Animals

Experiments were performed using adult (8–10 weeks old) male C57BL/6j mice purchased from Janvier labs (St Berthevin, France). They were housed in groups of five and maintained in standard conditions (controlled room temperature and humidity, 12 h/12 h light/dark cycle, with lights on at 8:00 AM, ad libitum access to dry food pellets and water) at the Pasteur Institute animal care facility, officially registered for experimental studies on rodents (Approval number for animal care facility: A 75-15-01-6 2014-720). All studies were performed in compliance with the French legislation (Decree of 1 February 2013, 2013-118) and the European Communities Council Directive of 22 September 2010 (2010/63/EEC). All animal experiments were designed according to the 3 R’s rules and approved by the French Ministry of Research (projects CETEA #2013-0062, #2016-0023). Sample size was chosen using the freely downloadable G power software, to ensure adequate statistical power all the while respecting the 3 R principle. Behavioral analyses included 10 subjects per group, cellular analysis included 6 to 8 samples per group and molecular analyses included 5 samples per group.

Unpredictable chronic mild stress (UCMS) procedure

Mice were chronically exposed to interchanging unpredictable mild stressors, administered daily in random order for 8 weeks, as previously described [3, 4, 27, 28]. Two stressors were applied daily, one in the morning and one in the evening, and none of the stressors involved food or water deprivation. The different stressors included cage shaking (5 min), cage tilting 45 °C (2 H), moist bedding (2 H), overnight illumination (12 H), reversing day and night (24 H), restraint (30 min), recurrent cage change (2 H) and exposure to predator odors (rat or fox urine). Behavioral experiments were performed over a period of 1 week, starting at week 8 of the UCMS protocol. At week 9, mice were euthanized and randomly allocated to histological or molecular analyses using a counter-balanced design (see Supplementary Table 1). The control animals were socially housed (5 animals/cage) and left undisturbed except for necessary procedures, including routine cleaning.

Treatments

The antibiotic (ABX) compounds were applied in drinking water during one week and consisted of a mixture of ampicillin (1 mg/ml), streptomycin (5 mg/ml), colistin (1 mg/ml), vancomycin (0.5 mg/ml) and amphotericin (0.1 mg/ml), as previously described [3]. Overall, we did not witness any significant ABX-induced changes in stool consistency/shape, presence of mucus or blood or diarrhoea-induced fur staining. One day prior microbiota transplantation, the ABX treatment was discontinued and replaced by sterile water. The fecal samples used for the transfer protocol were harvested from donor mice (CT or UCMS) the day of the inoculation. The fecal suspension was produced by dissolving 1 mg of feces in 5 ml sterile PBS. The fecal suspension (300 μl per mouse) was delivered by oral gavage 1 and 4 days following ABX discontinuation.

Subdiaphragmatic vagotomy

Subdiaphragmatic vagotomy was performed as previously described [7, 11]. Mice were anesthetized with ketamine/xylazine (10 mg/g BW, Sanofi). The stomach and lower esophagus were gently exposed following a mid-lateral incision of the skin and abdominal wall, and the intestine was retracted to allow access to the stomach. A ligature was placed around the esophagus at its entrance to the stomach to allow for gentle retraction and clearly expose both vagal trunks. These were dissected and all neural and connective tissue surrounding the esophagus below the diaphragm was removed to transect all small vagal branches. A 2-week recovery period was allowed before the behavioral experiments took place.

Experimental sets

A total of 280 mice were used in this study, allocated in six separate experimental sets. All of the data presented herein have been reproduced at least twice in the host laboratory, with some results (for instance, impact of microbiota inoculation on behavior and neurogenesis) being replicated up to four times. All subjects euthanized at belated time-points (3 and 7 weeks post-inoculation) were subjected to behavioral analyses and were then assigned to cellular or molecular analyses by simple randomization. The latter was performed by creating a list of subjects and treatments using the Microsoft Excel’s function RAND. Analyses were performed by an investigator who was blind to the experimental conditions and used numerical coding. Sample size was determined by power analysis, using the G Power software. At least 10 animals per group were included in the behavioral analyses and 5–8 samples per group were included in the molecular and cellular analyses. A detailed schematic representation of all the experimental sets used in this study can be found in Supplementary Table 1.

Behavioral assessment

For all behavioral tests, mice were transferred to the testing room at least 1 H before testing. GM donor mice were tested over a period of 1 week while the animals sustained the last week of UCMS. GM recipient mice were tested upon exit from the isolators. All analyses were performed with the experimenter blind to the experimental condition.

Open field

Animals were placed in white Plexiglas containers (43 × 43 cm) and their behavior was recorded by a video camera during 30 min. A tracking system (Noldus Ethovision 3.0) was used to map center and periphery zones and to calculate the time spent in each zone.

Elevated plus maze

The test was conducted using a plus-cross-shaped apparatus made of black Plexiglas that was elevated 58 cm above the floor and comprised two open and two closed arms (30 × 6 cm) that extended from a central platform (7 × 7 cm). The Noldus Ethovision 3.0 tracking system was used to record behavior for 6 min. The time spent and number of entries in the center, open arm, and closed arm zones were calculated.

