Animals

All methods were performed in accordance with the relevant guidelines and regulations of the University of Virginia and approved by the University of Virginia Animal Care and Use Committee. Male C57BL/6 and BALB/c (8 weeks old) were purchased from Jackson or Taconic laboratories as described in the text. The mice were maintained on a 12 hours light/dark cycle with lights on at 7am. Chronic stress was started after at least 1 week of acclimation. All behavioral interventions were performed between 4 pm and 7 pm and take-downs were performed between 11am and 2 pm. Sample size was selected to be similar to previously reported behavioral experiments. Animals were housed 2–3 per cage and cages were randomly assigned to control or experimental groups. In each experiment, every group consisted of at least 3 cages in order to minimize cage effects. Investigators were not blinded to the group allocation. Experiments were conducted in such a way as to make sure that all experimental groups were exposed to the same environments. Animals were excluded from the experiments if they developed illnesses (e.g. dermatitis) that might affect the outcome of the results. Certain measurements are occasionally not available for each mouse enrolled in the study due to technical issues (e.g. fecal sample not provided, sample loss).

Chronic mild stress protocol

Mice were subjected daily to an acute stressor (1–2 hours: restraint, loud white noise, crowded housing, strobe light) and an overnight stressor (12–24 hours: 45° cage tilting, repeated cage changes, wet bedding, dark deprivation) presented in a randomized fashion as described in the literature13. For restraint stress, mice were placed in clean 50 mL conical tubes with pierced holes for ventilation for 1 hour. For crowded housing stress, mice were placed on top of the cage wire for 2 hours, after which their cage was changed. For wet bedding stress, 200 mL of water was added to the bedding of a clean cage. All procedures used autoclaved, sterile materials (bedding, water) in order to prevent contamination. Food, water intake, and weights were monitored in initial experiments and no major changes were observed in the stressed group. For subsequent experiments, monitored parameters are indicated on matching experimental design figures. Sequencing results showed no new OTUs in the stressed microbiota, indicating the lack of contamination during the stress protocol.

Behavioral assessment

Despair behavior was assessed at time points of interest using the forced swim test, as described in the literature58. The last 4 minutes (out of 6 minutes total test) were scored for escape behavior, defined as active swimming, with all four limbs and tail moving. The test was conducted using autoclaved water and disinfected containers. Anxiety/compulsive behavior was assessed using the nestlet-shredding test16. The mice were singled housed 30–60 minutes before the beginning of the test. Each mouse then received a pre-weighed nestlet and allowed undisturbed activity for either 30 or 60 minutes. At the end of the test, the piece of nestlet that was still intact was weighed.

DNA isolation

Whole genomic DNA was isolated via phenol-chloroform extraction. Briefly, a fecal pellet was placed in a 2 mL tube containing 200 μL silica-zirconia beads (0.1 mm). The tube was filled with 750 μL extraction buffer, 200 μL 20% SDS and 750 μL phenol-chloroform-isoamyl alcohol (25:24:1). After disruption, the aqueous phase was separated by centrifugation and cleaned up with two washes of chloroform-isoamyl alcohol (24:1). The DNA was precipitated and resuspended in 10 mM Tris solution.

Fecal microbiota sequencing

For 16S rRNA sequencing, the V3-V4 region of the 16S rRNA gene was amplified for 25 cycles using specific primers with adapter overhangs as per the Illumina library preparation guide (Sup. Table 1). Following purification of the PCR products, individual indexes were added to the amplicons by PCR. The amplicons were purified, pooled in equal quantities, and then sequenced on the Illumina MiSeq platform. Reads with an average quality score below 25 (from any 10-bp window) or a mismatched barcode were removed. Paired-end reads were then merged using the software FLASH59. Merged reads were analyzed using the QIIME pipeline with default parameters to remove chimeric, pick 97%-identity OTUs and assign taxonomy60.

Selective culture of Lactobacilli and L. reuteri preparation

Fecal pellets were resuspended in 1 mL of deMan, Rogosa and Sharpe (MRS) broth supplemented with sodium azide (0.02% w/v) to select Lactobacillus19. After brief decanting of insoluble fecal material, samples were further diluted 1:1000 in MRS/azide and 50 μL of this dilution were spread on MRS/azide agar plates and grown overnight at 37 °C, in aerobic conditions. The plates were then imaged with a Bio-Rad gel imager and colonies were counted using the “particle counter” plugin in ImageJ.

