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Obese-type Gut Microbiota Induce Neurobehavioral Changes in the Absence of Obesity

      Abstract

      Background

      The prevalence of mental illness, particularly depression and dementia, is increased by obesity. Here, we test the hypothesis that obesity-associated changes in gut microbiota are intrinsically able to impair neurocognitive behavior in mice.

      Methods

      Conventionally housed, nonobese, adult male C57BL/6 mice maintained on a normal chow diet were subjected to a microbiome depletion/transplantation paradigm using microbiota isolated from donors on either a high-fat diet (HFD) or control diet. Following re-colonization, mice were subjected to comprehensive behavioral and biochemical analyses.

      Results

      The mice given HFD microbiota had significant and selective disruptions in exploratory, cognitive, and stereotypical behavior compared with mice with control diet microbiota in the absence of significant differences in body weight. Sequencing-based phylogenetic analysis confirmed the presence of distinct core microbiota between groups, with alterations in α- and β-diversity, modulation in taxonomic distribution, and statistically significant alterations to metabolically active taxa. HFD microbiota also disrupted markers of intestinal barrier function, increased circulating endotoxin, and increased lymphocyte expression of ionized calcium-binding adapter molecule 1, toll-like receptor 2, and toll-like receptor 4. Finally, evaluation of brain homogenates revealed that HFD-shaped microbiota increased neuroinflammation and disrupted cerebrovascular homeostasis.

      Conclusions

      Collectively, these data reinforce the link between gut dysbiosis and neurologic dysfunction and suggest that dietary and/or pharmacologic manipulation of gut microbiota could attenuate the neurologic complications of obesity.

      Keywords

      The etiology of most neuropsychiatric disorders is likely multifactorial and based on genetic and environmental risk factors (
      • Shih R.A.
      • Belmonte P.L.
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      A review of the evidence from family, twin and adoption studies for a genetic contribution to adult psychiatric disorders.
      ). One potentially important environmental driver of mental illness is obesity, which dramatically increases risk of depression, dementia, and stroke, and is associated with increased brain pathology and decreased brain function [reviewed in (
      • Bruce-Keller A.J.
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      Obesity and vulnerability of the CNS.
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      • Elias M.F.
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      Lower cognitive function in the presence of obesity and hypertension: The Framingham Heart Study.
      ,
      • Waldstein S.R.
      • Katzel L.I.
      Interactive relations of central versus total obesity and blood pressure to cognitive function.
      ) and likewise link obesity to enhanced depression and anxiety disorders (
      • Needham B.L.
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      Trajectories of change in obesity and symptoms of depression: The CARDIA study.
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      • Ma J.
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      Obesity and depression in US women: Results from the 2005-2006 National Health and Nutritional Examination Survey.
      ). However, there are contradictory reports that dispute these findings (
      • Atlantis E.
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      Obesity effects on depression: Systematic review of epidemiological studies.
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      • Rivenes A.C.
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      The relationship between abdominal fat, obesity, and common mental disorders: Results from the HUNT study.
      ), suggesting that the cause of obesity-associated mental illness is not obesity per se but rather one or more of the variable manifestations of obesity.
      One potential site whereby diet-induced obesity could affect physiology is the gut microbiome, as recent advances in 16S ribosomal RNA sequencing and informatics have revealed that modern diets high in fat and sugar trigger robust alterations in the core gut microbiome (
      • Kim K.A.
      • Gu W.
      • Lee I.A.
      • Joh E.H.
      • Kim D.H.
      High fat diet-induced gut microbiota exacerbates inflammation and obesity in mice via the TLR4 signaling pathway.
      ). The human gastrointestinal tract harbors as many as 100 trillion bacteria from up to 1000 distinct species, and this dynamic population of microbes participates in numerous physiologic functions including nutrition/digestion, growth, inflammation, immunity, and protection against pathogens (
      • Robles Alonso V.
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      Linking the gut microbiota to human health.
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      The adoptive transfer of behavioral phenotype via the intestinal microbiota: Experimental evidence and clinical implications.
      ,
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      Regulation of innate and adaptive immunity by the commensal microbiota.
      ). Accordingly, the varying combinations of bacteria within individuals have been suggested to underlie variable host susceptibility to illness (
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      Gut microbiota and gastrointestinal health: Current concepts and future directions.
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      The microbiome and cancer.
      ), including neuropsychiatric impairment (
      • Douglas-Escobar M.
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      Effect of intestinal microbial ecology on the developing brain.
      ,
      • Dinan T.G.
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      Probiotics in the treatment of depression: Science or science fiction?.
      ). For example, specific alterations in colon bacteria are associated with cognitive impairment in patients with hepatic encephalopathy (
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      • Heuman D.M.
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      • et al.
      Linkage of gut microbiome with cognition in hepatic encephalopathy.
      ), and clinical studies show that oral probiotics decrease anxiety and improve mental outlook (
      • Bested A.C.
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      Intestinal microbiota, probiotics and mental health: From Metchnikoff to modern advances. Part III - convergence toward clinical trials.
      ,
      • Chen X.
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      The role of gut microbiota in the gut-brain axis: Current challenges and perspectives.
      ). Furthermore, animal studies have shown that behavior and synaptic plasticity are altered in germ-free mice and that this phenotype is reversed by microbiome colonization (
      • Diaz Heijtz R.
      • Wang S.
      • Anuar F.
      • Qian Y.
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      • Samuelsson A.
      • et al.
      Normal gut microbiota modulates brain development and behavior.
      ). The aim of the present study was to test the hypothesis that the obesity-concomitant microbiome undermines behavior even in the absence of obesity. Nonobese, adult male C57BL/6 mice were conventionally housed and maintained on chow diet but subjected to a microbiome depletion paradigm followed by adoptive transfer of cecal plus colonic contents collected from donor mice fed either a high-fat diet (HFD) or control diet (CD). Recipient mice were subjected to a battery of neuropsychological tests, followed by sequencing of gut microbiota and thorough biochemical evaluation of intestine, blood, and brain samples.

