Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Margaret McCarthy ( mmccarthy@som.umaryland.edu ).

To generate sex-specific mixed glia cultures, rat pups were sexed on the day of birth and rapidly decapitated, and their brains were removed and placed into ice cold HBSS (without divalent ions), stripped of meninges, and minced with a razor blade. Individual brains were incubated for 15 min in 0.5% trypsin at 37°C, then dissociated by trituration with a pipette. The resulting suspension was pelleted by centrifugation at 400g for 4 min. Excess supernatant was aspirated and the suspension was resuspended in culture media (DMEM/F12 supplemented with 0.45% D-glucose, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum). One P0 rat brain was used to seed a T75 flask, which was incubated at 37°C with 5% CO 2 . One day after seeding, the culture media was completely replaced to remove unattached cells. Subsequently, 50% of the media was replaced every 4 days, and cultures were allowed to grow until DIV14. On DIV14, flasks were shaken on a rotating platform at 100 rpm for 1 h to detach microglia from the astrocyte layer. Media was removed from the flask and centrifuged at 400g for 4 min to pellet microglia, and resuspended in serum-free culture media (DMEM/F12, 0.45% D-glucose, 100 U/ml penicillin, 100 μg/ml streptomycin).

Adult Sprague-Dawley rats, obtained from Charles River Laboratories, were maintained on a 12:12h reverse light/dark cycle with ad libitum food and water. Animals were mated in our facility, and pregnant females were allowed to deliver naturally with the day of birth being designated as postnatal day 0 (P0). On P0, pups were sexed, treated, and culled to no more than 14 pups per dam. Male and female pups were used in these studies, and treatment groups and sexes were balanced across litters. All animal procedures were performed in accordance with the Animal Care and Use Committee’s regulations at the University of Maryland School of Medicine.

Method Details

Animal treatments The following drugs and doses were used in these studies: For studies involving hormonal modulation, drugs were dissolved in sesame oil and delivered subcutaneously. Testosterone propionate (100 μg; Sigma-Aldrich Cat#T1875), flutamide (100 μg; Sigma-Aldrich Cat#F9397), and estradiol benzoate (10 μg; Sigma-Aldrich Cat#E8515) were injected in a volume of 0.1 mL per pup per day. Cannabinoid receptor agonists ACEA (1 mg/kg; Tocris Cat#1319) and GP1a (1 mg/kg; Tocris Cat#2764) and the MAGL inhibitor KML29 (1 mg/kg; Tocris Cat#4872) were dissolved in ethanol at 5 mg/ml and further diluted in saline and delivered intraperitoneally in a volume of 0.1 mL per pup per day. Cannabinoid receptor antagonists AM281 (1 mg/kg; Tocris Cat#1115) and AM630 (1 mg/kg; Tocris Cat#1120) were dissolved in DMSO at 5 mg/ml and further diluted in saline and delivered intraperitoneally in a volume of 0.1 mL per pup per day. BrdU (50 mg/kg; Sigma-Aldrich Cat#B5002) was dissolved in saline and delivered intraperitoneally in a volume of 0.1 mL per pup per day. Liposomal clodronate (Encapsula Nanosciences Cat#CLD-8909) was delivered by bilateral intracerebroventricular injection (1 μL per hemisphere) at the following coordinates: −1.0 mm, caudal from bregma; ± 1.0 mm, lateral from midline; −3.0 mm, ventral from surface of the skull. Anti-CD11b antibody (0.5 mg/ml; OX-42 clone; Bio-Rad Cat#MCA275GA, RRID: AB_566455 ), anti-F4/80 antibody (0.2 mg/ml; Santa Cruz Cat#sc-52664, RRID: AB_629466 ) or phosphate buffered saline vehicle was delivered by intra-amygdalar injection at the following coordinates: −0.8 mm, caudal from bregma; ± 3.0 mm, lateral from midline; −5.0 mm, ventral from surface of the skull). Intracerebroventricular and intra-amygdalar injections were performed under cryoanesthesia using a 23 gauge Hamilton syringe attached to a stereotaxic instrument. The time the pups were separated from the dam was kept to a minimum, between 15 min to 1 h for all procedures.

