Influence of fecal microbial transplant (FMT) between male and female rats on methamphetamine-induced hyperthermia

Abstract Objective To investigate the effect of bidirectional fecal microbial transplant (FMT) between male and female rats on methamphetamine (MA)-induced hyperthermia. Methods FMT was performed between male and female rats prior to MA (10 mg/kg, sc) treatment. Core body temperature, plasma drug and norepinephrine (NE) levels were measured and compared between treatment groups. 16S rRNA gene sequencing of bacterial communities between male and female rats was performed. Results MA treatment resulted in significantly higher core body temperatures in male groups (control and FMT-treated) compared to MA-treated female groups (control and FMT-treated). Plasma concentrations of MA and amphetamine were higher in females than males. Whereas, plasma norepinephrine (NE) levels were not different between male and female rats 90 minutes after MA treatment. At the phyla level, the microbiome of male and female control rats were dominated by Firmicutes and Bacteroidetes. Males had a higher relative abundance of Firmicutes and lower relative abundances of Bacteroidetes than females. The FMT procedure changed the recipient group towards their donor with males getting closer to their donors than females. In the control groups following MA treatment, Firmicutes increased and Bacteroides decreased in females and males. Conversely, in the FMT treatment groups following MA treatment, Firmicutes decreased while Bacteroidetes increased in females and males. Conclusions Although definite differences in the structure and diversity of the gut microbiome were observed using 16S rRNA gene sequencing of bacterial communities between male and female rats, these differences do not seem to contribute to the sex-based differences in MA-induced hyperthermia.


Introduction
The recreational use and distribution of the phenethylamine, psychostimulant methamphetamine (MA) continues to be an extremely serious problem in the United States and globally [1][2][3][4].According to the 37th Annual Report of the American Association of Poison Control Centers' (AAPCC) National Poison Data System (NPDS) published in 2020, the majority of the chemical poisoning death cases from the previous year was related to MA exposure and consumption, a trend that was also reported previously by the National Institute of Drug Abuse [5,6].Additionally, MA was among the most frequently identified drugs in forensic cases by the National Forensic Laboratory Information System (NFLIS) for the NFLIS-Drug 2020 Midyear Report [1].The use of MA is linked to several serious health issues including: psychosis, convulsions, the possible transmission of human immunodeficiency virus (HIV), myocardial infarction and cardiomyopathy and hyperthermia that results in rhabdomyolysis, renal failure, liver failure and death [4,[7][8][9].
The hyperthermia mediated by MA is suggested to occur predominantly through the activation of hypothalamic-pituitary-adrenal (HPA) axis and subsequent neurotransmitter release [9,10].Peripherally, the activation of sympathetic nervous system (SNS) results in norepinephrine (NE) release that triggers vasoconstriction through the activation of a 1 -adrenergic receptors resulting in impaired heat dissipation.Additionally, NE activation of ꞵ 3 -adrenergic receptors in brown adipose tissue (BAT) and skeletal muscle (SKM) results in heat generation through the regulation of uncoupling proteins (UCP1 and UCP3) [9,11,12].Furthermore, gut microbiome modulation has been associated with phenylethylamine-induced hyperthermia (PIH) [13].Ridge et al. demonstrated that antibiotic (ABX) treatment via drinking water for 14 days prior to 3,4-methylenedioxymethamphetamine (MDMA) treatment diminished the gut microbiome of the recipient rats and attenuated MDMA-induced hyperthermia compared to control groups [13].Angoa-P erez et al. investigated the effect of multiple synthetic cathinones and amphetamine derivatives on gut microbiome in a mouse model [14].
Specifically, mephedrone, methcathinone, MA and 4-methylmethamphetamine led to time-dependent changes in the composition and diversity of the gut microbiome with the most significant changes observed in the two phyla: Firmicutes with methcathinone, 4-methyl-methamphetamine and Bacteroidetes with all sympathomimetic agents explored [14].Subsequently, Goldsmith et al. performed a bidirectional fecal microbial transplant (FMT) between methylone-induced hyperthermic tolerant (MHT) and methylone naïve (MN) rats to assess the possible transferal of the hyperthermic phenotypic response between treatment groups [15].MHT rats were treated with methylone once a week for four weeks until tolerance to methyloneinduced hyperthermia was attained while the MN group was treated with saline under the same conditions.Once MHT rats became tolerant to methylone-induced hyperthermia, a bidirectional FMT was carried out between both groups for seven days.The single dose treatment with methylone (10mg/kg sc) after the FMT period resulted in restoration of hyperthermic response in MHT rats while MN rats did not display a significant change in body temperature upon methylone treatment.The relative abundances of Gammaproteobacteria, Alphaproteobacteria, and Erysipelotrichia were altered in pre-versus post-FMT methylone tolerance phenotypes of recipients [15].
Sex-based differences in PIH have been documented in previous literature [16][17][18].Wyeth et al. demonstrated that MDMA treatment resulted in a lower hyperthermic response in female rats compared to male rats [16].Upon examination of the thermogenic mediators of PIH, female rats displayed lower plasma NE levels 30 min after MDMA challenge and lower skeletal muscle UCP3 protein levels compared to male rats treated under the same conditions [16].The differences in females response was partially explained by the reduced activation of the SNS after MDMA treatment, the lower sensitivity to a 1 -adrenergic receptor activation in females vasculature, higher sensitivity to nitric oxide (NO) in MDMA-treated female rats and lower UCP3 protein expression levels in females compared to males [16].Consistent to these findings, Nelson et al. found that a-pyrrolidinovalerophenone (a-PVP) resulted in dose and time-dependent differences in temperatures between male and female rats [17].We have further found that female rats are less responsive to the hyperthermic effects of methylone than male rats [18].
The results from these studies indicate that the hyperthermic response and the molecular mediators of sympathomimetic-induced thermogenesis differ greatly between male and female rats.Given the possible link of gut microbes in PIH [12,13,15] and studies demonstrating the existence of gender specific gut bacteria [19,20], we sought to determine if there is a gender specific role of the gut microbiome in MA-mediated hyperthermia utilizing an FMT between male and female rats.