      Methods and Materials

      Animals and Treatments

      The Pennington Biomedical Research Center Institutional Animal Care and Use Committee approved all experimental protocols, which were compliant with National Institutes of Health guidelines. To generate microbiota donor material, 8-week-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, Maine) were given regular chow diet (13% fat calories, Purina LabDiet 5001; LabDiet, St. Louis, Missouri) or high-fat diet (60% fat calories, Research Diets D12492; Research Diets, Inc., New Brunswick, New Jersey) for 10 weeks (see Table S1 in Supplement 1 for diet compositions). At the time of microbiota harvest, the high-fat fed mice weighed 37.0 ± 1.7 g and the chow-fed mice weighed 24.5 ± 1.2 g. Mice were euthanatized and cecal plus colonic contents were harvested, pooled, and diluted fortyfold (weight:volume) in sterile water. After centrifugation at 800 rpm, the supernatant was aliquoted under sterile conditions for storage at −80°C. Recipient 3-month-old male C57BL/6 mice (Jackson Laboratories) were group-housed under standard laboratory conditions with free access to water and chow diet (Purina LabDiet 5001). Mice were given a cocktail of ampicillin, gentamicin, metronidazole, and neomycin (all at .25 mg/day) and vancomycin (.125 mg/day) once daily for 14 consecutive days by oral gavage (
      • Reikvam D.H.
      • Erofeev A.
      • Sandvik A.
      • Grcic V.
      • Jahnsen F.L.
      • Gaustad P.
      • et al.
      Depletion of murine intestinal microbiota: Effects on gut mucosa and epithelial gene expression.
      ). Mice were re-colonized 72 hours later via daily oral gavage of donor microbiota (100 µL) for 3 days (
      • Heimesaat M.M.
      • Plickert R.
      • Fischer A.
      • Göbel U.B.
      • Bereswill S.
      Can microbiota transplantation abrogate murine colonization resistance against Campylobacter jejuni?.
      ,
      • Ubeda C.
      • Bucci V.
      • Caballero S.
      • Djukovic A.
      • Toussaint N.C.
      • Equinda M.
      • et al.
      Intestinal microbiota containing Barnesiella species cures vancomycin-resistant Enterococcus faecium colonization.
      ). To offset potential founder and/or cage effects (
      • McCafferty J.
      • Mühlbauer M.
      • Gharaibeh R.Z.
      • Arthur J.C.
      • Perez-Chanona E.
      • Sha W.
      • et al.
      Stochastic changes over time and not founder effects drive cage effects in microbial community assembly in a mouse model.
      ) and to reinforce the donor microbiota genotype, booster inoculations were given biweekly throughout the study. Body weight and composition were measured regularly, and all mice were euthanized following behavioral testing. Plasma, lymphocytes, intestines, intestinal contents, and brains were collected, with data compiled from 10 animals per group.

      Behavioral Testing

      All behavioral testing was conducted between 7:00 am and 1:00 pm and was recorded/analyzed using Any-Maze software (Stoelting Co., Wood Dale, Illinois) for unbiased quantification of body location, orientation, distance, speed, and mobility/immobility. Detailed methods on behavioral assays are provided in Supplement 1. Overall anxiety and exploratory behavior were assessed using elevated plus (
      • Walf A.A.
      • Frye C.A.
      The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.
      ) and open field assays (
      • Choleris E.
      • Thomas A.W.
      • Kavaliers M.
      • Prato F.S.
      A detailed ethological analysis of the mouse open field test: Effects of diazepam, chlordiazepoxide and an extremely low frequency pulsed magnetic field.
      ). Stereotypical behavior was assessed by quantifying marble burying during a 30-minute trial in a novel cage preloaded with 4 cm of clean bedding and 16 evenly spaced marbles (
      • Angoa-Pérez M.
      • Kane M.
      • Briggs D.I.
      • Francescutti D.M.
      • Kuhn D.M.
      Marble burying and nestlet shredding as tests of repetitive, compulsive-like behaviors in mice.
      ) (Figure 1D). Memory was measured using a video-based fear conditioning system (Med-Associates, St. Albans, Vermont) that pairs a unique context (scent and cage) and unconditioned stimulus (auditory tone) with a repeated foot shock (day 1) and then quantifies freezing behavior to the context (day 2) and to the tone (day 3) as measures of memory (
      • Freeman L.R.
      • Zhang L.
      • Nair A.
      • Dasuri K.
      • Francis J.
      • Fernandez-Kim S.O.
      • et al.
      Obesity increases cerebrocortical reactive oxygen species and impairs brain function.
      ). Behavioral tests were administered in the order listed above over 2 weeks, beginning 3 weeks after the end of antibiotic treatment (Figure 1A). To curtail carryover effects, the elevated plus, open field, and marble-burying assays were conducted during the first week of testing with 48 hours recovery between each task, while fear conditioning was tested the following week (
      • Paylor R.
      • Spencer C.M.
      • Yuva-Paylor L.A.
      • Pieke-Dahl S.
      The use of behavioral test batteries, II: Effect of test interval.
      ,
      • Võikar V.
      • Vasar E.
      • Rauvala H.
      Behavioral alterations induced by repeated testing in C57BL/6J and 129S2/Sv mice: Implications for phenotyping screens.
      ).
      Figure thumbnail gr1
      Figure 1High-fat diet-associated microbiota increases anxiety and stereotypical behaviors but decreases memory in mice. (A) Body weight during depletion (ABX), recolonization (microbiome transplant), and behavioral protocols shows no difference between mice transplanted with microbiota from high-fat diet (HFD) fed donors or control diet (CD) fed donors. (B) Time spent exploring the open arms of the elevated plus maze was significantly reduced in HFD as compared with CD mice. (C) Time spent in the inner zone of the open field (left panel) but not mean speed (center panel) or total distance traveled (right panel) was significantly decreased in HFD mice as compared with CD mice. (D) Marble-burying behavior was significantly increased in HFD versus CD mice, as shown by representative images of marble placement before (Init) or after the 30-minute trail with mice transplanted with CD- or HFD-associated microbiota and quantitative analysis (right panel). (E) Following fear conditioning, freezing behavior to context on training day 2 was not different between groups (left panel), but conditioned freezing to the tone on day 3 was significantly reduced in HFD as compared with CD mice (right panel). All data are presented as mean ± SEM of 10 mice per group and *p < .05 based on t tests or analysis of variance.

