u0026thinsp;=\u0026thinsp;0.05; unpaired t-test).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOpen field test\u003c/p\u003e \u003cp\u003eBased on the aforementioned results of the behavioral tests, the open field test was performed with 5.0 \u0026micro;g/mL MCAT-treated zebrafish. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, the total distance moved and mobility time increased during the test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G), and the time spent in the center zone increased at a higher speed in the zebrafish treated with MCAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I). This was likely due to the decreased time spent swimming in the center of the test box rather than reducing free exploration along the walls.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMCAT treatment altered the gut microbiota community\u003c/h2\u003e \u003cp\u003eTo confirm whether MCAT treatment altered the gut microbial community in zebrafish, we established a chronic MCAT exposure model. Based on the aforementioned results of the behavioral tests, the zebrafish were treated with 5.0 ug/mL of MCAT 10 min twice daily for 15 days, and fecal samples were collected and 16S rRNA sequencing was performed (Supplementary Fig.\u0026nbsp;1B). The operational taxonomic unit (OTU) count index decreased in the MCAT group compared to that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The Chao community richness at the OTU and specie levels decreased significantly in the MCAT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), although the Simpson index at the OTU level and Shannon index at the species level were comparable (Supplementary Fig.\u0026nbsp;2A). It indicated that α diversity decreased after MCAT treatment. Compared with the control group, principal coordinates analysis (PCoA) showed that the cluster of the MCAT group shifted on the OTU, genus, and specie levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), but no significant differences were observed at the phylum, class, order, and family levels (Supplementary Fig.\u0026nbsp;2B). These results indicated that MCAT treatment altered the gut microbiota community, with a significant decrease in both abundance and diversity of the gut microbiota.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiome profiles shift after MCAT treatment\u003c/h2\u003e \u003cp\u003eThe gut microbiota composition was assessed at each taxonomic level to evaluate the effect of MCAT treatment on the gut microbiota profile. Five domain phyla comprised\u0026thinsp;\u0026gt;\u0026thinsp;95% of the gut microbiota in zebrafish, including Proteobacteria, Actinobacteria, Fusobacteriota, Firmicutes, and Bacteroidetes (Supplementary Fig.\u0026nbsp;4B). Firmicutes and Bacteroidetes are the two most common phyla in humans and mice. Significant alterations in the ratio of Firmicutes to Bacteroidetes have been observed in patients with various chronic diseases, and they are sensitive markers of overall microbiota alteration in humans (Magne et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In zebrafish, Proteobacteria and Actinobacteria were the two major components of the gut microbiota, accounting for \u0026gt;\u0026thinsp;80% of the gut microbiota composition (Supplementary Fig.\u0026nbsp;4B). Our data showed an increasing trend in the ratio of Proteobacteria to Actinobacteriota and Firmicutes to Bacteroidetes, although no significant change was observed (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eCompared to the control zebrafish, the composition of the gut microbiota in the MCAT group shifted significantly at each taxon level. A Venn diagram showed the shared and unique bacteria in both groups. After the MCAT treatment, the proportions of three phyla, six classes, twelve orders, and twelve families decreased. In contrast, two orders and three families increased significantly in the MCAT group (Supplementary Fig.\u0026nbsp;4\u0026ndash;7).\u003c/p\u003e \u003cp\u003eAt the genus level, 289 genera were shared by both groups; 240 and 176 genera were unique to the control and MCAT group, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The proportion of 12 genera decreased and that of three genera (Tardiphaga, Sphingomonas, and Vagnococcus) increased significantly in the MCAT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). At the specie level, 334 species were shared; 373 and 293 species were unique to the control and MCAT groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Twelve species decreased, and three species (\u003cem\u003eRhodococcus erythropolis\u003c/em\u003e, uncultured bacterium Tardiphaga, \u003cem\u003eSphingomonas paucimobilis\u003c/em\u003e, and Sphingomonas) increased in the MCAT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Linear discriminant analysis effect size (LEfSe) was performed to identify specific bacteria that exhibited remarkably different proportions in the control and MCAT groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These data indicate that MCAT treatment shifts the gut microbiota profile and