For Lactobacillus supplementation experiments, L. reuteri was obtained from ATCC (23272) and cultured aerobically according to manufacturer instructions. Radiated food pellets were pulverized in a blender and kneaded with fresh L. reuteri (2 billion CFU/mouse/day) and water, or with MRS culture broth and water for control. The animals received fresh food prepared daily.

Intestinal transit time measurement

Large intestinal transit time was measured as previously described61. Briefly, mice were anesthetized with isofluorane and a 3 mm diameter glass bead was inserted 2 cm inside the rectum with a lubricated rod. The mice were then placed in an empty cage without bedding and the time to bead expulsion was measured.

Intestinal tissue analysis

The small intestine was dissected by excising under the stomach and before the cecum. Mesenteric fat and Peyer’s patches were carefully removed using fine forceps. The intestine was opened longitudinally and the contents were removed in two PBS washes. Excess liquid was gently absorbed using kimwipes and the tissue was weighed. Intestinal cells were then isolated as previously described62. Briefly, the mucus layer and epithelial cells were shook off in two washes with HBSS/5% FBS/2 mM EDTA. The tissue was then digested with collagenase VIII (Sigma, C2139) for 12–15 minutes, filtered, and the cells washed twice with HBSS/5% FBS/2 mM EDTA and finally resuspended in FACS buffer (0.01 M PBS, 1% BSA, 2 mM EDTA, 0.1% sodium azide). Cell counts and viability were determined using an acridine orange/propidium iodine assay on a Nexcelom cell counter.

Metabolite analysis

Blood was collected by cardiac puncture and centrifuged at 10,000 × g for 3 minutes in gel tubes for serum preparation. Frozen (−80 °C) serum samples were shipped to the University of Michigan Metabolomics Core for untargeted metabolomics analysis. Metabolites isolated by positive and negative ion selection were analyzed by mass spectrometry. Mass spectrometry peak intensities were further analyzed using the MetaboAnalyst online software. Peak values were filtered using the interquantile range, normalized to the group sum, then log transformed and auto-scaled for principal component analysis and further statistical tests63. Identifiable significant metabolites were analyzed through pathway analysis, followed by further manual pathway enrichment.

ROS quantification

Fresh fecal samples were collected in sterile 2 mL tubes, weighed, and resuspended in 1 mL sterile PBS. After brief sedimentation of insoluble particles, 500 μL of bacterial slurry were incubated at 37 °C for 30 minutes. Following bacterial culture centrifugation, 50 μL of supernatant was reacted using the Amplex Red hydrogen peroxide/peroxidase assay kit (Thermo Fisher, cat. A22188) according to manufacturer’s protocol. For ROS production by individual Lactobacillus species, fecal Lactobacilli were cultured as described above for 20 hours. Individual colonies were selected and dissociated in 200 uL MRS media and incubated for 30 minutes at 37 °C. The resulting ROS concentration was measured as described above. The identity of each colony was verified using specific primers (Sup. Table 1).

qPCR

For RNA quantification, frozen tissues (brain and intestine) were homogenized by bead beating in RNA TRI Reagent (Life technologies) and RNA was extracted according to manufacturer’s protocol. cDNA was synthethized with the High Capacity cDNA Reverse Transcription kit (Life Technologies). cDNA was amplified using the Sensifast Sybr NO-ROX kit (Bioline), according to manufacturer’s instructions. Gapdh was measured as a normalizer for each sample. Results were analyzed by the relative quantity (ΔΔCt) method64.

For total 16S rRNA quantification, we followed the BactQuant protocol described by Liu et al.65. Briefly, the 16S gene was amplified using primers directed to the V3-V4 rRNA region (Sup. Table 1). L. reuteri DNA was used for the standard curve. For relative quantification of total Lactobacillus or specific Lactobacillus species, the ΔΔCt method was used to compare Lactobacillus-specific amplification to that of the 16S rRNA gene. Reactions were performed using the Sensifast Sybr NoROX kit from Bioline (BIO-98005). Primer sequences are available in Sup. Table 1.

Statistical analysis

All statistical analyses were performed in Prism. The results of the statistical tests are presented within the results section. Analyses involving two groups were performed using a two-tailed t-test. If the variances between groups were significantly different, a Welch’s correction was applied. For experiments involving stress and another variable (e.g. L.reuteri treatment), data were analyzed with a two-way ANOVA. For the metabolomics experiments involving only 3 groups, a one-way ANOVA was utilized. Outliers were excluded if they fell more than two standard deviations from the mean. For all analyses, the threshold for significance was at p < 0.05. Repeats for each experiment, if performed, are specified in the figure legend corresponding to the respective panel.