Study design

Male and female BALB/c mice (breeding pairs), 6–8 weeks old, were acquired from Charles River (Montreal, QC, Canada) and allowed to acclimatize in the housing facility for at least 1 week. Breeding of mice was organized as follows: a single female mouse was placed in the male’s cage for 48 h. Pregnancy was confirmed by increased weight (>3 g within 8 days following mating). One week before delivery, pregnant females were housed singly with nesting material and treatment (Tx) was started (number of days of Tx before delivery=7.3±1.1). Pregnant females were treated with either drinking water (control group defined as CT, n=5) or penicillin V (antibiotic group defined as AB, n=4) or penicillin V and Lactobacillus rhamnosus JB-1 (defined as AB/JB1, n=3) until weaning of pups (postnatal day 21). Pups were therefore exposed to the treatment prenatally and during the early postnatal life. This period is referred to as ‘early life’ in the description of the results. Since the dams were the animals treated and not the pups, it was impossible to randomly assign the pups to a treatment group. At weaning, male and female offspring were separated from the dams and housed 3–5 per cage and received regular drinking water and standard rodent chow ad libitum. A battery of behavioural tests was started when the offspring reached 6-weeks old (postnatal day 42), with 2 days of rest between each test. The microdefeat was performed at the end of the experiment, only in males. Mice were killed by decapitation the day following the last test in order to collect trunk blood and tissue samples. A total of 72 mice were used in this study (Fig. 1). Sample size of this exploratory study could not be calculated here as the effect size was unknown. For the microdefeat paradigm, male CD-1 retired breeders were obtained from Charles River. As per our ethical approval, only male mice were used in the social defeat experiments. All animals were housed under 12 h light/12 h dark cycle. All experiments followed the guidelines of the Canadian Council on Animal Care and were approved by the McMaster Animal Research Ethics Board.

Treatment with antibiotic and L. rhamnosus JB-1

Dams received AB and L. rhamnosus JB-1 treatments through the drinking water in order to avoid stress induced by oral gavage. AB treatment consisted in penicillin V (Sigma-Aldrich, MO, USA) that is absorbable by the gastro-intestinal tract66 and found in breast milk39. Penicillin V crosses placenta and passes into fetal circulation47. Dams received a low dose of penicillin V that, in our opinion, is clinically relevant (50,000 U kg−1 per day or 31 mg kg−1 per day)47,67. Since water consumption increases significantly during the gestational and lactating periods and is influenced by the number of pups per litter, the dose of penicillin V was adjusted to body weight and water consumption of dams, measured twice weekly. L. rhamnosus JB-1 was obtained as a gift from Alimentary Health, Cork, Ireland and is the same bacterial strain we have used previously32. It was also administered in drinking water (109 c.f.u. per day) in which it remains viable for more than 12 h. Dams from the AB group received regular water during the day (9:00 to 18:00) and penicillin V during the night (18:00 to 9:00). Dams from AB/JB1 group received L. rhamnosus JB-1 during the day and penicillin V during the night. This schedule, reported in a previous study27, has been used to avoid the adverse effect of AB on the bacteria. We found using quantitative PCR that L. rhamnosus was detectable in faeces of all dams before treatment and only in dams of the AB/JB1 group after treatment (Supplementary Fig. 20), proving that the protocol using two separated drinking bottles alternately, is efficient in maintaining the presence of some L. rhamnosus in the gut. All bottles of water containing bacteria were shaken three times per day.

Behavioural testing

All behavioural tests were recorded by a video camera or connected to a computer and data analysis was performed by using Motor Monitor software (Kinder Scientific, Poway, CA) or EthoVision XT software (Noldus, Leesburg, VA). The experimenter was not blinded to the group allocation when assessing behaviour.

Open field. Mice were tested during the dark phase under dim-light conditions. After a 1 h period of habituation in the testing room, animals were placed in the enclosure for 30 min. Distance moved in the open field and rearing were recorded using Motor Monitor software. The apparatus was thoroughly cleaned with water and dried between each animal.