induces dysbiosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePredicted metabolic pathway analysis after MCAT treatment\u003c/h2\u003e \u003cp\u003eBased on the 16S rRNA sequencing data, we inferred the abundance of functional genes in the gut microbiota and predicted their metabolic pathways using the software package PICRUSt2 (Jiang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After investigating the level 3 KEGG functional classes, 41 metabolic pathways showing statistically different abundance between the two groups were identified. These 41 metabolic pathways belong to 19 superclasses. Interestingly, four pathways were related to antibiotics biosynthesis, including acarbose and validamycin biosynthesis, penicillin and cephalosporin biosynthesis, biosynthesis of vancomycin group antibiotics, and biosynthesis of enediyne antibiotics. This suggests that MCAT treatment influences the levels and composition of antibiotics produced by the gut microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The proportions of facultative anaerobic, gram-positive, and gram-negative bacteria were altered after the MCAT treatment (Supplementary Fig.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe network between the gut microbiota and metabolic pathways is shown based on the correlation coefficients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Focal adhesion, apoptosis, protein digestion and absorption, hematopoietic cell lineage, arrhythmogenic right ventricular cardiomyopathy (ARVC), regulation of the actin cytoskeleton, acarbose and validamycin biosynthesis, osteoclast differentiation, ferroptosis, staurosporine biosynthesis, and dilated cardiomyopathy were negatively correlated with Fusobacteriota. Parathyroid hormone synthesis, secretion, and action, biosynthesis of various secondary metabolites part 1, gonadotropin-releasing hormone (GnRH) signaling pathway, pancreatic cancer, AGE RAGE signaling pathway in diabetic complications, indole alkaloid biosynthesis, Fc gamma R-mediated phagocytosis, malaria, relaxin signaling pathway, chronic myeloid leukemia, and biosynthesis of vancomycin group antibiotics were positively correlated with Verrucomicrobiota. However, biosynthesis of the vancomycin group antibiotics was also positively related to Fusobacteriota, and dilated cardiomyopathy was negatively related to Verrucomicrobiota (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effect of MCAT on histopathology of intestinal tissue\u003c/h2\u003e \u003cp\u003eThis study examined the protective effects of MCAT on zebrafish colonic morphology. To investigate this, histological analysis of the zebrafish intestinal segments was performed following H\u0026amp;E and alcian blue-PAS staining. The control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) exhibited normal histological features with no morphological changes. However, the MCAT-treated group showed tissue inflammation and severe pathological changes, including high epithelial degradation, goblet cell hyperplasia, and lymphocyte accumulation. The MCAT-exposed group showed a greater number of goblet cells in the intestine than the other group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSubstance use disorders are characterized by chronic dependence on special substances (including alcohol, cocaine, amphetamine, opioids, nicotine etc.), and represent mental and physical disorders. Increasing evidence indicates that persistent intake of addictive substances alters the gut microbiota, contributing to the development of SUDs.\u003c/p\u003e \u003cp\u003eIn this study, we treated zebrafish with a popular novel psychoactive substance, MCAT, and found that at the behavioral level, MCAT induced dose-dependent changes in locomotion. Anxiety, aggressive, and social behaviors correlated with dosages increasing from 1.0 to 10.0 \u0026micro;g/mL. The effects of MCAT on zebrafish mimic several aspects in humans. MCAT induces a remarkable increase in locomotion in zebrafish, which is the most prominent behavioral effect reported in rodents (Gatch et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wojcieszak et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jones et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), similar to the hyperactive effects and higher psychomotor capacity produced by synthetic cathinones in humans. Characterized lap-running of the periphery in mice has also been reported for psychoactive synthetic cathinones, such as MDMA (van de Wetering et al., 2017; Nguyen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the novel tank and mirror biting tests, the lower and medium doses of MCAT-treated fish had lower endurance of anxious behavior but enhanced aggressiveness; a higher dose of MCAT led to a high level of anxiety and remained motionless. In the social preference test, the high-dose group swam frenetically along the wall at a high speed, completely ignoring the presence of the social partner in the tank (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, C, D). Lastly, enhancement of spontaneous locomotor activity in zebrafish occurred after exposure to MCAT at a medium dose (5.0 \u0026micro;g/mL) in the open field test for sure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F, G, H, I). It appears that MCAT produces hyperactive locomotion in an exaggerated state, and this behavior mimics the manic behavior reported in overdosing human users (Papaseit et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Papaseit et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Overall, the behavioral tests revealed that the effects of MCAT on zebrafish were similar to the psychostimulant effects reported by human users, suggesting that zebrafish are good animal models for studying the neuronal mechanisms of such drugs.