      16S Metagenomic Sequencing

      Fecal samples were collected under aseptic conditions from all mice during the final week of behavioral testing, while cecal samples were collected aseptically at euthanasia. Sequencing and bioinformatics were performed by the Louisiana State University Microbial Genomics Resource Center. DNA was isolated using QIAamp DNA Stool kits (Qiagen, Germantown, Maryland) modified to include a bead-beating step. After DNA isolation, 16S ribosomal DNA hypervariable regions V3 and V4 were polymerase chain reaction amplified using primers with the V3F CCTACGGGAGGCAGCAG and V4R GGACTACHVGGGTWTCTAAT gene-specific sequences, Illumina adaptors, and molecular barcodes as described in Kozich et al. (
      • Kozich J.J.
      • Westcott S.L.
      • Baxter N.T.
      • Highlander S.K.
      • Schloss P.D.
      Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.
      ) to produce 430 base pair (bp) amplicons. Samples were sequenced on an Illumina MiSeq (Illumina, San Diego, California) using V3 sequencing kit (300 bp paired end reads). The forward read files were processed through the UPARSE pipeline (drive5, Tiburon, California) (
      • Edgar R.C.
      UPARSE: Highly accurate OTU sequences from microbial amplicon reads.
      ), truncating reads to a uniform length of 250 bp, then removing reads with quality scores less than 16. Additional filtering removed reads that appeared only once throughout all samples (singletons) and remaining unique reads were clustered into operational taxonomic units (OTU) at 97% similarity. Chimeric OTUs were removed as identified by UCHIME drive5 run against a gold standard reference database of nonchimeric sequences. Finally, the original filtered reads (before dereplication) were mapped to the OTUs using USEARCH drive5 at 97% identity. QIIME 1.8 (open source, www.qiime.org) was used to pick and align a representative set. The Ribosomal Database Project classifier was used to assign a taxonomic classification to each read in the representative set and a phylogenetic tree was constructed from the representative sequences. Among samples, the minimum read count after filtering was 21,182, with a median read count of 57,537. Relative abundance of each OTU was examined at phylum, class, order, family, genus, and species levels. Alpha (within a community) and beta (between communities) diversity metrics as well as taxonomic community assessments were produced using QIIME 1.8 scripts.

      Plasma and Tissue Analyses

      Whole blood was collected by cardiac puncture of terminally anesthetized mice into ethylenediaminetetraacetic acid treated tubes, and plasma and lymphocytes were isolated and analyzed immediately or stored at −80°C. Endotoxin levels in plasma were measured using a kinetic limulus amebocyte lysate test (Lonza Group, Limited, Basel, Switzerland). Levels of bioactive lipids and hormones/adipokines were measured as previously reported (
      • Pepping J.K.
      • Freeman L.R.
      • Gupta S.
      • Keller J.N.
      • Bruce-Keller A.J.
      NOX2 deficiency attenuates markers of adiposopathy and brain injury induced by high-fat diet.
      ). Lymphocyte, colon, jejunum, and brain (medial prefrontal cortex) samples were homogenized and processed for Western blot with chemiluminescence as previously reported (
      • Pepping J.K.
      • Freeman L.R.
      • Gupta S.
      • Keller J.N.
      • Bruce-Keller A.J.
      NOX2 deficiency attenuates markers of adiposopathy and brain injury induced by high-fat diet.
      ). For accurate quantification across blots, samples from both treatment groups were included in each individual blot. Data were first calculated as a ratio of expression over tubulin expression, and then expression in mice with HFD microbiota was calculated/presented as percent expression in control (CD) mice.

      Statistical Analyses

      All behavioral and biochemical data were analyzed using Prism software (GraphPad Software, Inc., La Jolla, California) and are displayed as mean ± standard error of measurement. Body weight and fear conditioning behavior were analyzed with two-way repeated measures analysis of variance (ANOVA) to determine main effects of treatment and duration, followed by planned Bonferroni post hoc comparisons to determine differences between groups. All other behavioral and biochemical data were analyzed by unpaired t tests. Statistical significance for all analyses was accepted at p < .05.
      For sequencing data, alpha diversity rarefaction curves were produced by plotting several diversity metrics against the number of sequences considered from a sample. Subsequent analysis of diversity was performed at a depth of 20,000 sequences per sample. Statistical significance was compared using a nonparametric permutation test for a pairwise comparison of categories. The p values were Bonferroni-corrected for multiple testing. Beta diversity, principle coordinates analysis plots were produced, using both weighted (considers abundance of each species) and unweighted (considers presence/absence of species) UniFrac metrics. Plots were visualized using the Emperor 3D Viewer (open source, www.emperor.colorado.com). Statistical significance was assessed with a nonparametric permutation test to compare a chosen category. ANOVA was used to test for differences in relative abundance of specific OTUs for each group. An unweighted g-test was used to evaluate the statistical significance of the presence/absence of OTUs across categories. DESeq2 software (open source, www.bioconductor.org) was also used to test for differential representation of OTUs and also to identify in an unbiased manner all individual OTUs in which the group difference reached statistical significance using mean normalized sequence count level higher than 30 counts and false discovery rate (FDR) < .05 criteria (Wald statistics with Benjamini-Hochberg correction) in either the CD or HFD group.