Animals and study design
Adult, male (n ¼ 12, average weight ¼ 311.25 ± 5.2 g) and female (n ¼ 12, average weight ¼ 212.75 ± 4.5 g) Sprague-Dawley (Rattus norvegicus domesticus) were obtained from Envigo (Indianapolis, IN).The Bowling Green State University Animal Care and Use Committee approved all protocols and experimental procedures and animal maintenance and research were conducted in accordance with the eighth edition of the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health.Animals were housed one per cage (cage size: 21.0 Â 41.9 Â 20.3 cm) with sterilized bedding, maintained on a 12:12 h light/dark schedule and had ad libitum access to food and water.Animals were maintained at an ambient temperature of 27-28 C to maximize thermogenic response [21].An acclimation period of seven days was provided for all animals prior to the start of experimental procedures.The study design for the experiment is outlined in Figure 1.Male and female rats were randomly assigned into four treatment groups (n ¼ 6).One male group and one female group served as control groups, whereas the rest served as FMT groups.

Drugs and chemicals
Methamphetamine (MA) was obtained from Cayman Chemicals (Ann Arbor, MI, USA) as a hydrochloride salt.On the day of the study, MA solution was prepared fresh at a concentration of 10 mg/ml in 0.9% normal saline.All other chemicals and reagents were obtained from Sigma Chemical (St.Louis, MO, USA).

Feces collection for FMT and antibiotic (ABX) treatment
Prior to ABX treatment, fecal pellets from each animals were pooled for each of the two FMT groups on Day 1 and stored at 4-8 C until fecal slurries were made for the bidirectional FMT procedure.Approximately two grams of the fecal pool were stored at À80 C for bacterial community analysis.The FMT groups were treated with ABX from Day 2 onwards for five consecutive days via oral gavage [22,23].The antibiotic cocktail consisted of ampicillin, neomycin and metronidazole (100 mg/kg/day) and vancomycin (50 mg/kg/day).The ABX treatment period was followed by 48 h wash-out period prior FMT treatment.

Fecal slurry preparation and FMT treatment
On FMT treatment day, the fecal slurries were prepared according to the methods previously described [22,23].
Briefly, approximately 9 g of donor stool was mixed with 50 ml of normal saline and blended with a commercial blender for 2 to 4 min until a homogenous texture was attained.Next, the homogenous mixture was filtered twice using 4 Â 4 gauze and the collected solution was centrifuged at 6000 Â g for 15 min.After centrifugation, supernatant was discarded, and the remaining material was resuspended in 20 ml phosphate buffered saline (PBS) solution.The bidirectional FMT treatment between male and female FMT groups started immediately after the fecal slurries were prepared by administering approximately 1 ml/kg of fecal slurry via oral gavage in two rounds separated by 3 days.The second FMT round was performed to ensure complete transfer of the gut microbiome between FMT-treated groups.All FMT procedures were carried out at the same time of the day.

MA treatment and temperature measurement
The day following the second round of FMT treatment, all rats were weighed, and the core body temperature (T c ) was recorded prior to treatment and noted as baseline temperature.Rectal temperatures were measured in all animals using a Physiotemp Thermalert TH-8 thermocouple (Physitemp Instruments, Clifton, NJ) attached to a RET-2 (rat) rectal probe coated with white petrolatum prior to insertion.RET-2 probes were inserted 5 cm into the rectum, where they remained for at least fifteen seconds, until a stable temperature was obtained.All four groups of animals were treated with a single subcutaneous (sc) dose of MA (10 mg/kg) and T c was recorded for 90 min at 30 min intervals.After 90-min timepoint, rats were euthanized with CO 2 and blood samples collection was performed via cardiac puncture.The blood samples were collected in pre-heparinized vials and processed immediately to extract plasma.Briefly, blood samples were centrifuged at 6000 Â g for 5 min at 4 C then plasma layer was extracted and stored at À20 C until further analysis.