\u003c/p\u003e \u003cp\u003eSimilar to other SUDs, MCAT administration can alter the gut microbiota of the host. Both abundance and diversity decreased after two weeks of MCAT exposure in zebrafish. However, the mechanisms underlying gut microbiota dysbiosis following MCAT treatment remain unclear. Both aerobic and anaerobic bacteria colonize the gut, and oxygen consumption affects the growth of commensal bacteria. Our data suggest that MCAT administration altered the proportion of facultative anaerobic bacteria (Supplementary Fig.\u0026nbsp;8). Hypoxia-inducible factors (HIF) are a family of proteins that can sense oxygen concentration, and their production is increased in hypoxic tissues. Intestinal HIF-2α increases intestinal lactic acid levels by directly activating lactate dehydrogenase A, thereby influencing the intestinal microbiota composition (Wu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Methamphetamine (METH), an analog of MCAT, is one of the most widely consumed psychostimulants worldwide. Increasing evidence confirms that METH abuse is associated with hypoxia and ischemia. METH exposure induces chronic hypoxia and increases placental growth in pregnant women, increasing the risk of neurodevelopmental deficits (Carter et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Transient and persistent METH consumption can induce vascular changes and promote the development of hypoxia and hypoperfusion in the striatum, leading to dopamine reduction and an increased risk of Parkinson\u0026rsquo;s disease (Kousik et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). METH reward behaviors are regulated by iron homeostasis and HIF-1α (Yan et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). METH treatment enhances the production of monoamine neurotransmitters, including dopamine, serotonin, and norepinephrine. All of these act as vasoconstrictors involved in the regulation of blood pressure and blood flow. Monoamine neurotransmitters promote platelet aggregation and occlusion. As a result, METH treatment induces retinal hypoxia and increased HIF-1α expression (Lee et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, METH treatment enhances the expression of antimicrobial peptides in rat liver, which are transported to the intestine and influence the gut microbiota community (Qu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Based on previous studies, it is reasonable to hypothesize that MCAT treatment may induce hypoxia in the gut and enhance the production of antimicrobial peptides in the liver, leading to alterations in the gut microbiota of zebrafish. The potential mechanisms underlying gut microbiota dysbiosis after MCAT treatment require further investigation.\u003c/p\u003e \u003cp\u003eIncreasing evidence indicates bidirectional communication between the gut microbiota and brain. The alteration of gut microbiota and its metabolites may not only be a consequence of SUD, but may in fact play a role in mediating behavioral responses to drugs of abuse (Salavrakos et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The expression of central GABA receptors, which play a critical role in emotional regulation capacities, is partially dependent on gut microbiota colonization, and their production decreases in germ-free (GF) mice. The GF mice exhibit weak social cognition and fewer anxiety-like behaviors. Fecal microbiota transplantation from patients with major depressive disorders to GF mice leads to depression-like behavior, whereas FMT from healthy donors has no effect on mouse behavior (Zheng et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Recently, Cuesta et al. showed that cocaine exposure increases gut norepinephrine levels and facilitates Proteobacteria colonization in mice. Proteobacteria consume glycine from the host and facilitate cocaine-induced addition-like behaviors (Cuesta et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These studies demonstrate that altered microbiota communities can affect host behavior by modulating host metabolism. In this study, it was predicted that the parathyroid hormone and relaxin signaling pathways were altered after MCAT treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Hypoparathyroidism, a rare endocrine disorder where there is deficiency of parathyroid hormone, can cause emotional complaints, including anxiety and depression (Vokes et al., 2019). The relaxin signaling pathway can modulate social recognition in rats via effects within the amygdala, likely through interactions with GABA and oxytocin signaling (Albert-Gasco et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is reasonable to hypothesize that MCAT exposure modulates the behavior of zebrafish via the parathyroid hormone and relaxin signaling pathways, which requires further confirmation.