Three-chamber sociability test. Mice were tested in the three-chamber apparatus during the light phase, following a 30 min period of habituation in the testing room. The apparatus consists of three rectangular Plexiglas chambers whose dividing walls possess small openings that allow access into each chamber. The experimental mouse was first placed in the centre chamber while the doorways were closed and allowed to explore for a 5-min habituation period. In the second phase of the test, an unfamiliar mouse (strain- and sex-matched) was placed within an inverted wire cup in one of the outer chambers while an empty wire cup was placed in the other outer chamber. The doors to the outer chambers were opened and the sociability trial was conducted for 10 min during which the experimental mouse was allowed to explore the three chambers. Time spent in each chamber was recorded by a video camera positioned over the apparatus and analysed by using EthoVision XT software. Sociability is defined as the experimental mouse spending more time in the chamber containing the novel mouse than in the chamber containing the novel object. In the third phase of the test, a second novel mouse (stranger 2) is placed inside the previously empty wire cup while the initial novel mouse (stranger 1) remains inside its cup. The experimental mouse is given 10 min to explore all the three chambers. Preference for social novelty is defined as more time spent in the chamber with stranger 2 than time spent in the chamber with stranger 1. The apparatus was thoroughly cleaned with water and dried between each animal.

Elevated plus maze. Mice were tested in the EPM apparatus that is elevated at 76 cm off the ground, consists of four arms—two open arms and two closed arms made with black Plexiglas walls. Mice were transported to the behavioural testing room for a 30 min habituation period. The mouse was then placed in the intersection of the four arms, facing open arm, and allowed to explore for 5 min. The EPM was connected to a computer and behavioural data were analysed by using Motor Monitor software. Time spent, distance travelled and number of entries in the open arms were used to assess anxiety-like behaviour. The apparatus was thoroughly cleaned with water and dried between each animal.

Microdefeat stress and social avoidance test. The microdefeat paradigm is a short (acute) version adapted from the chronic social defeat described by Krishnan et al.68 and by Golden et al.50.The microdefeat is a model that was developed initially to measure increased susceptibility to stress. It can reveal susceptible phenotype if an animal’s stress threshold is shifted by the experimental manipulation. The microdefeat has been described in C57BL/6 male mice only and under control condition, this protocol does not induce social avoidance. There are currently no widely accepted versions of microdefeat in females. In our study, microdefeat has been performed in male BALB/c as follows. First, CD1 aggressors (retired breeders >4–5 months old) were screened for consistent attack latencies (<60 s on three consecutive screening sessions with BALB/c intruders called screeners). CD-1 mice were singly housed throughout the experiment. Successful application of social defeat stress is dependent on appropriate selection of CD-1 mice with consistent levels of aggressive behaviour. Within the same day, BALB/c mice are forced to intrude into the space territorialized by the larger and aggressive CD-1 mouse for three sessions of 3 min, followed by a rest period 15 min in the home cage between each exposure. This situation leads to intruder aggression and subordination. After the last resident–intruder session, BALB/c mice are housed singly (with free access of food and water) and a wound score (0: absent, 1: light, 2: moderate, 3: severe) was assigned. BALB/c mice were tested for social avoidance 24 h later. Social avoidance test was performed in a rectangular Plexiglass box, where an unfamiliar CD-1 aggressor was placed under a wire cage at one end of the arena. The box was virtually split into two parts, an interaction zone (close to the wire cage) and a non-interaction zone at the other end of the arena. The BALB/c mouse was allowed to explore the arena for a first trial of 150 s when the aggressor was absent (empty wire cage) and then for a second period of 150 s when the aggressor was present. A social interaction ratio was calculated as follows: 100 × (time spent in the interaction zone when the aggressor is present/time spent in the interaction zone when the aggressor is absent). Mice with an interaction ratio <100 were considered susceptible to stress while mice with an interaction ratio >100 were resilient to stress. Results were analysed by using EthoVision XT software.

