Animals
All animal procedures were approved by the Institutional Animal Care and Use Committee (covered by Animal welfare assurance No A3011-0), at Stony Brook University, and conducted in accordance with the guidelines of the National Institutes of Health “Guide for the Care and Use of Laboratory Animals”. Experiments were performed using adult (2–3 months old) male mice [C57BL/6J (wt), CX3CR1-GFP (Jackson Labs, 005582 model B6.129P-Cx3cr1tm1Litt/J), CX3CR1-CreERT2-eYFP (Jackson Labs, 021160 model B6.129P2 (Cg)-Cx3cr1tm2.1(cre/ERT2)Litt/WganJ), td-Tomato (Jackson Labs, 007905 model number B6;129S6-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze/J)], were used in the study. All the mouse lines were backcrossed to a C57BL/6J background, bred in-house, and genotyped by PCR. CD-1 retired-breeder male mice (Charles River Laboratories, CD-1 IGS mice, strain code:022) were used as aggressors. The CD-1 male mice exert aggression only towards male mice, therefore female mice were excluded in the study. For induction of recombination in the CX3CR1-CreERT2-eYFP::Rosa26-tdTom mice, tamoxifen (Sigma CAS # 10540-29‐1) dissolved in ethanol : sunflower seed oil was injected intraperitoneally (i.p.) starting at P30, at a daily dose of 75 µg/g body weight (from a 10 mg/ml stock), for 5 consecutive days, as in15,27. All animals were housed in 12-hr light/dark cycle. Food and water were provided ad libitum by the experimenters.
Repeated social defeat stress paradigm (RSDS)
Adult male mice were subjected to repeated bouts of physical aggression (‘‘defeats’’) from aggressive CD-1 mice for 10 consecutive days, as previously described15,18. The CD-1 aggressors were screened during a 3-day screening period for adequate aggressive behavior and then housed on one side of the divided mouse cage (known as the home cage), at least overnight prior to the start of defeat sessions. All defeat experiments were performed within that compartment, while the intruders were rotated across defeat days, so that the experimental animals would not habituate to a single aggressor. On day 0 (D0) of RSDS the ‘socially defeated-to-be’ (SD) mice were placed into the aggressor’s space for 10 min (physical stress), and after the end of the encounter they were returned to their side overnight. The mice could see and smell the aggressor through the clear perforated divider (sensory stress). The naïve mice (Con) were exposed to wt (C57BL/6J) mice, instead, for 1 minute. About 80% of the mice that were subjected to social defeat stress displayed depressive-like behavior (SD-Sus for Susceptible), whereas the rest 20% were the nonresponding mice (SD-Res for Resilient) did not, thus modeling the heterogeneity in individual responses to stress in humans56. At the end of RSDS paradigms all animals were singly housed. Socially defeated mice were categorized based on the SI output, and further behavioral analyses for each group followed this categorization. Both SD-Sus and SD-Res exhibited anxiety-like behavior, however the SD-Sus additionally demonstrated social avoidance, reduction of reward under stress, anhedonia, despair-like behavior, and elevated plasma corticosterone levels (~ 130 ng/ml)15,56. In addition to this paradigm, a short-term RSDS paradigm adaptation (miniSD; 3 days) followed by 3 days of behavioral tests (BH; D4-D6) was also performed in the results shown in Supplementary Fig. 2, which resulted in ~ 50% of the SD mice becoming SD-Sus, as we have previously demonstrated15. Although we find that the model produces very reproducible / reliable results, it does have the limitation that in the current format the model uses male mice only for the experiments.
Behavioral analyses
For endpoints D15 and D25, mice were behaviorally tested for 4 days (1 experiment/day). Behavioral testing and recordings were performed during the light phase. Both naïve and SD mouse groups were tested for behavioral alterations using the i) social interaction test (SI; measures social avoidance), ii) elevated plus-maze test (EPM; measures stress/anxiety), iii) forced swimming test (FST; measures despair-like behavior), iv) sucrose preference test (SPT; assesses anhedonia-lack of feeling pleasure), and v) novelty suppressed feeding test (NSF; assesses reward under stress). All experiments (except for SPT) were performed in a light-controlled and sound-isolated behavioral analysis room. The mice were acclimatized to the experimental room for 1 hour before the start of each experiment. The SI and EPM were performed under red light conditions; the remaining behavioral tests were performed with lights on. The software Noldus Ethovision XT16 was used for the automated tracking and scoring during the behavioral tests. White noise generator (70–75 dB) was used to mask intermittent disturbing sounds (from surrounding areas) that would potentially startle the animals.