High performance liquid chromatographyelectrochemical detection (HPLC-EC)
To evaluate NE levels in plasma samples, 200 mL collected from each rat were purified and extracted for HPLC-EC (Shimadzu, Canby, OR) analysis using procedures previously described by Holmes et al. [24].The HPLC-EC method was as follows: the mobile phase was comprised of 14% methanol, and an 86% mixture of 0.05 M phosphate, 0.03 M citric acid buffer, 0.6 mM octasulfonic acid, and 1.0 mM EDTA-disodium.The pump flow rate was 0.55 ml/min and was set to an operating temperature of 30 C. NE was separated using a PP-ODS II reverse phase C18-column (Shimadzu, Colombia, MD) and identified according to the retention time of the standard, and concentrations were quantified by comparison with peak heights of the standard calibration curve (10 4 -10 8 pg/ lL).The quantification of NE sample levels was performed using an Epsilon electrochemical (EC) detector connected to the HPLC.The detector sensitivity was 5 uA and the oxidation potential was fixed at þ700 mV using a glassy carbon working electrode versus an Ag/AgCl reference electrode.
Lab Solution software was used to integrate and analyze the raw data for determination of NE levels.

Amphetamine and methamphetamine (MA) levels assessment
To assess amphetamine and MA levels, plasma samples were prepared as follows: 200 mL of plasma sample were mixed with 600 mL crash solution (1% formic acid in acetonitrile solvent) in microcentrifuge tubes and vortexed for 5 min.The supernatant was drawn off and added to HybridSPE-Phospholipid filters (Sigma-Aldrich, St. Louis, MO, USA) and was extracted by vacuum.Using direct flow of nitrogen gas, most of the acetonitrile was evaporated until approximately 100 mL of the solvent was left.The remaining extractant was spiked with 30 mL of internal standard mixture (ISTD) and vortexed briefly for 30 s.The final extractant sample was injected into the LC-MS at an injection volume of 3 mL.The triple-quadrupole LCMS-8050 CL from Shimadzu USA.Manufacturing (Canby, OR, USA) was used for sample analysis with a gradient separation method utilizing water þ0.1 vol% acetic acid and 100% methanol for the mobile phases.The flow rate was 0.75 ml/min at 70 C over the 4.65 min gradient between 1 and 100% MeOH with a total method run time of 7 min.The stationary phase consisted of a Raptor 50 Â 2.1 mm, 2.7 mm, biphenyl column (Restek, Bellefonte, PA, USA) and a Raptor guard column to act as a filter to protect the biphenyl column.

Statistics of physiological data
GraphPad InStat v.6.0 software was used to complete all statistical analyses of rat organismal data.The results are presented as the mean ± SEM.Within the time course of a treatment group, statistical significance was determined using repeated measures ANOVA with a Student-Newman-Keuls post hoc test between groups at each time point.To calculate maximum change in temperature (maximum D C), the maximum elevation in core body temperature was compared to the animal's baseline temperature.A two-tailed t-test was performed when only two groups were compared.Significance was established at p < 0.05 a priori.

Microbial community analysis
To characterize the differences in gut microbiome before treatments and changes in response to treatments, 16 pooled fecal samples for microbial community analysis were collected at 4 timepoints from all 4 groups of rats as depicted in Figure 1.Feces collected for FMT groups for both sexes on Day 1 served as baseline samples (before any FMT or drug modulation), while fecal samples collected on Day 9 represented gut microbiomes at the end of ABX treatment period (immediately before FMT treatment).On Day 18, feces for the same two groups were collected both pre and post MA treatment.Feces collected immediately before the drug challenge represent microbial community present after FMT treatment period, while fecal samples were collected 90 min post MA treatment enable the determination of effects of MA treatment on the gut microbiome.Feces from control groups were collected on Day 1, Day 9 and Day 18 respectively without any interventions (i.e., baseline, ABX and FMT procedures) except for the second sample on Day 18 post-MA challenge.Cages were changed 24 h before each collection to avoid contamination from previous samples.All collected samples were stored at À80 C until DNA extraction.
For DNA extraction, $150 mg from pooled and ground fecal samples from each group were used.DNA was recovered from the pooled samples using a DNeasy Powersoil Kit (Qiagen Inc., CA, USA) according to manufacturer's instructions.The quality of DNA was assessed using 0.8% agarose gel electrophoresis and DNA concentration was measured using NanoDrop Spectrophotometer (Thermo, MI, USA).Library preparation and sequencing of 16S rRNA gene libraries were performed by Laragen Sequencing and Genotyping (Culver City, CA, USA) using primers BAC357TS-for (CCTACG GGNGGCWGCAG) and BAC806TS-rev (GACTACHVGGGTATC TAATCC).The resulting $450-bp amplicons spanned the V3 and V4 regions.Amplicon libraries were sequenced as 250bp paired-end reads on an Illumina MiSeq platform.Raw sequence reads were trimmed to remove primers and lowquality ends, then error-corrected and de-replicated using DADA2 1.18.0 [25] in R 4.0.5.This generated 2,574 amplicon sequence variants (ASVs) across the entire dataset.Taxonomies were assigned using Ribosomal Database Project (RDP) Classifier 16 [26].Prior to determination of alpha and beta diversities, all libraries were rarefied to 20,000 reads, with 2,334 total ASVs remaining after rarefaction.Principal coordinates analysis (PcoA) on Bray-Curtis (BC) dissimilarities was performed using the Vegan package 2.5-7 [27] in R.