Immunohistochemistry Rat pups were deeply anesthetized with Fatal Plus (Vortech Pharmaceuticals) and transcardially perfused with phosphate-buffered saline (PBS; 0.1M, pH 7.4) followed by 4% paraformaldehyde (PFA; 4% in PBS, pH 6.8). Brains were removed and postfixed for 24 h in 4% PFA at 4°C, then kept in 30% sucrose at 4°C until fully submerged. Coronal sections were cut at a thickness of 45 μm (for developmental studies) or 20 μm (for juvenile studies) on a cryostat (Leica CM2050S) and directly mounted onto silane-coated slides. For fluorescent imaging, slide-mounted sections were washed in PBS and blocked with either 5% normal goat serum (NGS) or 10% bovine serum albumin (BSA) in PBS + 0.4% Triton X-100 (PBS-T) for 1 h. Slides were incubated in primary antibody solution (either 5% NGS or 5% BSA in PBS-T) overnight. The following day, slides were incubated in secondary antibody solution (either 5% NGS or 5% BSA in PBS-T) for 2 h and coverslipped with ProLong Diamond Antifade (Thermo Fisher Scientific). For quantification of DNA material in phagocytic cups, sections were incubated in NucRed Dead 647 (Invitrogen Cat#R37113) or DAPI (Hoechst; Thermo Fisher Scientific Cat#62249) according to manufacturer’s instructions. Sections used for fluorescent Nissl imaging were stained with NeuroTrace (Thermo Fisher Scientific Cat#N21480) according to manufacturer’s instructions. For DAB staining, slide-mounted sections were washed in Tris-buffered saline (TBS; 0.05M, pH 7.6), incubated in 0.3% hydrogen peroxide in TBS for 30 min at room temperature. Sections were blocked with 5% NGS or 10% BSA in TBS + 0.4% Triton X-100 (TBS-T). Sections were incubated in primary antibody in solution (either 5% NGS in TBS-T or 5% BSA in TBS-T) overnight. The following day, sections were incubated in biotinylated secondary antibody for 1 h, followed by incubation in ABC reagent (1:500 dilution; Vectastain Elite ABC Kit, Vector Laboratories) in TBS-T for 1 h, and visualized using DAB chromagen or nickel-enhanced DAB chromagen (0.05% 3,3′-diaminobenzidine, 0.2% nickel (II) sulfate, 0.006% hydrogen peroxide; all from Sigma-Aldrich). The DAB reaction was allowed to proceed until completion, as confirmed under a microscope. Sections were counterstained with either hematoxylin or methyl green, dehydrated in an ascending ethanol series, defatted in xylene, and coverslipped with DPX mounting medium. Antigen retrieval (0.01M sodium citrate, pH 6.0 for 20 min at 99°C) was used prior to the blocking step to enhance immunolabeling for PCNA, ALDH1L1, and zif268. Primary antibodies used included the following: Rabbit anti-Iba1 (1:1000; Wako Cat#019-19741, RRID: AB_839504 ), goat anti-Iba1 (1:1000; Abcam Cat#ab5076, RRID: AB_2224402 ), mouse anti-BrdU (1:500; BD Biosciences Cat#347580, RRID: AB_400326 ), mouse anti-CD68 (1:500; Abcam Cat#ab31630, RRID: AB_1141557 ), mouse anti-CD11b (1:500; OX-42 clone; Abcam Cat#ab1211, RRID: AB_442947 ), rabbit anti-NeuN (1:1000; Abcam Cat#ab177487, RRID: AB_2532109 ), rabbit anti-GFAP (1:1000; Abcam Cat#7260, RRID: AB_305808 ), mouse anti-PCNA (1:1000; Abcam Cat#ab29, RRID: AB_303394 ), rabbit anti-cleaved caspase-3 (Asp175) (1:500; Cell Signaling Technology Cat#9661, RRID: AB_2341188 ), rabbit anti-ALDH1L1 (1:1000; Abcam Cat#ab87117, RRID: AB_10712968 ), rabbit anti-Egr1 (zif268) (1:1000; Cell Signaling Technology Cat#4153, RRID: AB_2097038 ). Secondary antibodies used included the following: biotinylated anti-rabbit (1:500; Vector Laboratories Cat#BA-1000, RRID: AB_2313606 ), biotinylated anti-mouse (1:500; Vector Laboratories Cat#BA-2000, RRID: AB_2313581 ), Alexa Fluor 488- or 594-conjugated antibodies to rabbit (488 Cat#A21206, RRID: AB_2535792 ; 594 Cat#A21207, RRID: AB_141637 ), goat (488 Cat#A11055, RRID: AB_2534102 ; 594 Cat#A11058, RRID: AB_2534105 ), or mouse (488 Cat#A21202, RRID: AB_141607 ; 594 Cat#A21203, RRID: AB_2535789 ) (1:500; Thermo Fisher Scientific, all raised in donkey).