      Results

      HFD-Derived Gut Microbiota Impair Behavioral Performance in Mice

      All animals tolerated the antibiotic regimen with no overt effects other than a mild, approximate 10% loss of body weight (Figure 1A). Quantitative real-time polymerase chain reaction based analyses of 16S RNA levels in fecal samples collected from mice midway through the antibiotic treatment revealed an approximate 90% to 95% reduction in fecal bacteria burden compared with matched but untreated mice (fecal DNA concentration 82,502.1 ± 18,255 µg/g in control samples, 3417.4 ± 1212 µg/g in samples following antibiotic exposure). Mice were subjected to thorough behavioral phenotyping starting 3 weeks after recolonization with either microbiota from high-fat or chow-fed mice (Figure 1A). Exploratory and anxiety-based behavior assessed using the elevated plus maze revealed that mice with HFD-associated microbiota spent significantly less time (t18 = 2.32, p < .05) in the open arms of the maze (Figure 1B). The open field assay likewise revealed that mice with HFD-associated microbiota spent significantly less time (t18 = 2.13, p < .05) in the inner zone of the open field (Figure 1C). Overall, locomotor activity assessed in the open field showed no differences in mean speed or total distance traveled between CD and HFD groups (Figure 1C), suggesting that decreased exploratory behavior in mice with HFD microbiota reflects increased anxiety not decreased motor function. Mice were tested for marble burying, a measure of compulsive, anxiety-like behavior (
      • Njung’e K.
      • Handley S.L.
      Evaluation of marble-burying behavior as a model of anxiety.
      ). HFD-shaped microbiota were associated with a significant increase (t18 = 2.64, p < .05) in marble burying (Figure 1D). Finally, the fear conditioning assay was used to measure memory. Significant differences in freezing behavior were observed on the third day of the fear conditioning test, when cued learning (freezing in a novel context in response to the tone) was assessed. Post hoc analyses revealed that freezing in response to tone was significantly decreased in mice with HFD microbiota as compared with mice with CD microbiota (Figure 1E). In addition, the averaged slopes of behavioral waveforms depicting freezing of individual mice in response to tone were significantly different between the groups (−11.8 ± 1.48 in CD mice; −5.5 ± .6 in HFD mice; t18 = 4.05, p < .001), suggesting attenuated within-session extinction of fear behavior in mice with HFD microbiota.

      Microbiota Transplantation Results in Distinct Phylogenetic Profiles

      Fecal and cecal microbiota compositions in recipient mice were analyzed by V3 16S ribosomal DNA phylogenetics as described in Methods and Materials. Analysis of cecal and fecal samples from all mice demonstrated that the adoptive transfer protocol was successful in producing distinct core microbiomes in the two groups of mice (Tables S2 and S3 in Supplement 1). Additionally, HFD microbiota demonstrated significantly reduced alpha diversity relative to CD samples, as well as greater evenness (cecal samples: Chao1 p = .0362, observed species p = .0302; fecal samples: Chao1 p = .0033, observed species p = .004; Figure 2A and Figure S1 in Supplement 1). Rarefaction curves of fecal and cecal diversity (Figures S1 and S2 in Supplement 1) also support this interpretation as well. Evaluation of beta diversity metrics based on unweighted UniFrac distances showed that the community structures observed in the HFD samples were significantly different (p = .0001) from the communities detected in the CD samples (Figure 2B). Visualization by principal coordinates analysis demonstrated that CD and HFD samples formed distinct clusters and that for each condition, cecal and fecal samples formed a cluster around the initial inoculum (Figure 2B). The taxonomical distribution within groups at phylum, family, and genus levels for the cecal (Figure 2C; Figure S3 in Supplement 1) and fecal samples (Figure S4 in Supplement 1) revealed the divergent composition of communities in CD compared with HFD samples.
      Figure thumbnail gr2
      Figure 2Effects of transplantation protocol on recipient gut microbiome diversity and population. Cecal and fecal microbiome populations from both donor and recipient mice were analyzed using 16S ribosomal RNA sequencing. (A) Box plots that were generated to depict differences in Chao1 α-diversity show that mice with high-fat diet (HFD) microbiota exhibited a statistically significant (*p = .0362) reduction in α-diversity compared with mice with control diet (CD) microbiota. Red line: median; black lines: range of values. (B) Scaled principal coordinate analysis to visualize the unweighted UniFrac distances of both cecal and fecal samples from individual recipient mice. Red and orange circles depict cecal and fecal samples from CD-treated mice; green and yellow circles represent cecal and fecal samples from HFD-treated mice. The pooled samples used as donor microbiota are shown in blue for the CD donor pool and in magenta for the HFD donor pool. β-diversity was found to be statistically significantly different between the CD- and HFD-treated groups (p = .0001). (C) Microbiota membership is reflected in bar diagrams depicting the taxonomic distribution within cecal samples within the CD and HFD groups at the phylum, family, and genus levels. Microbiota from the HFD-treated group show higher representation of Clostridiales (family: purple; genus: blue). Higher resolution images together with the detailed color codes are shown in and in . (D) Microbiome differences between CD- and HFD-treated mice at the level of Lachnospiraceae and Ruminococcaceae, two families within the order of Clostridiales. Statistically significant fold changes between the HFD-treated group versus the CD-treated group were determined in DESeq2. Significant fold changes for individual operational taxonomic units belonging either to Lachnospiraceae or to Ruminococcaceae (family level as maximum taxonomical depth for these operational taxonomic units) were log2-transformed and plotted relative to the CD group. This analysis demonstrated shifts in representation within each family. PC1, Principle Coordinate 1; PC2, Principle Coordinate 2.
      The UPARSE pipeline (
      • Edgar R.C.
      UPARSE: Highly accurate OTU sequences from microbial amplicon reads.
      ) was used to identify OTUs, and ANOVA revealed significant differences in the relative abundance of OTUs between HFD and CD microbiota (see Tables S4 and S5 in Supplement 1 for Bonferroni-corrected and FDR controlled values). To corroborate ANOVA data with statistical methods better suited for sequence count data, DESeq2 software was used to test for differential representation of OTUs in cecal samples. Individual OTUs in which the group difference reached statistical significance using mean normalized sequence count level higher than 30 counts and FDR <.05 criteria (Wald statistics with Benjamini-Hochberg correction) in either the CD or HFD group were identified (Table S6 in Supplement 1). Of the 104 OTUs passing these DESeq2 filters, 53 OTUs were higher in the HFD samples compared with CD, whereas 51 OTUs were higher in CD. Of the 104 significantly different OTUs, 91 belong to the phylum Firmicutes, with 90 of these coming from class Clostridiales. These unbiased analyses show that the overall distinction between HFD and CD is based on shifts in the representation of individual OTUs within Clostridiales rather than a binary shift from, for example, Bacteroidetes in the CD samples to Firmicutes in the HFD samples. Looking at specific orders within Clostridiales (Figure 2D; Figure S5 in Supplement 1), some orders are present at significantly higher levels in the HFD group (17 members of Lachnospiraceae; 9 members of Ruminococcaceae), whereas others are lower (21 members of Lachnospiraceae; 9 members of Ruminococcaceae). Similar findings were obtained in fecal samples (data not shown). Finally, the list of differently represented OTUs was queried for presumed beneficial bacteria, such as Akkermansia muciniphila. Akkermansia muciniphila was 5.4-fold lower in HFD samples compared with CD (FDR = .06), indicating that this species of bacteria may be associated with a healthier microbiome, as suggested previously (
      • Everard A.
      • Belzer C.
      • Geurts L.
      • Ouwerkerk J.P.
      • Druart C.
      • Bindels L.B.
      • et al.
      Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity.
      ). Likewise, the presumably detrimental Bilophila sp. (belonging to Desulfovibrionaceae) was strongly enriched (~ 300-fold; FDR = 2.5 × 10−25) in HFD microbiota, comprising .78% of the microbial community in this group. Conversely, Bilophila sp. was barely detectable in CD samples at .0024%.