Effect of MA treatment on body temperature in male and female rats
The treatment with MA (10 mg/kg sc) resulted in significantly higher T c in both male groups (control and FMT-treated) compared to MA-treated female groups (control and FMTtreated) at 60-and 90-min time points and the male treatment groups at 30 min (Figure 2  similar pattern as male groups showed higher maximum change temperature than female groups (Figure 2(B) -One way ANOVA with Student-Newman-Keuls post hoc between treatment groups: F (3,15) ¼ 7.99, p < 0.05).

Norepinephrine (NE) concentration in plasma
The assessment of plasma NE levels revealed no significant difference between male and female rats after 90 min of MA treatment (Figure 3).

Amphetamine and MA levels assessment
The analysis of MA and amphetamine (the main metabolite of MA) levels in plasma demonstrated a significantly higher levels of both MA and amphetamine in female ABX-FMT-treated rats compared to male ABX-FMT-treated rats (Figure 4 two-tailed t-test: p < 0.005).

Microbiome differences between male and female control rats
The 16S rRNA gene analysis of pooled fecal samples of control rats over different time points revealed that the gut microbiomes of male and female rats were distinct from one another.The b-diversity plot of control rat microbiomes at ASV level using PcoA based on BC Dissimilarity showed distinctly separated clusters of male (blue) and female (red) samples (Figure 5(a)).On Day 1 of the experimental study (following 7 days of acclimation), male and female samples were distinct with BC dissimilarity of 0.52.With the progress of study although changes in microbiome compositions in both male and female rats were observed, the dissimilarity between male and female microbiomes was consistent (BC dissimilarity of 0.53 and 0.51 respectively for Day 9 and 18 pre-MA treatment).Sex-specific differences in the makeup of rat microbiomes were also evident during analyses of a-diversity and taxa at various levels.Throughout the study, female control microbiomes had consistently higher a-diversity than male controls in time-matched samples with D ASVs of 16-52 and D Shannon's Diversity Index (SDI) of 0.05-0.16(Figure 6).Analyses of microbiomes at phyla levels (Figure 7(a)) showed that male and female control rats were both dominated by Firmicutes and Bacteroidetes.However, males had higher relative abundance of Firmicutes (92.5 ± 1.5 vs. 88.7 ± 0.7%) and lower relative abundances of Bacteroidetes (6 ± 1 vs. 9.2 ± 0.9%) than females.Both sexes had high contributions of families Ruminococcaceae, Lachnospiraceae, Lactobacillaceae, and Clostridiaceae_1 (Firmicutes); and Porphyromonadaceae, Bacteroidaceae, Rikenellaceae (Bacteroidetes) (Figures 7(a) and 8(a)).Of these, Ruminococcaceae was similarly and most abundant between the two sexes (38.3 ± 2.3%), while Lachnospiraceae was more abundant in male samples (49.1 ± 4.1%) compared to female counterparts (40.8 ± 3.7%).

Microbiome diversity decreases in response to ABX
Following antibiotic (ABX) treatment on the FMT groups (Day 9), considerable changes in the community richness and composition occurred.SDI substantially decreased from 5.37 for female group before (Day 1) to 1.68 after ABX treatment (Day 9; Figure 6(b)).Accordingly, observed ASVs decreased from 528 to 26 (Figure 6(b)).For the matched male groups, the SDI also dropped from 5.38 (Day 1) to 2.09 (Day 9) and ASVs from 552 to 47 (Figure 6).Concurrently, Bacteroidetes decreased to below detection in both sexes after ABX treatment and Firmicutes dramatically decreased to 50.8% in female rats and 73.2% in male rats from pprox.90% (Figure 7(b)).Ruminococcaceae, Lachnospiraceae, Porphyromonadaceae, Bacteroidaceae and Rikenellaceae, which make 76-95% of total bacterial families (belonging to dominant phyla Firmicutes and Bacteroidetes), were reduced by ABX treatment in both groups, while Lactobacillaceae (also Firmicutes) increased tremendously to 50.6% (from 4.8%) and 71.2% (from 5.8%) respectively in female and male groups (Figure 8(b)).In contrast, Enterobacteriaceae (Proteobacteria) became highly enriched after ABX treatment from 0.01 to 49.1% in female and from below detection level to 26.7% in male samples (Figure 8(b)).This dramatic change in microbiome composition following ABX is also evident in the PcoA plot (Figure 5(b)), which showed that the microbiomes of ABX-treated rats for both sexes (Day 9; Cluster II) were highly dissimilar from their pre-FMT baseline (Day 1; Cluster I).Post-ABX male and female samples both have BC dissimilarities of 0.99, to their corresponding pre-ABX samples (Male and Female Day 1), respectively.