RNA extraction and RT–qPCR analysis

After decapitation of animals, gut (distal ileum and colon) and brain (frontal cortex and hippocampus) were quickly dissected and put into RNAlater solution (Ambion, Life Technologies, CA, USA). Tissues were incubated overnight at 4 °C then transferred at −20 °C until further processed. Following tissue homogenization, total RNA was extracted using TRIzol Reagent (Ambion, Life Technologies). One microgram RNA was then converted into cDNA using SuperscriptIII First-Strand Synthesis Supermix (Invitrogen, CA, USA). Diluted or non-diluted cDNA was used as template for qPCR reaction using PowerUp SYBR Green Master Mix (Applied Biosystem, Life Techologies, TX, USA) containing ROX dye Passive Reference. The qPCR reactions were performed in the fast mode (UDG activation 50 °C, 2 min; Dual-Lock DNA polymerase 95 °C, 2 min; denaturation: 95 °C, 1 s; annealing/extension 60 °C, 30 s; number of cycles: 40–50) by using QuanStudio3 machine (Applied Biosystem). Data were normalized to the endogenous control GAPDH and the relative quantification was analysed using the ΔΔCt method. The experimental treatments did not affect GAPDH expression in any of the tested tissues (Supplementary Fig. 21). Primers were designed with Primer Express Software and used at a concentration of 300 nM. Primer sequences are listed in Supplementary Table 6.

Western blotting

Brain tissues (hippocampus and frontal cortex) were quickly dissected following decapitation and stored at −80 °C. Samples were homogenized and lysed (45 min on ice) in protein lysis buffer (100 μl per 10 mg; 0.05 M Tris-HCl, 0.15 M Nacl, pH 7.4) containing 1% Triton X-100, 1 mM EDTA, 10 mM NaF, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM Na 3 VO 4 and protease inhibitor cocktail (1 tablet per 10 ml, cOmplete Roche, Sigma-Aldrich) and centrifuged at 15,000g for 10 min at 4 °C. The supernatant was collected and protein concentration was measured by the Lowry method (DC Protein Assay, Biorad). Proteins (20 μg) were fractionated on a 12% SDS–polyacrylamide gel electrophoresis gel and transferred to 0.2 μm polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Germany). Membranes were blocked in 0.1% Tween-TBS 20 with 5% nonfat dry milk for 1 h at room temperature, then probed with rabbit antibodies to occludin (Invitrogen 40–4700, 1:1,000, overnight, 4 °C), claudin-5 (Invitrogen 34-1600, 1:1,000, overnight, 4 °C) or β-actin (Bioss bs-0061R, 1:5,000, 2 h, room temperature). After extensive washing, membranes were incubated for 1 h at room temperature with a goat anti-rabbit IgG-peroxidase secondary antibody (Invitrogen A0454, 1:20,000), then visualized with Amersham ECL western blotting detection reagents (GE Healthcare Life Sciences) and developed in the dark room. Densitometry was quantified with Image J69.

Assessment of intestinal barrier permeability

Evaluation of intestinal barrier integrity was performed by measuring the mRNA expression of tight junction proteins occludin and zonula-occludens 1 (ZO-1), two key markers of paracellular permeability70, in ileum and colon and by the faecal albumin concentration. The latter is a good indicator of disrupted intestinal barrier function and has been shown to be comparable and correlate well with the orally administered FITC-dextran method71. Faecal albumin was determined by ELISA (Bethyl Labs, TX, USA) following manufacturer’s guidelines.

Serum cytokine assays

Trunk blood was collected into sterile tubes, allowed to clot at room temperature, spun down for 10 min at 850g and serum was stored at −80 °C. Cytokines were analysed by using the mouse cytokine 32-plex discovery assay (Eve technologies, AB, Canada).