Social Interaction (SI) One day after the final social defeat stress interaction, a SI test was performed to determine whether the animals display social avoidance, as previously established15,18. Mice were placed into the social interaction open-field arena [(42 cm (w) × 42 cm (d) × 42 cm (h)], and the time spent in the interaction zone (< 8 cm from the wire-mesh enclosure) was monitored for the two 2.5-min phases (without or with a novel CD-1 aggressor present in the enclosure), separated by a duration of 30 s. The socially defeated mice with SI ratio [(Time in interaction zone with aggressor) / (Time in interaction zone without aggressor)] > 1 were classified as SD-Res, and mice with a ratio below 1 (social avoidance) were classified as SD-Sus15. The naïve mice were categorized as Control (C).
Elevated plus-maze test (EPM) The apparatus used for this test was cross-shaped with two open arms (30 cm x 5 cm) and two closed arms (30 cm x 5 cm x15 cm) that extended from a central platform (5 cm x 5 cm), as previously described15,57. The entire maze was elevated 40 cm above the floor. The enclosed arms offered safety when the mouse was stressed; on the other hand, the open arms offered the motive of exploration. Each mouse was placed in the central square of the apparatus, facing an enclosed arm. The mice were let to roam freely for 10 min totally; an arm entry was defined when all four paws entered an arm. The total mobility, time spent in open arms and time closed arms were recorded, as an index of stress/anxiety57.
Forced swimming test (FSM) Each mouse was placed in a transparent tank [inescapable Plexiglas cylindrical tank (height: 30 cm, diameter: 22.5 cm)] that was filled with water (to a depth of 15 cm and maintained at 25°C) and their escape related mobility behavior was measured. Once the mouse was in the water, it was left to swim for 6 min in total, assessing the behavior only during the last 4 min, as previously performed15. The mice that acquired an immobile posture, characterized by motionless floating in the water, were termed immobile (immobility time), making only the necessary movements to keep the head above the water. Before returning the animals to their home cages, they were dried gently using paper towels to prevent hypothermia.
Sucrose preference test (SPT) The SPT was performed as previously15. The experimental groups were habituated for 72 h to 2% sucrose, in which the bottles were alternated to avoid bias for a specific cage side. The day before the testing the mice underwent an 18 h water deprivation period. The day of the testing bottles filled with sucrose 2% or water were weighed, and consumption was determined for a 24 h period. Bottles were weighed again at the end of the 24 h period. Sucrose preference was expressed as (Δweightsucrose) / Δweightsucrose + Δweightwater) x100. Sucrose preference score less than 70% was displaying anhedonia.
Novelty suppressed feeding test (NSF) The NSF test measures the time that mice take to approach and eat food in a novel environment following an extended period of food deprivation. Mice were food-deprived for 24 h and moved in freshly prepared home-cages (to avoid any pellets remaining on the bedding). The next day, each mouse was placed at the corner of an open-field arena [(42 cm (w) × 42 cm (d) × 42 cm (h)] with a pellet of chow already positioned at the center of it. The time until the first bite of the chow pellet was recorded, with maximum trial time the 10 min. After the trial, the mice were returned to their home cage which contained a pre-weighed food pellet, and food consumption was measured for a period of 5 min, as described15.
BrdU labeling
For the labeling of proliferating cells, 5-Bromo-2′-deoxyuridine (BrdU;Sigma-B5022) was dissolved in drinking water (1 mg/ml), and all mouse groups were given access to the water ad libitum throughout the 10 days of the RSDS paradigm15.
Microglial depletion with PLX5622
Experimental groups were fed with colony stimulating factor-1 receptor (CSF1R) inhibitor, PLX5622 (PLX56, provided by Plexxikon Inc.), formulated in AIN-76A standard chow at 1200 mg/kg of food weight (Research Diets, Inc.), as previously 58. Control animals were fed AIN-76A standard chow (Research Diets, Inc.). Seven days of PLX administration can achieve > 90% microglial ablation 58,59. Exact treatment duration varied depending on the experimental design and is defined on each section.
Peripheral macrophage depletion with PLX73086
Experimental groups were fed with colony stimulating factor-1 receptor (CSF1R) inhibitor, PLX73086 (PLX73, provided by Plexxikon Inc.), formulated in AIN-76A standard chow at 200 mg/kg of food weight (Research Diets, Inc.), as previously59. Control animals were fed AIN-76A standard chow (Research Diets, Inc.). Exact treatment duration varied depending on the experimental design and is defined on each section.