Microbiome composition is altered by FMT procedure
After FMT, recipient communities (Day 18 pre-MA administration) acquired similarities to donor communities but retained distinct differences at the ASV level.The a-diversity (ASV richness; Figure 6) of the FMT recipients for both sexes increased to 428 (Female) and 471 (Male), yet post-FMT community richness remained lower than those of their respective donors (Male donors 552 and Female donors 528).BC dissimilarity between the donor female group (Day 1) and recipient male group (Day 18 pre-MA) was 0.64, decreasing from 0.99 of the male recipient post-ABX/pre-FMT (Day 9), an increase of 35.4% similarity.Similarly, the BC dissimilarity between male donor (Day1) and female recipient (Day 18 pre-MA) was 0.71, compared to 0.99 between male donor (Day1) and female recipient post-ABX/pre-FMT (Day 9) (increase of 28.3%).Together, this indicates that the FMT procedure shifted the recipient group toward their donor with males getting closer to their donors than females The taxonomic profile following FMT treatment (Figures 7(b) and 8(b)) showed considerable recovery from ABX disturbance.In female rats (Day 18 pre-MA), Firmicutes rebounded to 82.3% though still reduced compared to baseline female (Day 1; 90%) and donor males (Day1 89.7%).In male rats (Day 18 pre-MA), Firmicutes rose to 90% after FMT, approximately the same relative abundances as its donor females (Day1;90%) and baseline males (Day 1; 89.7%).As prior to ABX, Ruminococcaceae, Lachnospiraceae, Lactobacillaceae, Rikenellaceae, and Clostridiaceae_1 were primary Firmicutes families in both the groups.Here, Lactobacillaceae drastically decreased in both male and female recipients compared to their post-ABX treatment abundance on Day 9 (Females: 50.6% and Males: 71.2%) reaching almost back to their baseline values (Females 7%, Day 1 ¼ 4.8% and Males 5.8%, Day 1 ¼ 5.8%).Interestingly, Ruminococcaceae decreased (Females Day1; 45.6% to 24% and Males Day 1; 38.2-25.9%)and Lachnospiraceae increased (Females Day 1;36.1% to 48.6% and Males Day 1; 42.61-54.1%)in both groups of recipients.In females, Bacteroidetes (consisting mainly Porphyromonadaceae, Bacteroidaceae and Rikenellaceae families) reemerged to 16.8%, which was higher than donor males (Day 1; 8.16%) and also their baselines of 9%.Proteobacteria was reduced back to 0.35% after the FMT (baseline of 0.85%, Day 1).In males, Bacteroidetes was recovered to 5.8% (8.2% in baseline, Day 1) and Proteobacteria declined to 0.37% (0.83% in baseline, Day 1).

Microbiome changes in response to MA treatment
MA dosage induced an effect on microbiomes (Female vs Male, control & FMT) seen within 90 min from pre-MA to post-MA treatment on Day 18.The MA effects on alterations in the control microbiomes was observed as decrease in  a-diversity (Figure 6) with D ASVs and SDI values of 60 and 0.3 in females, whereas 88 and 0.42 for males.The PcoA plot Figure 5(a) of microbiome also showed change, where males (pre-MA vs. post-MA) had BC dissimilarity value of 0.41, while in females this value (pre-MA vs. post-MA) was 0.36.At phyla level, Firmicutes increased from 89.3% to 93.5% and Bacteroides decreased from 8.2 to 3% in females.Also, in males Firmicutes increased slightly from 92.1 to 94.4%, whereas the Bacteroides decreased to from 7 to 4% (Figure 7(a)).At the family level (Figure 8), relative abundances of Firmicutes families Lactobacilaceae increased (Females: 3.7-14.2%and Males: 4.7-20.4%)and Lachnospiraceae decreased in both male and female groups (Females: 44.3-42% and Males: 51.1-33.6%),whereas Ruminococcus decreased in females (38% to 33.5%) and increased in males (34.9-38.2%).Bacteroidetes families consisting mainly Rikenellaceae, Porphyromonadaceae and Bacteroidaceae were reduced from a combined 8.2-3% in females and 7 to $4% in males.
In FMT treatment groups as in control groups, a-diversity measures D ASVs and SDI values declined in both groups on Day 18 pre-MA and post-MA.In females, D ASVs and SDI values were 61 and 0.32 whereas these values were 70 and 0.07 in males.The PcoA plot (Figure 5(b)) shows the difference of FMT groups pre-and post-MA treatment (Clusters III for Females and IV for Males) on Day 18. BC dissimilarity of female FMT group after MA (pre-MA vs. post-MA) was 0.47 while in female it was 0.29.At the phyla level, in contrast to both control male and female groups Firmicutes decreased (Females: 82.3-68.4% and Males: 90-85%) while  Bacteroidetes increased (Females: 16.8-29.9%and Males: 5.8-6.8%) in both groups.At the family level, Lactobacillaceae (Firmicutes) markedly increased similarly to control groups in both female and male groups (Females: 7-11.6% and Males: 5.8-8.8%).The two most abundant families (Ruminococcaceae, Lachnospiraceae) belonging to Firmicutes in both groups however decreased from combined 74.7 to 46.2% in females and 80 to 71.4% in males.Bacteroidetes (Porphyromonadaceae, Rikenellaceae, Bacteroidaceae), in contrast to control groups, were enriched from 16.3 to 31.7% in females and 6 to 7.3% in males.