16S rRNA gene sequence analysis

Stool samples were collected in sterile tubes and stored at −80 °C until further analysis. DNA from 176 stool samples was extracted using the PowerSoil HTP DNA Isolation Kit (MoBio, USA) according to the manufacturer’s instructions with a beadbeater (BioSpec, USA) set on high for 2 min. Following DNA extraction, the V4 variable region of the bacterial 16S rRNA gene was amplified by PCR using the 515F and 806R (each well received a separate 515F barcoded primer). 515F- (barcode) 5′-AATGATACGGCGACCACCGAGATCTACACGCTAGCCTTCGTCGCTATGGTAATTGTGTGYCAGCMGCCGCGGTAA-3′ and 806R 5′-CAAGCAGAAGACGGCATACGAGATAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT-3′ (ref. 72). PCR reactions were carried out with the Primestar taq polymerase (Takara, Japan) for 30 cycles of denaturation (95 °C), annealing (55 °C) and extension (72 °C), and a final elongation at 72 °C. Products were purified using AMPure magnetic beads (Beckman Coulter, USA) and quantified using Pico-green dsDNA quantitation kit (Invitrogen, USA). Samples were then pooled at equal concentrations (50 ng μl−1), loaded on 2% E-Gel (Thermo Fisher, USA) and purified using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Germany). Purified products were sequenced using the Illumina MiSeq platform (Genomic Center, Faculty of Medicine, BIU, Israel). Data analysis was performed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline version 1.8.0 (refs 72, 73). Paired-end sequences were joined using fastq-join, demultiplexed and quality filtered with an average quality threshold of 25. Chimeric sequences were identified using USEARCH and removed, and reads were clustered into OTUs using the open reference UCLUST method against the GreenGenes 08/13 database74, with a cutoff of 97% sequence identity. Core OTUs were calculated by filtering for OTUs presents in at least 50% of subjects in the same treatment group. Analyses were performed on the core OTUs using a rarefied table of 10,200 sequences per sample. In addition, alpha diversity was estimated using Faith’s phylogenetic diversity (PD whole tree) and beta diversity was calculated using weighted and unweighted UniFrac75. Significance between groups for distances was assessed using t-tests as implemented in ‘make_distances_boxplots.py’ script in QIIME 1.8.0.

Statistical analysis

Results of behavioural tests were analysed by a two-way ANOVA with sex (male versus female) and treatment (CT, AB, AB/JB1) as between factors. Homogeneity of variance was tested with Levene’s test. When the sex × treatment interaction was significant, post hoc tests using Bonferroni correction were performed. Analysis of the bacterial relative abundance in dams and offspring was performed by using mixed ANOVA with time (T0, T1, T2 or PND21, PND42) as a within-factor (repeated measure) and treatment (CT, AB, AB/JB1) as a between-factor. For the dams, assumption of sphericity was checked with Mauchly’s test and Greenhouse–Geisser correction was applied to produce a valid F-ratio when condition of sphericity was not met. For the offspring, homogeneity of variances for each combination of the groups (time and treatment) was checked with Levene’s test. If the time × treatment interaction was significant, post hoc tests were performed with Bonferonni correction (when homogeneity of variances was met) or with Games–Howell procedure (when homogeneity of variances was not assumed). Significant outliers were identified graphically (with boxplot) and by using the ‘studentized residual’. Since males have been subjected to the social defeat while females have not been stressed, males and females were analysed separately regarding the biological assays (serum, gut and brain samples) by using one-way ANOVA (or Kruskal–Wallis test if assumptions of normality and homoscedasticity were not met). Tukey post hoc tests were performed for pairwise comparisons. Results were considered statistically significant when P<0.05. All tests were two-tailed. Statistics were performed with SPSS 23.0.

Data availability

The 16S rRNA gene sequence data have been deposited in the European Bioinformatics Institute (EBI) database with accession code ERP021539. The authors declare that all other relevant data supporting the findings of this study are available within the paper and its Supplementary Information files, or from the corresponding authors on request.