      HFD-Derived Gut Microbiota Increase Intestinal Permeability, Systemic Inflammation, and Brain Inflammation

      Postmortem studies were conducted to identify potential pathways whereby altered gut microbiota impaired behavior. Analysis of blood glucose and bioactive hormones/lipids in plasma, as well as body weight and body composition, revealed no significant group differences, demonstrating that obesity and metabolic syndrome/dysfunction were not induced by HFD microbiota (Table S7 in Supplement 1).
      To determine if transplantation with HFD-shaped microbiota altered intestinal barrier function, the expression of markers of intestinal inflammation and permeability and also circulating endotoxin and inflammatory markers were assessed (see Figure S6 in Supplement 1 for representative Western blots). Compared with CD, mice with HFD microbiota had significantly decreased occludin (t18 = 4.95, p < .001) expression in the jejunum (Figure 3A). Additionally, expression of inducible nitric oxide synthase (t18 = 3.70, p < .01) and phosphorylation of the p65 subunit of nuclear factor kappa B (t18 = 4.13, p < .001) were increased, while occludin (t18 = 3.32, p < .01) and claudin-3 (t18 = 4.13, p < .001) were decreased, in colons of HFD mice (Figure 3A), indicating increased intestinal inflammation and permeability in mice with HFD microbiota. In addition, data showed significantly increased plasma endotoxin (t18 = 2.64, p < .05) in mice with HFD microbiota (Figure 3B), while evaluation of isolated lymphocytes revealed increased expression of the macrophage marker ionized calcium-binding adapter molecule 1 (t16 = 2.59, p < .05) and toll-like receptor 4 (t16 = 2.73, p < .05) in mice with HFD microbiota (Figure 3B).
      Figure thumbnail gr3
      Figure 3Transplantation with microbiota shaped by high-fat diet disrupts intestinal barrier proteins and increases systemic and brain inflammation. (A) Relative expression of tight junction proteins occludin, claudin-2, and claudin-3 in jejunum (left). Expression of inducible nitric oxide synthase (iNOS), phosphorylated p65 (phos-p65), and tight junction proteins occludin, claudin-2, and claudin-3 in colon (right) in mice with high-fat diet (HFD) microbiota relative to control-diet (CD) mice. (B) Levels of plasma endotoxin (Endtxn) and lymphocyte expression of macrophage markers (ionized calcium-binding adapter molecule 1 [Iba1]) and toll-like receptor (TLR) 4 in mice with HFD microbiota as compared with CD mice. (C) Markers of inflammation, cerebrovascular integrity, and synaptic density in tissue homogenates prepared from the medial prefrontal cortex. Graphs depict increased microgliosis (Iba1) and TLR2 and TLR4 expression, increased matrix metalloproteinase (MMP) 9 expression, and decreased expression of endothelial tight junction proteins (zona occludens protein 1 [ZO-1] and claudin-5) and phosphorylated synapsin-1 (P-Synap) in HFD mice. All data depict mean ± SEM expression in mice with HFD microbiota presented as % CD mice (100% line on graph). *p < .05, **p < .01, and ***p < .001, based on t tests. See in for representative images of all Western blot data. GFAP, glial fibrillary acidic protein; P-synap, phosphorylated synapsin I; SAP97, synapse-associated protein 97; Synap, synapsin I.
      To determine the effects of HFD-shaped microbiota on brain, protein markers of brain injury and inflammation were quantified (see Figure S6 in Supplement 1 for representative Western blots). Analyses were thematically split into evaluations of inflammation/gliosis, cerebrovascular integrity, and synaptic density and were conducted in the medial prefrontal cortex, a brain structure involved in both anxiety and cognitive behaviors in mice (
      • Han S.
      • Hong S.
      • Lee D.
      • Lee M.H.
      • Choi J.S.
      • Koh M.J.
      • et al.
      Altered expression of synaptotagmin 13 mRNA in adult mouse brain after contextual fear conditioning.
      ). Compared with mice with CD-shaped microbiota, expression of the microglial marker ionized calcium-binding adapter molecule 1 (t18 = 3.48, p < .01), toll-like receptor 2 (t18 = 2.72, p < .05), and toll-like receptor 4 (t18 = 2.83, p < .05) were increased in HFD mice (Figure 3B). Additionally, mice with HFD-associated microbiota had decreased levels of the tight junction proteins zona occludens protein 1 (ZO-1) (t18 = 2.32, p < .05) and claudin-5 (t18 = 4.11, p < .001) and increased expression of matrix metalloproteinase 9 (t18 = 2.29, p < .05; Figure 3B). While overall expression of the synaptic marker proteins synapse-associated protein 97 and synapsin 1 were similar in both groups, levels of phosphorylated synapsin 1 were significantly reduced (t18 = 2.26, p < .05) in mice with HFD-shaped microbiota (Figure 3B). Finally, levels of brain-derived neurotrophic factor were assessed in the medial prefrontal cortex, but there were no significant differences in soluble brain-derived neurotrophic factor in mice with HFD microbiota as compared with mice with CD microbiota (Table S7 in Supplement 1).