Discussion
The current study examined the effects of a bidirectional FMT between male and female rats on MA-induced hyperthermia and gut microbiome diversity and composition.The core body temperature measurements revealed significant difference in the hyperthermic response at 60-and 90-min timepoints between male and female rats after MA treatment with the male rats showing significantly higher core body temperature at those time points in addition to higher maximum change in body temperature.Fonsart et al. demonstrated that the hyperthermic response to MDMA treatment (20 and 40 mg/kg, sc) was higher in males than the female Sprague-Dawley rats during the 5 h monitoring period following drug treatment [28].Furthermore, Wyeth et al. demonstrated that MDMA (20 mg/kg, sc) resulted in higher hyperthermic response in male Sprague-Dawley rats compared to female rats under the same conditions [16].In contrast, Colado et al. showed that the intraperitoneal (ip) administration of MDMA (10 mg/kg) resulted in more pronounced hyperthermic response in female Dark Agouti rats compared to male rats [29].The Dark Agouti rat model was used as it resembles poor metabolism phenotype in humans with the females displaying even lower metabolism activity than male rats whereas other rat strains are analogous to human extensive metabolizer phenotype [29].Fukumura et al. concluded that there were no significant differences in maximum D C between male and female Sprague-Dawley rats upon MA repeated (every 2 h for 6 h) treatment (5 and 10 mg/kg, sc) [30].The variability in PIH between male and female models presented in these latter papers may be attributed to the animal model used, drug dosage, route of administration and housing conditions including the ambient temperature of the treatment room.
In PIH, NE prevents heat dissipation through a 1 -adrenergic receptors mediated vasoconstriction and generates heat through b 3 -adrenergic receptors in BAT and SKM ultimately activating UCP1 and UCP3 [9,11,12].In the present study also examines the differences in NE concentrations in the plasma 90 min after MA treatment in male and female rats.We saw no significant differences in NE levels between male and female rats at this timepoint whereas NE levels were significantly higher in males as compared to females 30 min after MDMA [16].The different timepoints used for NE measurement more than likely contributed to these dissimilarities.
In rodents, MA is N-demethylated by CYP2D6 to amphetamine [31].In the present study, plasma concentrations of MA and amphetamine were higher in female than male rats.The sex differences in the pharmacokinetic profiles of MA and amphetamine between male and female Sprague-Dawley rats have been noted in the literature [32,33].Milesi-Hall e et al. found that females have a lower total and renal clearance of MA than males [32].Rambousek et al. found that plasma and brain levels of MA and amphetamine were significantly higher in females compared to male rats following treatment with MA (1 and 5 mg/kg, sc) [33].Given these pharmacokinetic differences, females may be expected to have an augmented hyperthermic response to MA.However, studies with MDMA showed that females have lower NE and UCP3 levels and less vasoconstriction following sympathomimetic treatment [16].These differences in the molecular mediators of PIH have been suggested to contribute to the attenuated hyperthermic response seen in female rats [16].
Using16S rRNA gene analysis, we saw differences in the relative abundance of microbiomes that were commonly shared between male and female control matched groups over time at various taxa levels (phylum and family).BC based PcoA plot also showed that male control groups from different time points clustered closer to each other compared to clusters of their matched female groups.Sex specific difference in gut microbes has been suggested to occur to adjust various sex-specific host systems and processes through different stages of life [34].Except for the feces after MA treatment, female controls at different timepoints had higher alpha diversity compared to matched male control groups.Higher alpha diversity in females compared to males have been reported in post-pubescent mice [35] and human [36,37].
Given the differences between female and male rat microbiomes and the hypothesized link between microbiome composition and hyperthermic response, a bidirectional FMT procedure was undertaken after ABX treatment.Microbial communities were then characterized at critical time points in order to track associated changes and contribution of gut microbiome to the differences observed in MA-induced hyperthermia between male and female rats.Microbiomes of rats in the treatment (FMT) group had high similarity (0.37 BC dissimilarity) with sex-matched controls on Day 1, therefore these samples provided a suitable baseline for subsequent microbiome differences.The use of ABX treatment to attenuate the gut microbiome richness and diversity prior to FMT has been established in previous literature [38][39][40].Consistent with the results reported by Manichanh et al. the ABX treatment in this study resulted in significant decrease in the relative abundance of Firmicutes and almost complete abolishment of Bacteroidetes phyla while Proteobacteria levels elevated significantly [41].This was also supported by the severe drop in a-diversity (shown by SDI and ASV Richness) after the ABX treatment, similar to other studies [38].The increase in Proteobacteria upon ABX treatment is proposed to be as a result of major phyla depletion, thus creating more favorable environment for Proteobacteria to populate.Thereafter, Proteobacteria have been usually found to be gradually replaced by other phyla/taxa after FMT procedures [42].Proteobacteria remained the least abundant phyla (<1%) across all timepoints and groups.