Light and dark box

A two-compartment box containing a dark chamber (black walls with upper lid) and a light chamber (300 lux, white Plexiglas walls, no upper lid) was used. The chambers were connected by a 10 × 10 cm door in the middle of the wall. Animals were placed in one corner of the light chamber facing the wall and were allowed to freely explore for 10 min. The Noldus Ethovision 3.0 tracking system was used to record behavior. The number of entries and the total time spent in the light chamber were estimated as a proxy of anxiety-like states.

Sucrose preference test

Mice were given for 24 H a free choice between two bottles, one filled with a sucrose solution (1%, diluted in drinking water) and another one filled with drinking water. To avoid potential bias of side preference in drinking behavior, the position of the bottles was switched after 12 H. The consumption of water and sucrose solution was calculated by weighing the bottles. Sucrose preference was measured as the percentage of consumed sucrose solution to the total amount of solution consumed (sucrose and water).

Novelty suppressed feeding test

The test container used was a white plastic box (50 × 50 × 20 cm) whose floor was covered by wooden bedding. Twenty-four hours prior testing, all food was removed from the home cage. A single food pellet (regular chow) was adhered on a piece of Whatman paper and positioned in the center of the container that was brightly illuminated. The latency to eat was measured during 10 min. At the end of each session, the animals were transferred to their home cage and the amount of food consumed over the subsequent 5-min period was measured as a control of feeding drive.

Tail suspension test

Mice were suspended at approximately one-third from the end of the tail, using regular tape, to an aluminum bar connected to a strain gauge. Tape was affixed to the mouse’s tail 2 cm from the tip and the mouse was suspended from a metal rod at a height of 30 cm. The test was recorded during a 5-min period. Upon viewing of the video recordings, the total time spent in an immobile posture was measured. Mice were considered immobile when they stopped struggling to escape and hung passively, motionless, by the tail.

Forced swim test

Mice were placed into a clear Plexiglas cylinder (25 cm in height and 10 cm in diameter) filled with water (22 °C) for a 5-min test session. The duration of immobility was measured upon viewing of the video recordings. Immobility was defined as the lack of active movements except from those required for floating.

Tissue collection

Mice allocated to immunofluorescence studies were deeply anesthetized with a mixture of xylazine-ketamine (10 mg/g bw, Sanofi) and were perfused transcardially with a solution containing 0.9% NaCl, followed by 4% paraformaldehyde (PFA, 4 °C) in phosphate buffer (pH 7.3). Animals were sacrificed in randomized order to minimize experimental bias. Mice were then decapitated and the brain was carefully removed. Mice allocated to molecular biology analysis, were deeply anesthetized with xylazine-ketamine (10 mg/g bw, Sanofi). The whole HPC was carefully dissected, immediately snap-frozen in liquid-nitrogen, and maintained at −80 °C until further processing. Fecal samples were harvested at the end of the UCMS protocol in donor mice and upon exit from the isolators in recipient mice.

Quantitative RT-qPCR

Total RNA was extracted from brain tissue and cDNA was synthesized using a Cells-to-Ct Kit (Applied Biosystems) according to the manufacturer’s instructions. Real-time PCR was performed using the SYBR Green Master Mix (Applied Biosystems) and products were detected on an Applied Biosystems ViiA 7 Real-Time PCR System. Relative expression of Th, Tph2, Ddc, GluR1, Gls1, Gls2, Grid1, Gad1, Gad2, Gabra2 and Gabrb2 was calculated using the 2(11 C.t/) method. Conditions for real-time PCR were: initial denaturation for 10 min at 95 °C, followed by amplification cycles with 15 s at 95 °C, and 1 min at 60 °C.