Immunoblot analysis
Mouse subjects were euthanized with isoflurane overdose, and freshly extracted brains were micro-dissected using a McIlwain tissue chopper to obtain ~ 500-µm thick brain coronal sections. Medial PFC was dissected out (with the help of a dissecting microscope) from + 1.2 mm to + 2.5 mm anterior to bregma slices. Protein lysate was prepared using RIPA buffer (120–150 µl) with protease and phosphatase inhibitors (200 mM PMSF, 100 mM sodium orthovanadate, and protease inhibitor cocktail; Santa Cruz, sc-24948A). Lysates were shaken for 15 min at 4°C, cleared by centrifugation at 15,000 g for 10 min at 4°C, and protein concentration was determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific; 23225). Samples (15–20 µg) were boiled for 5 min with 5X SDS-PAGE sample loading buffer (Thermo Fisher Scientific; 39000), separated by SDS-PAGE, and transferred to PVDF membranes (ThermoFisher Scientific; 88518) at 30 V for 16–18 h at 4°C. Membranes were blocked with 5% w/v nonfat dry milk (Cell Signal; 9999S) and incubated with primary antibodies (Supplementary Table 2a) for 16–18 h at 4°C. Washes with TBST (Cell Signal; 9997S) were followed by incubation with HRP-coupled secondary antibodies (Supplementary Table 2b). Signal was visualized by Sapphire Biomolecular Imager (Azure Biosystems) using a chemiluminescent substrate mixture (Supersignal West Pico Plus; ThermoFisher Scientific, 34580 or Immobilon Western; Millipore, WBKLS0500). Optical densities were collected with Fiji ImageJ software60. Where indicated, protein levels were expressed as fold change (versus Control) of the arbitrary units (A.U.) following normalization to the corresponding loading controls (β-Actin, GAPDH). Samples were normalized to the total protein for each sample, as determined by the BCA assay. For a detailed antibody list used see Supplementary Table 2a.
Immunohistochemistry
Deeply anesthetized mice (isoflurane) were transcardially perfused with cold 1X PBS (15 ml) followed by cold PFA fixative solution (4% paraformaldehyde in 1X PBS; 20ml) and brains were dissected and post-fixed for an additional 24 hours. Then brains were cryoprotected in 30% sucrose for 24 hours and sectioned with a sliding microtome. Free-floating brain sections (30–40 µm thick) containing the mPFC, were blocked with 10% goat serum (ThermoFisher Scientific; 16210072) in 0.3% TritonX-100 in 1X PBS for 1–2 hour at room temperature. Tissue sections were incubated with primary antibodies overnight at 4°C at the indicated concentrations (Supplementary Table 2a). The following day, sections were washed in PBS-Triton and incubated with the appropriate cross-absorbed secondary antibodies (Table II-3). After 4 additional washes, nuclei were stained with DAPI (Sigma; D9542) and sections were mounted using MOWIOL mounting media. For BrdU IHC, sections were pretreated in 2M HCl for 40 minutes at 37°C, followed by 2-3x 5-minute washes in 0.1 M Boric acid (pH 8.5). For a detailed antibody list used see Supplementary Table 2c.
Microscopy and histological quantification
The confocal laser-scanning microscopes Leica TCS SP8X and Leica TCS-SP5 were used for imaging of FITC, EGFP, EYFP, Alexa-488, Alexa-555, Alexa-594, Alexa-647 and CY3 fluorophores. Optical sections (z = 1.0µm; total stack of 10–16µm for 1 cell layer) of confocal epifluorescence images were sequentially acquired using 10x, 20x, 40x (N.A. 1.30), 63x (N.A. 1.20) and 100x (N.A. 1.40) objectives, with LAS AF software. Fiji ImageJ software60 was used for image reconstruction, cell counting, integrated density analysis and colocalization analysis. For mPFC analysis, 6 fields (located between + 1.2mm to + 2.5mm anterior to bregma), containing the cingulate cortex (Cg1), prelimbic (PL), infralimbic (IL) and medial orbital cortex (MO) were taken from each animal, for quantitative analysis. Typically, 3–4 brain slices were analyzed per animal, and at least 3 different animals for each experimental condition were evaluated. Cell counts were presented as the number of marker + cells/section (section corresponding to 0.2mm2). Each section quantification depicts the sum of the marker + cells from all the analyzed fields. For the integrated density analysis, the IsoData thresholding was applied to each image, and the integrated density [is defined as the product of pixel intensity (255 = the maximum pixel intensity for an 8-bit image) x area] was measured. For the colocalization analysis, the IsoData thresholding was applied to each image, images were merged, compartmentalized, and the integrated density was measured for the colocalized signal. Each section with integrated density quantification is depicted by the mean of the integrated density from all the analyzed fields. All experimental group comparisons were conducted on sections stained and imaged with identical exposure and acquisition settings. All analyses were performed on raw images, prior to any image processing by Adobe Illustrator CS6 for representation purposes. The cell counting was performed in a blinded manner by 4 experimenters collectively, and tissue sections were matched across samples.