      Discussion

      The present findings represent the first definitive evidence that high-fat diet-induced changes to the gut microbiome are sufficient to disrupt brain physiology and function in the absence of obesity. Specifically, data show that transplantation of microbiota shaped by high-fat diet, but not control low-fat diet, caused significant and selective disruptions in exploratory, cognitive, and stereotypical behavior in conventionally housed, nonobese, diet-naïve mice. Overall, these data are in agreement with the extensive body of literature describing the sensitivity of the brain to diet-induced obesity (
      • Freeman L.R.
      • Zhang L.
      • Nair A.
      • Dasuri K.
      • Francis J.
      • Fernandez-Kim S.O.
      • et al.
      Obesity increases cerebrocortical reactive oxygen species and impairs brain function.
      ,
      • Stranahan A.M.
      • Norman E.D.
      • Lee K.
      • Cutler R.G.
      • Telljohann R.S.
      • Egan J.M.
      • Mattson M.P.
      Diet-induced insulin resistance impairs hippocampal synaptic plasticity and cognition in middle-aged rats.
      ) and the growing number of studies linking gut microbiota to central nervous system health and behavior (
      • Diaz Heijtz R.
      • Wang S.
      • Anuar F.
      • Qian Y.
      • Björkholm B.
      • Samuelsson A.
      • et al.
      Normal gut microbiota modulates brain development and behavior.
      ,
      • Neufeld K.M.
      • Kang N.
      • Bienenstock J.
      • Foster J.A.
      Reduced anxiety-like behavior and central neurochemical change in germ-free mice.
      ,
      • Bercik P.
      • Denou E.
      • Collins J.
      • Jackson W.
      • Lu J.
      • Jury J.
      • et al.
      The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice.
      ). For example, there is a reported high comorbidity between psychiatric syndromes, including depression and anxiety, with gastrointestinal disorders, while conversely, recent studies link probiotics to positive changes in mood and behavior [reviewed in (
      • Dinan T.G.
      • Cryan J.F.
      Melancholic microbes: A link between gut microbiota and depression?.
      )]. Furthermore, changes in microbiota appear to mediate weight gain commonly associated with antipsychotic administration (
      • Davey K.J.
      • O’Mahony S.M.
      • Schellekens H.
      • O’Sullivan O.
      • Bienenstock J.
      • Cotter P.D.
      • et al.
      Gender-dependent consequences of chronic olanzapine in the rat: Effects on body weight, inflammatory, metabolic and microbiota parameters.
      ,
      • Davey K.J.
      • Cotter P.D.
      • O’Sullivan O.
      • Crispie F.
      • Dinan T.G.
      • Cryan J.F.
      • O’Mahony S.M.
      Antipsychotics and the gut microbiome: Olanzapine-induced metabolic dysfunction is attenuated by antibiotic administration in the rat.
      ), which has been likewise linked to improvements in core schizophrenia symptoms, depression, and overall mental functioning (
      • Ascher-Svanum H.
      • Stensland M.
      • Zhao Z.
      • Kinon B.J.
      Acute weight gain, gender, and therapeutic response to antipsychotics in the treatment of patients with schizophrenia.
      ). It should be pointed out, however, that reports have shown that high-fat diet consumption can allay anxiety and depressive-like behaviors in mice subjected to chronic social stress (
      • Finger B.C.
      • Dinan T.G.
      • Cryan J.F.
      High-fat diet selectively protects against the effects of chronic social stress in the mouse.
      ). Thus, these data underscore the strong but complex influence of diet-induced changes to the gut microbiome on stress-induced behaviors and emphasize the clinical utility of the gut-brain axis as a target for future therapeutic intervention.
      The significant behavioral phenotype of the mice described in this report, combined with the established association between psychiatric conditions and gastrointestinal symptoms, support the concept of a microbiome-gut-brain axis (
      • Montiel-Castro A.J.
      • González-Cervantes R.M.
      • Bravo-Ruiseco G.
      • Pacheco-López G.
      The microbiota-gut-brain axis: Neurobehavioral correlates, health and sociality.
      ,
      • Ochoa-Repáraz J.
      • Mielcarz D.W.
      • Begum-Haque S.
      • Kasper L.H.
      Gut, bugs, and brain: Role of commensal bacteria in the control of central nervous system disease.
      ), but the mechanisms whereby gut microbes affect behavior are not understood. Gut microbial metabolism is known to produce catecholamines, histamine, and/or other neuroactive mediators that can directly stimulate the local enteric nervous system and/or primary afferent fibers of vagal or dorsal root origin (
      • Neufeld K.M.
      • Kang N.
      • Bienenstock J.
      • Foster J.A.
      Reduced anxiety-like behavior and central neurochemical change in germ-free mice.
      ,
      • Lyte M.
      Microbial endocrinology in the microbiome-gut-brain axis: How bacterial production and utilization of neurochemicals influence behavior.
      ). Indeed, reports have shown that the probiotic bacterium Lactobacillus rhamnosus can directly increase single- and multi-unit firing rates of the mesenteric nerve bundle and can decrease stress-induced corticosterone and anxiety/depression in mice (
      • Bravo J.A.
      • Forsythe P.
      • Chew M.V.
      • Escaravage E.
      • Savignac H.M.
      • Dinan T.G.
      • et al.
      Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve.
      ,
      • Perez-Burgos A.
      • Wang B.
      • Mao Y.K.
      • Mistry B.
      • McVey Neufeld K.A.
      • Bienenstock J.
      • Kunze W.
      Psychoactive bacteria Lactobacillus rhamnosus (JB-1) elicits rapid frequency facilitation in vagal afferents.
      ). Moreover, the positive behavioral effects of Lactobacillus rhamnosus are abolished by vagotomy (
      • Bravo J.A.
      • Forsythe P.
      • Chew M.V.
      • Escaravage E.
      • Savignac H.M.
      • Dinan T.G.
      • et al.
      Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve.
      ). In addition to direct interactions with neural processes, immune activation and inflammation participate in nearly all neurologic/psychiatric disorders (
      • Raison C.L.
      • Miller A.H.
      Is depression an inflammatory disorder?.
      ,
      • Lyman M.
      • Lloyd D.G.
      • Ji X.
      • Vizcaychipi M.P.
      Neuroinflammation: The role and consequences.
      ), and gut dysbiosis might alter brain function via this pathway. Indeed, the increases in gram-negative Proteobacteria within the gut, endotoxins in the blood, and inflammatory markers in the brain collectively suggest that intestinal permeability and inflammation link HFD microbiota to behavioral dysfunction. In further support of this scenario, transplantation of microbiota from obese donors to germ-free recipients has been shown to disrupt intestinal tight junction protein and increase translocation of bacteria into the bloodstream (
      • Duca F.A.
      • Sakar Y.
      • Lepage P.
      • Devime F.
      • Langelier B.
      • Doré J.
      • Covasa M.
      Replication of obesity and associated signaling pathways through transfer of microbiota from obese prone rat.
      ). In relation to neurologic impairment, alterations to gut microbiota and disrupted intestinal barrier function are seen in mouse models of autism spectrum disorder (
      • Hsiao E.Y.
      • McBride S.W.
      • Hsien S.
      • Sharon G.
      • Hyde E.R.
      • McCue T.
      • et al.
      Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders.
      ). Collectively, these data suggest that unhealthy diet-induced alterations to gut microbiota could boost the prevalence and/or severity of numerous neurologic conditions that involve inflammation, including autoimmune disease, autism, and Alzheimer’s disease.