Unbiased stereological cell counting Ashwell and Paxinos, 2008 Ashwell K.W.S.

Paxinos G. Atlas of the Developing Rat Nervous System. Stereological cell counts were performed using StereoInvestigator (MBF Bioscience) on a computer interfaced with a Nikon Eclipse E600 microscope and MBF Bioscience CX9000 camera. Every third section (45 μm thick) was used for analysis for a total of four sections, and the amygdala, prefrontal cortex, nucleus accumbens, and paraventricular nucleus of both hemispheres were quantified. The boundaries of each region were drawn with a 4x objective, referencing a neonatal rat atlas (), and the optical fractionator method was used to quantify microglia and BrdU+ cells at 40x magnification, using a 100 μm x 100 μm counting grid with a 250 μm x 250 μm sampling grid for microglia and a 50 μm x 50 μm counting grid with a 250 μm x 250 μm sampling grid for BrdU+ cells. The optical dissector height was set to 12 μm with a 2 μm guard zone on the top and bottom for both quantifications. Microglia were counted based on the presence of an observable cell body within the designated counting region and determined to be phagocytic if the microglia contained an observable phagocytic cup that was distinctly identifiable from the cell body. BrdU+ cells were counted if the nuclear staining was uniformly dark and was within the designated counting region.

Image acquisition For all experiments, confocal fluorescence images were acquired with the following: Zeiss LSM 710 microscope equipped with 488, 561, and 633 lasers, using a 20x (1.0 NA) water-immersion or 100x (1.46 NA) oil-immersion objective and Zeiss Zen software; Nikon CSU-W1 or A1 microscope equipped with 405, 488, 561, and 647 lasers, using a 20x (0.75 NA), 60x (1.49 NA) TIRF oil-immersion, or a 100x (1.45 NA) oil-immersion objective and Nikon Elements software. Widefield fluorescence and brightfield images were captured on a Keyence BZ-X700 microscope using a 4x (0.2 NA), 10x (0.45 NA) or a 20x (0.75 NA) objective and BZ-X Viewer software.

Quantification of microglial activation state Schafer et al. (2012) Schafer D.P.

Lehrman E.K.

Kautzman A.G.

Koyama R.

Mardinly A.R.

Yamasaki R.

Ransohoff R.M.

Greenberg M.E.

Barres B.A.

Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. 45 μm thick fixed coronal sections were immunolabeled for Iba1 and CD68 and imaged with a Zeiss 710 confocal microscope. Six fields of view (1 per hemisphere from 3 sections total) were taken with a 20x objective using 2 um z steps through the entire tissue thickness. Subsequent maximum intensity projections were used to quantify microglia morphology and CD68 expression. The activation state was categorized on a 0 (lowest activation) to 5 (highest activation) according to the following criteria: morphology was scored as 0 (5+ processes with at least secondary branches), 1 (1 - 4 processes with at least secondary branches), 2 (1+ processes with no secondary branches), and 3 (round with no clear processes). CD68 expression was scored as 0 (no clear expression), 1 (punctate expression) or 2 (aggregated expression throughout the cell). For each cell, the morphology and CD68 scores were combined to produce a final 0-5 score, as described in

