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: Gut dysbiosis; Intestinal permeability; Mental health; Neurobehavior; Neuroinflammation; Obesity; Psychiatric disease.

Figure 1

High 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 fed donors (HFD) or control diet fed donors (CD). (B) Time spent exploring the open arms of the Elevated Plus Maze was significantly reduced in HFD as compared to 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 to CD mice. (D) Marble burying behavior was significantly increased in HFD vs. 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 to CD mice (right panel). All data are presented as mean ± SEM of 10 mice per group, and *p < 0.05 based on t-tests or ANOVA.

Figure 2

Effects 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 rRNA sequencing, and (A) Box Plots were generated to depict differences in Chao1 α-diversity show that mice with HFD microbiota exhibited a statistically significant (p = 0.0362) reduction in α-diversity compared to mice with CD mirobiota. 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 show in blue for the CD donor pool, and in magenta for the HFD donor pool. α- diversity was found to be statistically significantly different between the CD- and HFD-treated groups (p = 0.0001). (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 Supplementary Figures S3 and S4. (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 vs. the CD-treated group were determined in DESeq2. Significant fold-changes for individual OTUs belonging either to Lachnospiraceae or to Ruminococcaceae (family level as maximum taxonomical depth for these OTUs) were log2-transformed and plotted relative to the CD group. This analysis demonstrated shifts in representation within each family.

Figure 3

Transplantation 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 HFD microbiota relative to CD mice. (B) Levels of plasma endotoxin, and lymphocyte expression of macrophage markers (Iba1) and TLR4, in mice with HFD microbiota as compared to 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 9 (MMP9) expression, and decreased expression of endothelial tight junction proteins (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), and *, **, and *** depict p < 0.05, p < 0.01, and p < 0.001, respectively, based on t-tests. See Supplementary Fig. S6 for representative images of all Western blot Data.