      While previous reports have shown that gut microbiome transplantations into germ-free mice can replicate many aspects of the obese phenotype (
      • Turnbaugh P.J.
      • Ley R.E.
      • Mahowald M.A.
      • Magrini V.
      • Mardis E.R.
      • Gordon J.I.
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      ,
      • Turnbaugh P.J.
      • Bäckhed F.
      • Fulton L.
      • Gordon J.I.
      Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome.
      ), this is the first demonstration that high-fat shaped gut microbiota can intrinsically and adversely affect neurologic function/physiology in conventionally housed mice, even in the absence of altered diet, adiposity, or metabolic syndrome. A variety of tools and techniques have been developed to study the gut microbiome; microbiota, including introduction into germ-free recipients; antibiotic use; administration of prebiotics and probiotics; and specific gastrointestinal infection. Indeed, the use of gnotobiological methods on experimental animals has been indispensable in establishing the significance of gut microbiota to mammalian physiology (
      • Yi P.
      • Li L.
      The germfree murine animal: An important animal model for research on the relationship between gut microbiota and the host.
      ). However, there are several characteristics of germ-free mice, outside of changes in cecal size and bowel motility, that undermine their utility and physiologic relevance. For example, germ-free mice are well known to be smaller than conventional mice, with decreased cardiac output and notably underdeveloped immune systems (
      • Luckey T.D.
      Effects of microbes on germfree animals.
      ,
      • Berg R.D.
      The indigenous gastrointestinal microflora.
      ). As any of these confounds could affect the development and function of the brain, we opted instead to use a strategy whereby donor microbiota were adoptively transferred to conventionally housed mice following antibiotic-based microbial depletion. While both the depletion and recolonization protocols are based on established methods (
      • Reikvam D.H.
      • Erofeev A.
      • Sandvik A.
      • Grcic V.
      • Jahnsen F.L.
      • Gaustad P.
      • et al.
      Depletion of murine intestinal microbiota: Effects on gut mucosa and epithelial gene expression.
      ,
      • Heimesaat M.M.
      • Plickert R.
      • Fischer A.
      • Göbel U.B.
      • Bereswill S.
      Can microbiota transplantation abrogate murine colonization resistance against Campylobacter jejuni?.
      ,
      • Ubeda C.
      • Bucci V.
      • Caballero S.
      • Djukovic A.
      • Toussaint N.C.
      • Equinda M.
      • et al.
      Intestinal microbiota containing Barnesiella species cures vancomycin-resistant Enterococcus faecium colonization.
      ,
      • McCafferty J.
      • Mühlbauer M.
      • Gharaibeh R.Z.
      • Arthur J.C.
      • Perez-Chanona E.
      • Sha W.
      • et al.
      Stochastic changes over time and not founder effects drive cage effects in microbial community assembly in a mouse model.
      ), there are limitations of the antibiotic-based model that could have affected the outcome of our study. It is likely that the antibiotic regimen did not entirely deplete the recipient microbiome, which could differentially affect recolonization by specific bacteria. It is also possible that repeated gavage and/or sustained systemic antibiotic exposure could contribute to some of the behavioral alterations obseved. However, as both groups were treated equally, the impact of such potential artifacts is minimized.
      Alterations in microbiome composition following manipulation have been evaluated in the past in an attempt to identify beneficial core microbiota. For example, studies on high-fat diet-induced gut microbiota have reported a shift in the relative abundance of two major phyla, a reduction in Bacteroidetes and an increase in Firmicutes (
      • Turnbaugh P.J.
      • Ley R.E.
      • Mahowald M.A.
      • Magrini V.
      • Mardis E.R.
      • Gordon J.I.
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      ,
      • Turnbaugh P.J.
      • Bäckhed F.
      • Fulton L.
      • Gordon J.I.
      Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome.
      ). Furthermore, abundance of these two phyla was shifted in the opposite direction after weight loss or gastric bypass surgery (
      • Zhang H.
      • DiBaise J.K.
      • Zuccolo A.
      • Kudrna D.
      • Braidotti M.
      • Yu Y.
      • et al.
      Human gut microbiota in obesity and after gastric bypass.
      ,
      • Sweeney T.E.
      • Morton J.M.
      The human gut microbiome: A review of the effect of obesity and surgically induced weight loss.
      ); thus, it has been proposed that the balance between these two phyla might reflect the balance of unhealthy and healthy microbiota. However, our data suggest that this binary distinction does not sufficiently reflect the complexity of diet-induced changes to the gut microbiome, as has been suggested in previous investigations of diet-induced obesity and gut microbiota (
      • Murphy E.F.
      • Cotter P.D.
      • Hogan A.
      • O’Sullivan O.
      • Joyce A.
      • Fouhy F.
      • et al.
      Divergent metabolic outcomes arising from targeted manipulation of the gut microbiota in diet-induced obesity.
      ). Specifically, our data indicate that shifts within the Firmicute phylum drive the overall distinction of HFD from CD, rather than phylum-wide shifts from Bacteroidetes to Firmicutes (Figure 2; Figure S5 in Supplement 1). While it is currently not possible to identify the specific alterations or population shifts that drive the observed behavioral/biochemical alterations, the relative abundance of purportedly beneficial and harmful species in each group was probed. Akkermansia muciniphila, a presumed beneficial species, was 5.4-fold lower in HFD samples compared with CD, indicating that this species of bacteria may be associated with a healthier microbiome, as suggested previously (
      • Everard A.
      • Belzer C.
      • Geurts L.
      • Ouwerkerk J.P.
      • Druart C.
      • Bindels L.B.
      • et al.
      Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity.
      ). Likewise, the presumed detrimental Bilophila sp. (belonging to Desulfovibrionaceae) was strongly enriched (~ 300-fold; FDR = 2.5 × 10−25) in HFD microbiota, comprising .78% of the microbial community in this group. Conversely, Bilophila sp. was barely detectable in CD samples at .0024%. As member(s) of the phylum Proteobacteria, Bilophila sp. may be partially responsible for the increase in endotoxin observed in the serum from HFD-treated mice compared with CD-treated mice (Figure 3). Indeed, higher Bilophila wadsworthia have been repeatedly found in human patients suffering from intestinal diseases (
      • Jia W.
      • Whitehead R.N.
      • Griffiths L.
      • Dawson C.
      • Bai H.
      • Waring R.H.
      • et al.
      Diversity and distribution of sulphate-reducing bacteria in human faeces from healthy subjects and patients with inflammatory bowel disease.
      ,
      • Baron E.J.
      Bilophila wadsworthia: A unique Gram-negative anaerobic rod.
      ). The collective identification of specific bacterial species/populations driving adverse physiologic responses to diet will facilitate the future design of personalized microbiomes that optimize physiologic function in the context of modern diets/lifestyles. Overall, these data strongly suggest that therapeutic manipulation of the microbiome, which should be highly responsive compared with existing clinical targets, could dramatically mitigate the prevalence and/or severity of neuropsychiatric disorders.