Quantification of phagocytic cup contents, diameter, and three-dimensional rendering of microglia For three-dimensional rendering, confocal z stacks were taken with a 100x objective to achieve an x-y pixel size between 0.012 - 0.070 μm and a z-step size between 0.050 - 0.200 μm. Images were deconvolved using the automatic deconvolution algorithm in Nikon Elements, and were reconstructed in three dimensions in Imaris (Bitplane, RRID: SCR_007370 ). Individual microglial cells were reconstructed using the Surfaces module to create a volumetric boundary of the cell. The resulting microglial surface was then used as a mask to process the channels containing the material to be colocalized. After masking, the Surfaces module was again used to generate a new volume of the engulfed material, and the two surfaces (microglia and engulfed material) were merged to create the final rendering. For phagocytic cup analyses, single confocal images were taken with a 20x objective through the middle of the phagocytic cup. Images were then assessed for colocalization or measured for diameter in ImageJ (RRID: SCR_001775 ).

Quantification of local cell-microglia interactions (“Microglia Interactome”) Three-dimensional renderings of microglia were generated using the Surfaces module in Imaris as described above. The Spots module was used to mark the location of surrounding cells (DAPI+, PCNA+, cCasp3+), and the DistanceTransform function was used to calculate the distance of each spot (cell) from the microglia surface. Spots within a 10 μm distance from the microglia surface were included in analyses. Additionally, the total number of PCNA+ and cCasp3+ cells were quantified using 20x field of view images of the amygdala using ImageJ (RRID: SCR_001775 ).

Quantification of cell fate Quantification of the number of BrdU+ cells across the four MeA subregions was performed using Neurolucida software (MBF Bioscience, RRID: SCR_001775 ) on a computer interfaced with a Nikon Eclipse E600 microscope and MBF Bioscience CX9000 camera. Contours outlining the boundaries of each subregion were drawn at 4x magnification, and the area of each subregion was recorded. Sections from three alternate series were used to quantify the total number of BrdU+ cells and the number colocalized with either GFAP, NeuN or Iba1. Cells in each subregion were counted at 20x magnification across both hemispheres of the MeA. Additionally, the total number of GFAP+ cells was quantified only in the MePD. The data were normalized to the area of the subregion to account for any volumetric differences in the subregions between the sexes and averaged across hemispheres to generate a density estimate. In all cases, BrdU+ cells were counted if the nuclear staining was uniformly dark and present within the boundaries of the designated subregion. BrdU+ cells were counted as colocalized if a well-defined BrdU+ nucleus was associated with an immunopositive (i.e., GFAP+, NeuN+, Iba1+) cell body. For a subset of samples, colocalization criteria was confirmed by confocal microscopy.

Juvenile social play testing Animals were weaned on P21 and housed in same-sex, same-treatment sibling pairs. On P26, animals were tested for 10 min in an open field (78 × 78 cm, 40 cm high), underlaid with a grid delineating perimeter and center regions. Total locomotion and center time were analyzed to rule out confounds due to differences in activity or anxiety-like behavior on play. Once per day from P27-30, same-sex, same-treatment non-sibling pairs of animals were placed in an enclosure (49 × 37 cm, 24 cm high) with TEK-Fresh cellulose bedding (Harland Laboratories). Animals were allowed to acclimate for 2 min, then video recorded for 10 min. Behavior testing took place during the dark phase of the animal’s light/dark cycle under red light illumination. Videos were scored offline to determine the number of pounces, pins, and boxing behaviors.

Quantification of zif268 expression following a single social exposure Juvenile animals were given access to a novel sex-, treatment-, and age-matched play partner for 10 min as described above. Immediately following, animals were returned to their original cages. After 1 h, they were euthanized and brains were collected for histological analysis. Sections were labeled for zif268 and imaged using a Keyence BZ-X700 microscope at 4x magnification. Using the Hybrid Cell Count module in BZ-X Analyzer software (Keyence), contours were drawn to outline each region of interest, images were thresholded to account for background signal, and zif268+ cells were quantified. The number of zif268+ cells was normalized to the area of each contour to account for any volumetric differences.