Microglial morphological analysis
To quantitatively examine microglial morphology, unprocessed 30µm z-stack confocal images (100x objective; 20 individual images, step size: 1µm) of cells for Iba1 activity in the mPFC area were imported into Neurolucida software, as previously described15. Cell bodies and processes were manually traced (6 cells/mouse), and a 3D rendering was created. The 3D reconstructions were imported into Neurolucida Explorer for branched structure analysis and Sholl analysis (5µm starting radius, 50µm ending radius, and 5µm step size). Number of process intersections, cell surface area, and cell complexity were measured. Microglial complexity = [Sum of the terminal orders + Number of terminals] * [Total dendritic length / Number of primary dendrites]. Terminal orders: Number of "sister" branches encountered as proceeding from the terminal to the cell body (calculated for each terminal); Terminal: Refers to process endings.
Flow cytometry
Flow cytometry was performed as previously, with minor modifications61. Mice were deeply anesthetized (isoflurane) and transcardially perfused with 20 ml of ice cold 1X HBSS (pH 7.4). The mPFC of CX3CR1-GFP mice was collected in ice-cold Hibernate-A Medium (ThermoFisher Scientific; A1247501) isolated and enzymatically digested in a pre-warmed (10 min) 1.54 mg/ml papain solution (Worthington; LS003127), containing 1.1 mM EDTA, 0.067 mM β-mercaptoethanol and 5.5 mM cysteine-HCl, for 20 minutes at 37°C with manual inversion every 5 minutes. Further processing was performed at 4°C. Tissue was triturated with a p100 tip, followed by syringe G20 and then G25 trituration (5 times each). Tissue debris was removed by passing the cell suspension through a 40-µm cell strainer and centrifuged for 5 minutes at 2000 g at 4°C to remove papain solution. Myelin was removed using a discontinuous Percoll gradient. The cell pellet was resuspended in 30% Percoll (Sigma; P1644) in 1X HBSS, layered over a 37% and a 70% Percoll cushion, and centrifuged at 800 g for 40 min (800 x g) at 10°C. The supernatant containing myelin was removed, and cells were collected at the 37–70% Percoll interface. The remaining solution was diluted with 1X HBSS, and samples were centrifuged for 10 minutes at 1000 g, 4°C to pellet the cells. The pellet was then resuspended in flow cytometry (FC) buffer [0.5% bovine serum albumin (BSA) in 1X PBS]; cells were counted using trypan blue exclusion. Non-specific binding was blocked using anti- CD16/32 (1:50 in FC buffer; Tonbo biosciences, TB-70-0161-U500) for 30 minutes on ice and then stained with CD11b-APC, CD45-PerCP/Cy5.5, CD86-Pacific Blue, CD206-PE, antibodies (1:100 in FC buffer, Biolegend) in various combinations for 30 minutes on ice in dark conditions. The staining solution was removed by centrifugation for 5 minutes at 1000 g, and cells were washed twice with FC buffer before resuspending for flow cytometric analysis on a BD LSR Fortessa. The FC analysis was performed in the endpoint population of 15,000 CX3CR1-GFP+ cells/mouse. Data were further analyzed on the FlowJo software.
RNA isolation and quantitative PCR
Mouse subjects were euthanized with isoflurane overdose, and freshly extracted brains were micro-dissected using a McIlwain tissue chopper to obtain ~ 500-µm thick brain coronal sections. RNA from the mPFC (+ 1.2 mm to + 2.5 mm anterior to bregma) tissue or cells was isolated using Qiazol (Qiagen; 79306). The total RNA was separated from the aqueous phase using a RNeasy MicroKit (Qiagen; 74004). cDNA was reverse transcribed using random primers and Superscript IV (Invitrogen; 18090200). Quantitative PCR was performed from cDNA using Phusion Flash High-Fidelity PCR Master Mix (ThermoFisher Scientific; F548L) as per the manufacturer’s protocol on an Applied Biosystems 7900HT real time PCR system (Ct values above 35 cycles were not considered for the analysis). Fold changes were calculated using the ∆∆Ct method. For the primer sequences, see (Supplementary Table 2d). All gene expression levels were normalized to Gapdh mRNA levels.
Statistical analysis
No statistical methods were used to predetermine sample size a priori, but the sample sizes used were similar to those reported in previous studies15. Furthermore, representative data from each experiment were examined by Shapiro-Wilk’s test (p > 0.05)62 and a visual inspection of their histograms to confirm normal data distribution63,64. The majority of the experimental analyses was performed by parametric ordinary one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison post-hoc tests. For the glial morphological analyses two-way ANOVA was performed, followed by Tukey’s multiple comparison post-hoc tests. For the PLX5622 repopulation behavioral tests a paired t-test was performed. The GraphPad Prism 8 and Excel (Microsoft) were used for all statistical analyses. The data were reported as mean ± S.D. with symbols indicating the following P value ranges: *P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001).