Based on BC dissimilarity values, the recipients of FMT for both genders did not shift highly toward their donors from opposite genders.The male BC dissimilarity value of 0.99 for male recipient after ABX treatment on Day 9 (just before start of FMT procedure) with its female donor (FMT group Day 1) decreased to 0.64 after FMT (Day18 pre-MA), whereas the similar BC value of 0.99 for female recipient (Day 9) from its male donor (FMT group Day 1) dropped to 0.71 after the FMT process (Day18 post-MA).The a-diversity values for male recipients were also higher than female recipients after FMT process.This suggests that the microbes from the female donors were established more successfully in the male gastrointestinal tract.At the phyla level, we noticed that Firmicutes were established in male recipient relatively more abundantly than in female recipients, while this was opposite for the second abundant phylum Bacteroides, where females were enriched with more Bacteroidetes compared to males.This higher Firmicutes and lower Bacteroidetes pattern was also observed in male controls and FMT baseline, while the higher Bacteroidetes were seen in both female controls and FMT baselines.
At family level, the most abundantly established bacterial families in the FMT recipients from both genders were Lachnospiraceae and Ruminococcaceae (both Firmicutes), where Lachnospiraceae was more abundantly repleted in males compared to females, while the later was similarly abundant.Studies applying FMT procedures following preantibiotic treatment such as for correcting Clostridium difficile infection have reported more abundant establishment of these families (Ruminococcaceae and Lachnospiraceae) in responders compared to non-responders where Enterobacteriaceae are more abundantly established [43].Although these studies may have different intent from ours, the procedures involving pre-ABX treatment followed by FMT from a healthy/donor of interest are basically the same.
The changes in the dominant phyla Firmicutes and Bacteroidetes and the minor phyla Proteobacteria and Verrucomicrobia upon sympathomimetic treatments and/or FMT treatment were also reported by our lab and others [13][14][15].Angoa-P erez et al. reported that the post-treatment with different sympathomimetic agents resulted in distinct effects across different timepoints on the phyla levels of Firmicutes, Bacteroidetes and Verrucomicrobia compared to controls [14].The taxonomic analysis after bidirectional FMT from methylone-naïve to methylone hyperthermic tolerant rats (and vice versa) reported by Goldsmith et al. showed similar results as the relative abundance of Proteobacteria and Firmicutes varied between both groups pre-and post-FMT treatment [15].Additionally, Yang et al. confirmed that chronic MA abuse in human subjects led to significant changes in these phyla compared to control subjects and that these changes were linked to behavioral and psychopathology changes in MA users [44].In agreement with these data, the current study presents supporting evidence that MA challenge resulted in alteration of Firmicutes, and Bacteroidetes compared to pretreatment samples.
Interestingly, while some studies employing animal models concluded the significance of sex-based differences in gut microbiome and phenotypic behaviors, others did not find a significant correlation between gender and the gut microbiome [45][46][47].In the study performed by Markle et al. the gavage transfer of adult male mice gut microbiome to immature female mice resulted in significant hormonal and metabolic changes in the recipients suggesting that gut microbiome composition influences sex hormone levels and autoimmune diseases [47].On the other hand, despite the clear sex-based differences in gut microbiome richness and composition, other studies have demonstrated that higher differences were reported within the species and strain of the animals under investigation compared to the sex of the overall animals in general [20,46].These findings were attributed to multiple factors including genetics and environmental factors, gender specific hormones and diet.
Increasing similarity of the bacterial community toward its donor after FMT may not necessarily establish the expected phenotype/clinical outcomes [48], while desired phenotypic changes in recipient have also been achieved without the recipient of FMT highly shifting toward donor [43].While some studies related to FMT in Clostridium difficile infections have also suggested that transplant of key bacteria associated with the phenotype/clinical outcome of interest may be sufficient for a successful FMT than complete shift of microbiota toward the donor [49,50].Further, the establishment of microbial diversity and their functional potential could be more important in influencing host phenotype and gut [51].Besides, there can be various other types of microbes and/or microbial components being transferred during the FMT process [52,53] whose potential effect on the phenotype may be another dimension not included in our results.While this data overall suggests that the FMT caused a disturbance to the microbiome composition, we did not see complete shift in microbiome of transplanted groups toward their respective donors.Subsequently, the hyperthermia phenotypic response was not transferred between sexes.Additionally, one limitation of the current study is that in order to have sufficient sample amounts for all the necessary procedures (e.g., FMT, sequencing), fecal samples from the treatment groups were pooled.