In vitro phagocytosis assay Microglia harvested from sex-specific mixed glia cultures were plated into 6 well plates at a density of 50,000 cells per well and kept in serum-free culture media at 37°C with 5% CO 2 . After 24 h, carboxylated latex beads (FluoSpheres, 1 μm; Thermo Fisher Scientific Cat#F8816) were added to each well in a 1:100 cell:bead ratio and allowed to incubate for 1 h at 37°C with 5% CO 2 . Immediately following this, plates were placed on ice to halt any further phagocytosis, washed with ice-cold PBS containing 2 mM EDTA, and vigorously pipetted to detach cells from the plate. Cells were centrifuged at 400g for 4 min, resuspended in FACS buffer (1.0% BSA, 0.1% sodium azide, in HBSS), and incubated with propidium iodide for flow cytometry analysis. Flow cytometry was performed on a LSR II (BD Biosciences) with FACSDiva software, and analysis performed using FlowJo X (FlowJo). Live cells were gated based on propidium iodide staining and bead fluorescence determined using live cells that were not exposed to beads.

Flow cytometry for complement proteins On P4, male and female pups were rapidly decapitated, and the amygdala of both hemispheres was dissected out from the brain on ice. Tissue was immediately dissociated using the Neonatal Neuronal Dissociation (P) Kit (Miltenyi Bioscience Cat#130-092-628). The resulting single cell suspension was fixed in 4% paraformaldehyde for 20 min and stored in a 1:1 ratio of glycerol:HBSS at −20°C until staining. Cells were permeabilized with saponin and stained with a combination of the following antibodies: rabbit anti-C3b (1:50; Abcam Cat#ab11887, RRID: AB_298669 ), rabbit anti-C1qA (1:50; Abcam Cat#ab189922), and mouse anti-Ki-67-647 (1:10; BD Biosciences Cat#558615, RRID: AB_647130 ). Unconjugated primary antibodies were labeled using Alexa Fluor goat anti-rabbit-FITC (Cat#11-4839-81, RRID: AB_1210845 ) or Alexa Fluor goat anti-mouse-PE (Cat#12-4010-82, RRID: AB_11063706 ) (1:100; Thermo Fisher Scientific). Flow cytometry was performed on a LSR II (BD Biosciences) with FACSDiva software, and analysis performed using FlowJo X. Debris was eliminated based on forward and side scatter gating. Appropriate gates for C3b, C1qA, and Ki-67 were determined using an “empty channel” with the appropriate FMO samples to determine the negative population and autofluorescence.

Mass spectrometry Morena et al., 2015 Morena M.

De Castro V.

Gray J.M.

Palmery M.

Trezza V.

Roozendaal B.

Hill M.N.

Campolongo P. Training-associated emotional arousal shapes endocannabinoid modulation of spatial memory retrieval in rats. Qi et al., 2015 Qi M.

Morena M.

Vecchiarelli H.A.

Hill M.N.

Schriemer D.C. A robust capillary liquid chromatography/tandem mass spectrometry method for quantitation of neuromodulatory endocannabinoids. Qi et al., 2015 Qi M.

Morena M.

Vecchiarelli H.A.

Hill M.N.

Schriemer D.C. A robust capillary liquid chromatography/tandem mass spectrometry method for quantitation of neuromodulatory endocannabinoids. Lipid extraction and mass spectrometry was performed as previously described (). On P4, rat pups were rapidly decapitated and the amygdala dissected out and immediately flash frozen until analysis. Brain tissue was weighed and placed into borosilicate glass culture tubes containing 2 mL of acetonitrile with 5 pmol of AEA and 5 nmol of 2-AG for extraction. Tissue was homogenized with a glass rod, sonicated for 30 min in ice water, and incubated overnight at −20°C to precipitate proteins. Subsequently, samples were then centrifuged at 1500g to remove particulates, and the supernatants transferred to a new glass tube and evaporated to dryness under nitrogen gas. The samples were reconstituted in 200 μL of acetonitrile and stored at −80°C until further analysis. Analysis of AEA and 2-AG was performed by liquid chromatography tandem mass spectrometry as previously described ().