\u003c/p\u003e \u003cp\u003eIn conclusion, we established an MCAT exposure and reward behavioral response model in zebrafish. We also demonstrated that MCAT treatment alters the behavioral response and gut microbiota composition in zebrafish. It has been predicted that a series of metabolic pathway changes are associated with shifts in the gut microbiota profile, which may contribute to neurobehavioral responses to MCAT exposure.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" style=\"margin-right: calc(5%); width: 95%;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eMCAT\u003c/p\u003e\n \u003cp\u003eSUDs\u003c/p\u003e\n \u003cp\u003eGABA\u003c/p\u003e\n \u003cp\u003eFMT\u003c/p\u003e\n \u003cp\u003eMDMA\u003c/p\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003cp\u003eET\u003c/p\u003e\n \u003cp\u003eOTU\u003c/p\u003e\n \u003cp\u003ePcoA\u003c/p\u003e\n \u003cp\u003eLEfSe\u003c/p\u003e\n \u003cp\u003eARVC\u003c/p\u003e\n \u003cp\u003eGnRH\u003c/p\u003e\n \u003cp\u003eHIF\u003c/p\u003e\n \u003cp\u003eMETH\u003c/p\u003e\n \u003cp\u003eGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 359px;\"\u003e\n \u003cp\u003eMethcathinone\u003c/p\u003e\n \u003cp\u003eSubstance use disorders\u003c/p\u003e\n \u003cp\u003eGamma-aminobutyric acid\u003c/p\u003e\n \u003cp\u003eFecal microbiota transplantation\u003c/p\u003e\n \u003cp\u003e3,4-methylenedioxymethamphetamine\u003c/p\u003e\n \u003cp\u003eConspecific tank\u003c/p\u003e\n \u003cp\u003eEmpty tank\u003c/p\u003e\n \u003cp\u003eOperational taxonomic unit\u003c/p\u003e\n \u003cp\u003ePrincipal coordinates analysis\u003c/p\u003e\n \u003cp\u003eLinear discriminant analysis effect size\u003c/p\u003e\n \u003cp\u003eArrhythmogenic right ventricular cardiomyopathy\u003c/p\u003e\n \u003cp\u003eGonadotropin-releasing hormone\u003c/p\u003e\n \u003cp\u003eHypoxia-inducible factors\u003c/p\u003e\n \u003cp\u003eMethamphetamine\u003c/p\u003e\n \u003cp\u003eGerm-free\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and the protocols were approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology (Permit Number: S814, Wuhan, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available in the NCBI repository, [BioProject: PRJNA1253566].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2022YFC2305100 and 2024YFC3306604).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAijia Zhang: methodology, validation, formal analysis, investigation, data curation, writing original draft, software. Shuang Ye: methodology, validation, formal analysis, investigation, data curation. Liqi Wang: methodology, validation, formal analysis, investigation, data curation, software. Balibuli Bahetibieke: methodology, validation, formal analysis, investigation, data curation, software. Junzhong Wang: conceptualization, project administration, visualization, supervision, writing, review and editing. Man Liang: conceptualization, project administration, visualization, supervision, writing, review and editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the participants in Majorbio Company (Shanghai, China) for their contribution, and assistance with this work. And we also would like to thank Editage (www.editage.cn) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbert-Gasco H, Sanchez-Sarasua S, Ma S, Garc\u0026iacute;a-D\u0026iacute;az C, Gundlach AL, Sanchez-Perez AM, et al. Central relaxin-3 receptor (RXFP3) activation impairs social recognition and modulates ERK-phosphorylation in specific GABAergic amygdala neurons. Brain Struct Funct. 2019;224(1):453\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00429-018-1763-5\u003c/span\u003e\u003cspan address=\"10.1007/s00429-018-1763-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBajaj JS. Alcohol, liver disease and the gut microbiota. Nat Rev Gastroenterol Hepatol. 2019;16(4):235\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41575-018-0099-1\u003c/span\u003e\u003cspan address=\"10.1038/s41575-018-0099-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBajaj JS, Gavis EA, Fagan A, Wade JB, Thacker LR, Fuchs M, et al. A Randomized Clinical Trial of Fecal Microbiota Transplant for Alcohol Use Disorder. Hepatology. 2021;73(5):1688\u0026ndash;700. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/hep.31496\u003c/span\u003e\u003cspan address=\"10.1002/hep.31496\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaumann MH, Volkow ND. Abuse of New Psychoactive Substances: Threats and Solutions. Neuropsychopharmacology. 