Conclusion
Here, we employed bidirectional FMT and 16S rRNA gene sequencing analysis techniques to investigate the influence of sex-based differences in gut microbiome on MA-triggered hyperthermia.Although distinct differences in gut microbiome richness and composition and significant differences in hyperthermic response between male and female rats were observed, the observed microbiome differences at least in this experiment do not appear to be contributing to the sex-based differences in MA-mediated changes in temperature.

Figure 1 .
Figure1.Animal study design for the fecal microbial transplant (FMT) based hyperthermia study experiment carried out for 18 days where male and female rats were randomly divided into 4 groups (female and male controls and FMT groups).The male and female FMT groups participated first in ABX (Day 9) treatment followed by FMT treatment (Day 18), while the remaining two groups served as controls without any treatments until final day (Day 18) where all 4 groups were treated with MA (10 mg/kg, sc).ABX was carried out for 5 days (starting Day 2-6, followed by washout period of 3 days) and bidirectional FMT process (between male and female FMT groups) in two rounds of 3 days (Day 9-11 and Day 15-17 with gap of 3 days) via oral gavage.Feces were collected and pooled for all 4 groups at 4 time points (maroon dots) on Day 1(baseline), Day 9 (after ABX treatment) and Day 18 (pre-and post-MA treatment).Figure made with BioRender.
(A) -Repeated measures ANOVA with Student-Newman-Keuls post hoc test between groups at each time point: F (11,71) ¼ 10.75, p < 0.05).The maximum change in temperature calculations showed a

Figure 2 .
Figure 2. Core body temperature (T c ) change upon MA (10 mg/kg sc) treatment in male and female groups.(A) The difference from baseline calculations for both male and female groups.(B) maximum temperature change following MA administration.Each value represents the mean ± SEM, n ¼ 6. Ã indicates significant differences between MA-treated female groups (control and FMTtreated) at 30, 60-and 90-min time points and the male treatment groups (control and FMT-treated) at the 30 min time point (p < 0.05).

Figure 3 .
Figure 3. NE levels measurements in plasma samples at 90-min time point.All treatment groups (control, ABX-FMT-treated) showed comparable levels of NE 90 min after MA (10 mg/kg, sc) administration.Each value represents the mean ± SEM, n ¼ 6. Ã indicates significant differences between treatment groups (p < 0.05).

Figure 4 .
Figure 4. MA and amphetamine levels assessment in plasma in ABX-FMTtreated groups (Two-tailed t-test within same drug-assessed groups).Ã indicates significant differences between treatment groups.Each value represents the mean ± SEM, n ¼ 6. Ã indicates significant differences between treatment groups (p < 0.05).

Figure 5 .
Figure 5. Bray Curtis dissimilarity based Principal Coordinates Analysis (PCoA) showing b-diversity for a) Control male and female groups and b) FMT groups at 4 different time points: Day 1(D1, baseline), Day 9 (D9, after ABX treatment) and on Day18 (D18, pre-and post MA treatment).In figure b, Cluster I and II represent male and female samples from Day 1 and 9, Cluster III and IV for female and male groups on Day 18 (pre-and post-MA treatments).

Figure 6 .
Figure 6.The a-diversity plots showing a) ASV richness and b) Shannon Diversity Index plotted for control vs FMT groups for both male and female groups at 4 different timepoints.

(
Figure 5(b), Cluster III and IV).However, this change did not considerably revert the microbiome toward their donors (Figure 5(b), Cluster I).

Figure 7 .
Figure 7.Figure showing relative abundance (%) based stacked bar plot of the bacterial phyla (>0.1%) in (a) control and (b) FMT groups for both male and female groups at 4 different timepoints.
Figure 7.Figure showing relative abundance (%) based stacked bar plot of the bacterial phyla (>0.1%) in (a) control and (b) FMT groups for both male and female groups at 4 different timepoints.

Figure 8 .
Figure 8.Figure showing relative abundance (%) based stacked bar plot of the bacterial families (>0.1%) in (a) control and (b) FMT groups for both male and female groups at 4 different timepoints.
Figure 8.Figure showing relative abundance (%) based stacked bar plot of the bacterial families (>0.1%) in (a) control and (b) FMT groups for both male and female groups at 4 different timepoints.