      Acknowledgments and Disclosures

      This work was supported by the National Institutes of Health ( DK047348 to HRB) and also used Pennington Biomedical Research Center (Animal Phenotyping) and Louisiana State University (Microbial Genomics Resource Center), which are funded, in part, by the National Institutes of Health ( P20-RR021945 , P30-DK072476 , and P60-AA009803 ).
      We thank Dr. Barry Robert for expert veterinary assistance related to antibiotic administration.
      The authors declare no biomedical financial interests or potential conflicts of interest.

      Appendix A. Supplementary Materials

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      Linked Article

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          Human metagenomics is the genomic study of the microbial communities that inhabit Homo sapiens. Why should biomedical researchers and clinicians whose primary focus is psychiatric disorders, and who typically lack interest and expertise in microbiology, pay attention to a fast-moving literature on metagenomics? The past decade has seen important advances in research, notably concerning the gut microbiome (aggregate collection of gut microbial genomes and genes) which is the focus of the article by Bruce-Keller et al.
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      • High-Fat Diet–Induced Dysbiosis as a Cause of Neuroinflammation
        Biological PsychiatryVol. 80Issue 1
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          In their outstanding article, Bruce-Keller et al. (1) demonstrated for the first time that gut microbiota altered by a high-fat (HF) diet decrease memory and increase anxiety and stereotypical behaviors in mice in the absence of obesity. Obesity and related metabolic comorbidities are characterized by a low-grade chronic inflammatory state accompanied by abnormal cytokine production (2). Gut microbiota dysbiosis may be implicated in the pathophysiology of these diseases through their impact on local and systemic inflammation (3).
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