Western blots

Brain tissue was homogenized in RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Pierce Thermo Scientific), supplemented with protease (cOmplete, Sigma) and phosphatase (phosSTOP, Sigma) inhibitors. Protein concentration was measured with a Pierce BCA protein Assay Kit (ThermoFischer Scientific) prior to the Western blot assay. Tissue lysates were mixed with 4× NuPage LDS loading buffer (Invitrogen) and reducing agent (Invitrogen NP0004), and proteins were separated on a 12% SDS-polyacrylamide gel (Invitrogen NP0329) and subsequently transferred by semi-dry or liquid transfer onto a PVDF membrane (Trans-blot Turbo Mini PVDF, Biorad). The blots were blocked in 5% BSA in Tris-buffered saline with Tween (TBS-T) and incubated with the primary antibodies, including rabbit polyclonal anti-cFos (Merck, Cat No. ABE457), goat anti-COX2 (abcam, Cat No. ab179800), mouse monoclonal anti-CREB-1 (Santa-Cruz, Cat No. sc-240), rabbit polyclonal anti-phosphoCREB (Merck, Cat No SAB1306301), rabbit polyclonal anti-Cx3cr1 (abcam, Cat No. ab8020), chicken polyclonal anti-DCX (abcam), rabbit polyclonal anti-DCX (abcam, Cat No ab153668), rabbit polyclonal anti-Iba1 (Wako, Cat No 019-19741), mouse polyclonal anti-IL1β/IL-1F2 (R&D Systems, Cat No AF-401-SP), rabbit polyclonal anti-IL6 (abcam, Cat No. ab208113), rabbit polyclonal anti-Ki67 (abcam, Cat No. ab15580), mouse monoclonal anti-Sox2 (abcam, Cat No. ab79351), rabbit polyclonal anti-TGFβ 1 (abcam, Cat No. ab92486), rabbit polyclonal anti-TNFα (abcam, Cat No. ab66579). To detect protein signal, the following Horseradish peroxidase–conjugated secondary antibodies were used: Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (1:6000, Biorad, Cat No. 1706515) and Goat Anti-Mouse IgG1 heavy chain (HRP) (1:6000, abcam, Cat No. ab97240) and rabbit anti-goat IgG (H + L)-HRP (1:6000, Invitrogen, Cat No. 31402). Chemiluminescence detection of proteins was performed with Luminata Crescendo Western HRP Substrate (Merck Millipore) in a Chemidoc Imaging System (Biorad). Bands were quantified using the Image Lab software.

Immunofluorescence

Perfused brains were cut at 40-micron thick coronal sections using a vibrating microtome (VT1000S, Leica). Immunostaining was performed on free-floating sections. Non-specific staining was blocked by 0.25% Triton and 10% donkey serum albumin (Sigma-Aldrich). Sections were incubated overnight with the following primary antibodies at 4 °C: rabbit anti-DCX (1:400, abcam, Cat No. ab18723), chicken anti-DCX (1:400, abcam, Cat No. ab1536668), rabbit anti-Ki67 (1:200, abcam, Cat No. ab15580), rabbit c-Fos (1/1500, Merck-Millipore, Cat No. ABE457). Sections were incubated with secondary antibodies (Alexa-conjugated secondary antibodies at 1:800 dilution, Jackson ImmunoResearch Laboratories) for 1 H at room temperature. Fluorescent sections were stained with the nuclear dye Hoechst and then mounted using the Fluoromount aqueous mounting medium (Sigma-Aldrich).

Imaging and quantification

Images were acquired using a confocal laser-scanning microscope (LSM 710, Zeiss) and an Apotome, with Zen Imaging softwares (Zeiss). Z-stacks of the dentate gyrus were obtained (step size: 0.5–1 μm) using sequential scanning. Cell counting was performed either manually or by using the Icy open-source platform (http://www.icy.bioimageanalysis.org). Values are expressed as the mean number of immune-positive cell counts in 8–10 sections per animal.

Microbial extraction and gut microbiota profiling

Fecal samples were harvested 7 weeks post-inoculation. They were collected in autoclaved Eppendorf tubes and stored at −80 °C until further processing. Cellular DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Cat# 51504) according to the manufacturer’s instructions and including a step of bead beading to optimize the DNA outcome. The DNA was amplified by means of PCR, using primers specific to the V3 region of the 16 S rRNA gene. Amplicons were randomized and thereafter analyzed by denaturing gradient gel electrophoresis (DGGE) using an acrylamide gel containing a 25–65% chemical gradient (urea and formamide) that enabled separation of the PCR amplicons based on sequence differences in the V3 region. The DGGE profiles were analyzed in Bionumerics Version 4.5 (Applied Maths) using a mix of DNA profiles as a marker.

16S sequencing data analysis

Sequences were clustered into operational taxonomic units (OTU) and annotated with the MASQUE pipeline (https://github.com/aghozlane/masque). The final list of 16S rRNA targeted amplicons were converted into the negative logarithm of the expressions of the bacterial taxa (phylum and family). Final data were expressed as the average of the negative logarithm of OTU expression across experimental conditions. All data indexing, segregation and plotting were performed using custom-made scripts in Matlab (Mathworks Inc., 2015 release). Further statistical tests were conducted in the Prism software (GraphPad, version 6, San Diego, USA). The microbiota profiles obtained were further characterized using principal coordinate analysis (PCoA), relative abundance plots and Shannon Index as previously described [3]. Codes are available upon request: https://github.com/SohamSahaNeuroscience/Microbiota-analysis.

Statistical analysis

All data were expressed as mean ± SEM. Statistical analyses were performed using the Prism software (GraphPad, version 9), with p < 0.05 considered statistically significant. Data were analyzed using unpaired two-tailed Mann-Whitney test or the two-way ANOVA repeated measures followed by Bonferroni post-hoc test when appropriate. PCA analysis was performed using the Permanova test. Data met the assumptions of the tests applied and variance was similar across the different groups in different experimental conditions. Subjects were excluded from behavioral assessment when they were not fit or able to finish the trial (for instance risk of drowning in the swim tank or managing to detach the tail from the tail suspension test). Outliers were excluded using the ROUT method (GraphPad Prism, version 9). A detailed statistical analysis is presented in the Supplementary Materials.