Animals

Six C57Bl/6 Jdams with litters (3 male and 3 female 14-day-old pups) were purchased from Charles River Laboratories. Mice were housed in ventilated transparent OptiMouse plastic cages with Bed-O-Cobs® and AlphaDri bedding (35.6 × 48.5 × 21.8 cm; at Georgia State University). Lights were set to a 14 h:10 h light:dark cycle (lights off at 0900 ET), and ambient temperature was maintained at 23 °C. Food (Purina rodent chow no. 5001) and water were available ad libitum. On postnatal day 21 (P21), mice were weighed and placed in a plastic container for approximately 20 minutes to collect feces for later analysis. Mice were put into a new cage such that each experimental group contained mice from all litters and that offspring from all six dams were used in all test and measures performed, except for one male in the water-treated group who developed a severe mal-occulsion and needed to be euthanized during the course of the experiment (Supplement Fig. 1). Cages were given reverse-osmosis treated Atlanta city drinking water with sodium carboxymethylcellulose (CMC; Sigma, St. Louis, MO), or with polysorbate-80 (P80; Sigma) (1% in each case), or with no additives. The drinking water and emulsifier solutions were changed weekly. Body weights were measured weekly and expressed relative to the body weight on P21. All procedures were in accordance with the Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee at the Georgia State University (protocol number A15002).

Behavioral Testing

Starting on P70, behavioral tests were conducted once a week for 6 weeks, with a week between each test (Supplemental Fig. 1). Behavioral tests were conducted in the following order: Open Field Test, Elevated Plus Maze, Light/Dark Box, Marble Burying Task, Three-Chambered Sociability Task, and Porsalt Forced Swim Test. As behavioral responses can be altered by prior test history, the tests were conducted in order of least to most invasive or disruptive41,42. In order to avoid shifts in the circadian rhythms of the experimental animals, behavioral testing occurred within the first 4 h after the start of the dark and active phase and was conducted under dim red light (between 20–30 lux) except for the Light/Dark box, which was illuminated by overhead lights (between 300–400 lux). Arenas were cleaned with 70% ethanol between trials. Behavioral tests were videotaped using a Sony camcorder for later analysis by AnyMaze version 4.96 (Stoelting, Co., Wood Dale, IL) or The Observer version XT11 (Noldus Information Technology Inc., Wageningen, The Netherlands). An experimenter blinded to the treatment conditions scored behavioral tests in the Observer.

Open Field Test

Locomotor behavior was assessed in a 43.2 × 43.2 × 30.5 cm (WxLxH) Plexiglas arena (Med Associates, Inc., St. Albans, VT) containing 2 arrays of infrared transmitters strips (16 beams each) located on the bottom of the arena (in the X and Y plane). The center zone of the arena was defined as square containing the center 8 beams (e.g., beams 4–12) in the X and Y plane. Each mouse was placed into the arena with its nose facing the wall and allowed to freely investigate for 10 min. The total distance traveled, the total time spent in the center of the arena, and circling behaviors, which are defined as movements below a preset ambulatory threshold, were calculated by Activity Monitor (Med Associates, Inc.,) on a computer connected to the open field arenas.

Elevated Plus Maze

An elevated plus maze with two open and two closed arms was used. Arms were 10 cm W × 50 cm L, connected by a 10 × 10 cm2 center chamber. Closed arms had 40 cm H walls. The maze was elevated 50 cm from the floor. Mice were placed in the center of the arena and allowed to explore for 5 min. All trials were video-recorded from a digital camera mounted above the maze and connected to a computer. The number of entries into and the total time spent in the open arms, closed arms, or center were quantified by AnyMaze.

Light/dark Box

A 14.5 cm W × 30 cm L × 14 cm H chamber divided into a light and dark compartment was used. The light compartment (20 cm L) was made of white acrylic, and the dark compartment (10 cm L) of opaque black acrylic and covered. An opaque insert with a 5 cm W × 5 cm H opening connected the compartments. Mice were placed in the light compartment facing away from the entry into the dark chamber and allowed to freely investigate the chamber for 5 min. All trials were video recorded from a digital camera mounted above the Light/Dark box and connected to a computer. The number of entries into the dark chamber and total time spent in the light compartment were quantified in AnyMaze.

Marble Burying

A Plexiglas arena (24 cm W × 46 cm L) was filled 4 cm deep with Alpha-dri bedding (Shepherd Specialty Paper, Fibercore, Cleveland, OH, USA). Mice were placed into the arena, and after a 5 min habituation period, mice were removed and twenty marbles (17 mm) were evenly spaced on top of the bedding. Mice were placed in the center of the arena and video-recorded for 10 min. The number of marbles buried, as defined by being ½ or more covered with bedding, and the latency to bury the first marble were quantified using the Observer.

Three Chambered Sociability

A 24 × 74 × 24 cm (L × W × H) polycarbonate apparatus was divided into three equally sized chambers with openings 9 cm W to allow free movements between compartments. At either end of the apparatus was an (9 cm W × 10 cm H) opening beside which the stimulus cages were placed. The stimulus cages were 10 cm W × 10 cm L × 10 cm H polycarbonate cage with grid (10 × 10) of small holes 0.5 cm in diameter to allow transfer of visual and olfactory cues, while limiting physical interaction to nose contact or whisking.