2016;41(3):663\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/npp.2015.260\u003c/span\u003e\u003cspan address=\"10.1038/npp.2015.260\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlough BE, Decker AM, Landavazo A, Namjoshi OA, Partilla JS, Baumann MH, et al. The dopamine, serotonin and norepinephrine releasing activities of a series of methcathinone analogs in male rat brain synaptosomes. Psychopharmacology. 2019;236(3):915\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00213-018-5063-9\u003c/span\u003e\u003cspan address=\"10.1007/s00213-018-5063-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaumann MH, Solis E Jr, Watterson LR, Marusich JA, Fantegrossi WE, Wiley JL. Baths salts, spice, and related designer drugs: the science behind the headlines. J Neurosci. 2014;34(46):15150\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/JNEUROSCI.3223-14.2014\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.3223-14.2014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarter RC, Wainwright H, Molteno CD, Georgieff MK, Dodge NC, Warton F, et al. Alcohol, Methamphetamine, and Marijuana Exposure Have Distinct Effects on the Human Placenta. Alcohol Clin Exp Res. 2016;40(4):753\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/acer.13022\u003c/span\u003e\u003cspan address=\"10.1111/acer.13022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuesta S, Burdisso P, Segev A, Kourrich S, Sperandio V. Gut colonization by Proteobacteria alters host metabolism and modulates cocaine neurobehavioral responses. Cell Host Microbe. 2022;30(11):1615\u0026ndash;e16295. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chom.2022.09.014\u003c/span\u003e\u003cspan address=\"10.1016/j.chom.2022.09.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavies RA, Baird TR, Nguyen VT, Ruiz B, Sakloth F, Eltit JM, et al. Investigation of the Optical Isomers of Methcathinone, and Two Achiral Analogs, at Monoamine Transporters and in Intracranial Self-Stimulation Studies in Rats. ACS Chem Neurosci. 2020;11(12):1762\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acschemneuro.9b00617\u003c/span\u003e\u003cspan address=\"10.1021/acschemneuro.9b00617\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing Z, Huang G, Wang T, Duan W, Li H, Wang Y, et al. Genetic Ablation of GIGYF1, Associated With Autism, Causes Behavioral and Neurodevelopmental Defects in Zebrafish and Mice. Biol Psychiatry. 2023;94(10):769\u0026ndash;79. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopsych.2023.02.993\u003c/span\u003e\u003cspan address=\"10.1016/j.biopsych.2023.02.993\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGatch MB, Rutledge MA, Forster MJ. Discriminative and locomotor effects of five synthetic cathinones in rats and mice. Psychopharmacology. 2015;232(7):1197\u0026ndash;205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00213-014-3755-3\u003c/span\u003e\u003cspan address=\"10.1007/s00213-014-3755-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo W, Zhou X, Li X, Zhu Q, Peng J, Zhu B, et al. Depletion of Gut Microbiota Impairs Gut Barrier Function and Antiviral Immune Defense in the Liver. Front Immunol. 2021;12:636803. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2021.636803\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2021.636803\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamamah S, Aghazarian A, Nazaryan A, Hajnal A, Covasa M. Role of Microbiota-Gut-Brain Axis in Regulating Dopaminergic Signaling. Biomedicines. 2022;10(2):436. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biomedicines10020436\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines10020436\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang P, Green SJ, Chlipala GE, Turek FW, Vitaterna MH. Reproducible changes in the gut microbiome suggest a shift in microbial and host metabolism during spaceflight. Microbiome. 2019;7(1):113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40168-019-0724-4\u003c/span\u003e\u003cspan address=\"10.1186/s40168-019-0724-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones S, Fileccia EL, Murphy M, Fowler MJ, King MV, Shortall SE, et al. Cathinone increases body temperature, enhances locomotor activity, and induces striatal c-fos expression in the Siberian hamster. Neurosci Lett. 2014;559:34\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neulet.2013.11.032\u003c/span\u003e\u003cspan address=\"10.1016/j.neulet.2013.11.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiraly DD, Walker DM, Calipari ES, Labonte B, Issler O, Pena CJ, et al. Alterations of the Host Microbiome Affect Behavioral Responses to Cocaine. Sci Rep. 2016;6:35455. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep35455\u003c/span\u003e\u003cspan address=\"10.1038/srep35455\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKousik SM, Graves SM, Napier TC, Zhao C, Carvey PM. Methamphetamine-induced vascular changes lead to striatal hypoxia and dopamine reduction. NeuroReport. 2011;22(17):923\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/WNR.0b013e32834d0bc8\u003c/span\u003e\u003cspan address=\"10.1097/WNR.0b013e32834d0bc8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee M, Leskova W, Eshaq RS, Harris NR. Retinal hypoxia and angiogenesis with methamphetamine. Exp Eye Res. 2021;206:108540. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.exer.2021.108540\u003c/span\u003e\u003cspan address=\"10.1016/j.exer.2021.108540\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagne F, Gotteland M, Gauthier L, Zazueta A, Pesoa S, Navarrete P, et al. The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese Patients? Nutrients. 2020;12(5):1474. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/nu12051474\u003c/span\u003e\u003cspan address=\"10.3390/nu12051474\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen JD, Aarde SM, Cole M, Vandewater SA, Grant Y, Taffe MA. Locomotor Stimulant and Rewarding Effects of Inhaling Methamphetamine, MDPV, and Mephedrone via Electronic Cigarette-Type Technology. Neuropsychopharmacology. 2016;41(11):2759\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/npp.2016.88\u003c/span\u003e\u003cspan address=\"10.1038/npp.2016.88\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapaseit E, P\u0026eacute;rez-Ma\u0026ntilde;\u0026aacute; C, Mateus JA, Pujadas M, Fonseca F, Torrens M, et al. Human Pharmacology of Mephedrone in Comparison with MDMA. Neuropsychopharmacology. 2016;41(11):2704\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/npp.2016.75\u003c/span\u003e\u003cspan address=\"10.1038/npp.2016.75\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapaseit E, Olesti E, P\u0026eacute;rez-Ma\u0026ntilde;\u0026aacute; C, Torrens M, Fonseca F, Grifell M, et al. Acute Pharmacological Effects of Oral and Intranasal Mephedrone: An Observational Study in Humans. Pharmaceuticals (Basel). 2021;14(2):100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ph14020100\u003c/span\u003e\u003cspan address=\"10.3390/ph14020100\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu D, Zhang K, Chen L, Wang Q, Wang H. RNA-sequencing analysis of the effect of luteolin on methamphetamine-induced hepatotoxicity in rats: a preliminary study. PeerJ. 2020;8:e8529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7717/peerj.8529\u003c/span\u003e\u003cspan address=\"10.7717/peerj.8529\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScorza C, Piccini C, Mart\u0026iacute;nez Busi M, Abin Carriquiry JA, Zunino P. Alterations in the Gut Microbiota of Rats Chronically Exposed to Volatilized Cocaine and Its Active Adulterants Caffeine and Phenacetin. Neurotox Res. 2019;35(1):111\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12640-018-9936-9\u003c/span\u003e\u003cspan address=\"10.1007/s12640-018-9936-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrandwitz P. Neurotransmitter modulation by the gut microbiota. Brain Res 1693(Pt B). 2018;128\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.brainres.2018.03.015\u003c/span\u003e\u003cspan address=\"10.1016/j.brainres.2018.03.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpiller HA, Ryan ML, Weston RG, Jansen J. Clinical experience with and analytical confirmation of bath salts and legal highs (synthetic cathinones) in the United States. Clin Toxicol (Phila). 2011;49(6):499\u0026ndash;505. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3109/15563650.2011.590812\u003c/span\u003e\u003cspan address=\"10.3109/15563650.2011.590812\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalavrakos M, Leclercq S, De Timary P, Dom G. Microbiome and substances of abuse. Prog Neuropsychopharmacol Biol Psychiatry. 2021;105:110113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.pnpbp.2020.110113\u003c/span\u003e\u003cspan address=\"10.1016/j.pnpbp.2020.110113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomaz AC, Iyer V, Woodward TJ, Hohmann AG. Fecal microbiota transplantation and antibiotic treatment attenuate naloxone-precipitated opioid withdrawal in morphine-dependent mice. Exp Neurol. 2021;343:113787. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.expneurol.2021.113787\u003c/span\u003e\u003cspan address=\"10.1016/j.expneurol.2021.113787\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan de Wetering R, Schenk S. Repeated MDMA administration increases MDMA-produced locomotor activity and facilitates the acquisition of MDMA self-administration: role of dopamine D2 receptor mechanisms. Psychopharmacology. 2017;234(7):1155\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00213-017-4554-4\u003c/span\u003e\u003cspan address=\"10.1007/s00213-017-4554-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVokes T. Quality of life in hypoparathyroidism. Bone. 2019;120:542\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bone.2018.09.017\u003c/span\u003e\u003cspan address=\"10.1016/j.bone.2018.09.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Zhou X, Li X, Guo W, Zhu Q, Zhu B, et al. Fecal Microbiota Transplantation Alters the Outcome of Hepatitis B Virus Infection in Mice. Front Cell Infect Microbiol. 2022;12:844132. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcimb.2022.844132\u003c/span\u003e\u003cspan address=\"10.3389/fcimb.2022.844132\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWojcieszak J, Andrzejczak D, Wojtas A, Gołembiowska K, Zawilska JB. Methcathinone and 3-Fluoromethcathinone Stimulate Spontaneous Horizontal Locomotor Activity in Mice and Elevate Extracellular Dopamine and Serotonin Levels in the Mouse Striatum. Neurotox Res. 2019;35(3):594\u0026ndash;605. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12640-018-9973-4\u003c/span\u003e\u003cspan address=\"10.1007/s12640-018-9973-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Q, Liang X, Wang K, Lin J, Wang X, Wang P, et al. Intestinal hypoxia-inducible factor 2α regulates lactate levels to shape the gut microbiome and alter thermogenesis. Cell Metab. 2021;33(10):1988\u0026ndash;e20037. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmet.2021.07.007\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2021.07.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao F, Zhu W, Yu Y, Huang J, Li J, He Z, et al. Interactions and Stability of Gut Microbiota in Zebrafish Increase with Host Development. Microbiol Spectr. 2022;10(2):e0169621. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/spectrum.01696-21\u003c/span\u003e\u003cspan address=\"10.1128/spectrum.01696-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015;161(2):264\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2015.02.047\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2015.02.047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan PJ, Ren ZX, Shi ZF, Wan CL, Han CJ, Zhu LS, et al. Dysregulation of iron homeostasis and methamphetamine reward behaviors in Clk1-deficient mice. Acta Pharmacol Sin. 2022;43(7):1686\u0026ndash;98. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41401-021-00806-1\u003c/span\u003e\u003cspan address=\"10.1038/s41401-021-00806-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Deng W, Chen C, Kang J, Yang X, Dou Z, et al. Methcathinone Increases Visually-evoked Neuronal Activity and Enhances Sensory Processing Efficiency in Mice. Neurosci Bull. 2023;39(4):602\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12264-022-00965-z\u003c/span\u003e\u003cspan address=\"10.1007/s12264-022-00965-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Q, Xia P, Zhou X, Li X, Guo W, Zhu B, et al. Hepatitis B Virus Infection Alters Gut Microbiota Composition in Mice. Front Cell Infect Microbiol. 2019;9:377. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcimb.2019.00377\u003c/span\u003e\u003cspan address=\"10.3389/fcimb.2019.00377\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng P, Zeng B, Zhou C, Liu M, Fang Z, Xu X, et al. Gut microbiome remodeling induces depressive-like behaviors through a pathway mediated by the host's metabolism. Mol Psychiatry. 2016;21(6):786\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/mp.2016.44\u003c/span\u003e\u003cspan address=\"10.1038/mp.2016.44\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Drug addiction, gut microbiota, behavioral response, metabolic pathways, zebrafish model","lastPublishedDoi":"10.21203/rs.3.rs-6395322/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6395322/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Drug addiction can lead to gut microbiota dysbiosis, which further alters host metabolism and regulates host behavior through the brain-gut axis. Methcathinone (MCAT) is a novel addictive drug, and its effects on the gut microbiota and brain-gut axis are still unclear. This study aimed to determine the alteration of host behavior and gut microbiota community in zebrafish under methcathinone exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e In this study, MCAT was administeredto zebrafish, and their behavioral characteristics were evaluated. Changes in the gut microbiota and metabolic pathways in zebrafish were also analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We found that after the administration of MCAT, the zebrafish was anxious and aggressive, with hyperlocomotion, and the abundance and diversity of the gut microbiota significantly decreased. Additionally, the composition of the gut microbiota shifted. These alterations in the composition of the gut microbiota further led to changes in their metabolic pathways, some of which might be related to the behavioral response induced by MCAT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our findings provide insights into alteration of behavior and gut microbiota composition of zebrafish induced by MCAT which may further contribute to its neurobehavioral responses.\u003c/p\u003e","manuscriptTitle":"Methcathinone exposure alters host behavior and gut microbiota community in zebrafish","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-20 12:10:55","doi":"10.21203/rs.3.rs-6395322/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7acec54a-8172-4a40-af92-ee57eda3ec6b","owner":[],"postedDate":"May 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-24T06:24:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-20 12:10:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6395322","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6395322","identity":"rs-6395322","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}