Sociability test

Following a 5 min habituation period in which the mouse was allowed to explore the entire three-chambered apparatus, the experimental animal was removed, and an unfamiliar sex- and age-matched C57Bl6/J mouse was placed inside one of the stimulus cages beside one of the side chambers. An identical stimulus cage containing a novel object was placed beside the opposite chamber. The test animal was returned to the middle chamber and allowed to freely investigate the apparatus for 10 min.

The location of the novel mouse and the novel object were alternated between left and right chambers on consecutive sessions. The time spent and the numbers of entries into each chamber were measured using AnyMaze. The time spent sniffing or actively investigating the stimulus chambers over the 10 min test was scored in The Observer. A preference score was calculated by dividing the time spent investigating the novel mouse by the total time spent investigating the novel mouse and the novel object.

Social Preference test

Immediately following the 10 min sociability test, the experimental mouse was removed from the three-chambered apparatus, and the novel object was replaced with an unfamiliar stimulus sex- and age- matched C57Bl6/J mouse. The original stimulus mouse used in the sociability portion of the test remained in its cage beside one chamber of the apparatus. Identical measures were scored as in the sociability test: time spent in each chamber, entries between chambers, and time spent investigating each stimulus mouse.

Porsolt Forced Swim Test

A vertical Plexiglas cylinder (40 cm H × 18 cm diameter) was filled with 3 L of 30 °C (±2 °C) water. Mice were placed in the center of the cylinder and video recorded for 5 mins. The latency and duration of immobility were quantified in the Observer. Immobility was defined by the absence of movement or only small movements of posterior paws that did not result in displacement of the water. At the end of the test, mice were removed from the cylinder and placed in a recovery cage on a heating pad until they were dry and then returned to their home cage.

Euthanasia and Tissue Collections

One day following completion of behavioral testing (P105), mice were deeply anesthetized under isoflurane (5%v/v) and body weight was recorded. Blood was collected from the retrobulbar intraorbital capillary plexus. Mice were euthanized by cervical dislocation, and the colons, spleens, livers, adipose, feces, and brains were collected for subsequent analysis. Hemolysis-free serum was generated by centrifugation blood samples using serum separator tubes (Becton Dickinson, Franklin Lakes, NJ). The weight and length of the colon and weights of the spleen, liver, and perigonadal adipose fat depot were recorded and normalized to the body weight. Brains were removed and fixed in a 5% acrolein in sodium phosphate buffer (0.1 M, pH 7.4) at 4 °C, followed by cryoprotection in 30% sucrose in phosphate buffered saline (PBS: 0.05 M, ph7.4).

Immunohistochemistry

Brains were sectioned (30 µm) in the coronal plane with a cryostat and stored in a cryoprotectant solution (ethylene glycol/sucrose in sodium phosphate buffer) until immunostained. Free-floating sections were rinsed three times in Tris-buffered saline (TBS; 0.05 M Tris, 0,9% NaCl, pH 7.6), then incubated for 30 min in 0.05 M sodium citrate in TBS. After rinsing in TBS sections were places for 30 min in 0.1 M glycine in TBS, rinsed again, and placed into blocking solution (10% normal goat serum (NGS), 0.4% Triton-X and 1% H2O2 in TBS) for 30 min. Sections were incubated overnight in one of the following primary antibodies diluted in 2% NGS and 0.4% Triton-X in TBS; anti-agouti-related peptide (AgRP; Phoenix Pharmaceuticals; 1:250000), anti-alpha-melanocyte stimulating hormone (MSH; Millipore; 1:100000), and anti-ionized calcium-binding adaptor protein (Iba1; Wako Laboratoy; 1:30000). The next day, sections were rinsed three times in TBS containing 1% NGS and 0.02% Triton-X and incubated in biotinylated secondary antiserum [goat anti-rabbit for AgRP, and Iba1 immunoreactivity; rabbit anti-sheep for MSH (Vector Laboratories, Burlingame, CA)] diluted 1:800 in TBS with 2% NGS and 0.32% Triton-X for 1 h. This was followed by rinses in TBS containing 0.4% Triton X, incubated in avidin-biotin complex (Vectastain Elite ABC Kit; Vector Laboratories) diluted to 1:800 in TBS for 1 h, followed by three TBS rinses and three sodium acetate buffer rinses. Finally, the staining was visualized using nickel-enhanced diaminobenzidine (DAB) Substrate Kit (Vector Laboratories). Sections were mounted onto gelatin-coated slides and coverslipped with Permount.

Image Analysis

Slides were anatomically-matched and analyzed by an investigator blinded to the experimental groups. Sections were imaged using a Zeiss Axio Imager M2 microscope connected to an ORCA-R2 CCD digital camera (Hamamatsu Photonics). Gray-scale images of cell bodies, when present, and fibers immunopositive for AgRP, MSH, and AVP (Supplemental Fig. 6) were analyzed in Image J 1.43 u (National Institutes of Health, Bethesda, MD). Iba-immunoreactivity was analyzed as in43. The region of analysis was outlined in each section. Subjects for which the relevant sections were damaged or unavailable were dropped from a given analysis. All immunoreactivity is quantified by the mean area stained in pixels.

Fecal microbiota 16s rRNA gene sequencing and sequences analysis

16S rRNA gene amplification and sequencing were done using the Illumina MiSeq technology following the protocol of Earth Microbiome Project with their modifications to the MOBIO PowerSoil DNA Isolation Kit procedure for extracting DNA (www.earthmicrobiome.org/emp-standard-protocols)44,45. Bulk DNA was extracted from feces collected on P21 and P105 using a PowerSoil-htp kit from MoBio Laboratories (Carlsbad, CA, USA) with mechanical disruption (bead-beating). The 16S rRNA genes, region V4, were PCR amplified from each sample using a composite forward primer and a reverse primer containing a unique 12-base barcode, designed using the Golay error-correcting scheme, which was used to tag PCR products from respective samples45. We used the forward primer 515 F 5′-AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGT GTGCCAGCMGCCGCGG TAA -3′: the italicized sequence is the 5′ Illumina adapter B, the bold sequence is the primer pad, the italicized and bold sequence is the primer linker and the underlined sequence is the conserved bacterial primer 515 F. The reverse primer 806 R used was 5′-CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT -3′: the italicized sequence is the 3′ reverse complement sequence of Illumina adapter, the 12X sequence is the golay barcode, the bold sequence is the primer pad, the italicized and bold sequence is the primer linker and the underlined sequence is the conserved bacterial primer 806 R. PCR reactions consisted of Hot Master PCR mix (Quantabio, Beverly, MA, USA), 0.2 μM of each primer, 10–100 ng template, and reaction conditions were 3 min at 95 °C, followed by 30 cycles of 45 s at 95 °C, 60 s at 50 °C and 90 s at 72 °C on a Biorad thermocycler. PCRs products were purified with Ampure magnetic purification beads (Agencourt, Brea, CA, USA), and visualized by gel electrophoresis. Products were then quantified (BIOTEK Fluorescence Spectrophotometer) using Quant-iT PicoGreen dsDNA assay. A master DNA pool was generated from the purified products in equimolar ratios. The pooled products were quantified using Quant-iT PicoGreen dsDNA assay and then sequenced using an Illumina MiSeq sequencer (paired-end reads, 2 × 250 bp) at Cornell University, Ithaca.

Forward and reverse Illumina reads were joined using the fastq-join method46,47, sequences were demultiplexed, quality filtered using Quantitative Insights Into Microbial Ecology (QIIME, version 1.8.0) software package48. QIIME default parameters were used for quality filtering (reads truncated at first low-quality base and excluded if: (1) there were more than three consecutive low quality base calls (2), less than 75% of read length was consecutive high-quality base calls (3), at least one uncalled base was present (4), more than 1.5 errors were present in the bar code (5), any Phred qualities were below 20, or (6) the length was less than 75 bases). Sequences were assigned to operational taxonomic units (OTUs) using UCLUST algorithm49 with a 97% threshold of pairwise identity, and classified taxonomically using the Greengenes reference database 13_850. A single representative sequence for each OTU was aligned and a phylogenetic tree was built using FastTree51. The phylogenetic tree was used for computing the unweighted UniFrac distances between samples52,53. rarefaction were performed and used to compare abundances of OTUs across samples. Principal coordinates analysis (PCoA) plots were used to assess the variation between experimental group (beta diversity). Alpha diversity curves were determined for all samples using the determination of the number of observed species. LEfSE (LDA Effect Size) was used to investigate bacterial members that drive differences between groups54. The threshold on the logarithmic LDA score for discriminative features was set to 2.0, and the alpha values for the factorial Kruskal-Wallis test and pairwise Wilcoxon test between subclasses were set to 0.05.

In addition to fecal samples collected from the animals used in this current study, the 16s sequences previously generated from26 were reanalyzed by combining gene sequences from both male and female mice treated with water (male: 12; female: 12), CMC (male: 11; female: 12), and P80 (male: 10; female: 9).

Statistical analyses

Data were analyzed using IBM SPSS Statistics Version 21 (IBM) and visualized using GraphPad Prism 7.0c (GraphPad Software, La Jolla, CA). Body weights were analyzed by a repeated measure ANOVA, with sex and treatment as factors, followed by Fishers’ LSD as post hoc analyses. Anxiety-like and social behaviors were analyzed by a two-way ANOVA with treatment and sex as the factors, followed by Fishers’ LSD as post hoc analyses. Data were also analyzed by MANOVA, following by discriminant analysis in order to reveal patterns in the aggregate behavioral changes. Differences in the post-hoc comparisons were noted as significant *p < 0.05. For clustering analysis on principal coordinate plots, categories were compared and statistical significance of clustering was determined via Permanova.