Animals

All experimental procedures were approved by the Institutional Animal Care and Use Committees at either the University of Maryland School of Medicine or The Ohio State University, and followed all guidelines put forth in the Guide for Care and Use of Laboratory Animals. Sprague Dawley rats purchased from Harlan Laboratories or bred in-house from animals originally purchased from Harlan. Animals were housed on a reversed 12 h light/dark cycle in standard group cages, except when breeding, with food and water ad libitum. Adult females were paired with males and separated when vaginal lavage was sperm-positive. Once sperm-positive, pregnant females were isolated and allowed to deliver naturally. Rat pups used in experiments were birthed in-house, and pups from multiple litters were randomly assigned to experimental treatment and then randomly distributed back to dams to control for differences in maternal care. Cages were checked daily to determine the day of birth (designated postnatal day (PN) 0).

Intraperitoneal injections

Lipopolysaccharide from E. coli (LPS; strain K-235, cat#L2143, Sigma; dose: 200 μ/kg ip in 0.05 ml pyrogen-free saline) was given as an immune challenge via intraperitoneal injection on PN0, and control animals received an equivalent injection of saline vehicle. LPS-challenged animals were euthanized on PN4 and tissue collected for western blot (detailed below).

Gestational allergic challenge

Prior to pregnancy, adult females assigned randomly to the experimental group were sensitized with a subcutaneous injection of 1 mg ovalbumin (OVA grade V, Sigma) prepared at 4 mg/ml in pyrogen-free 0.9% saline and precipitated at a 1:1 ratio with Al(OH) 3 (Thermo Scientific) according to manufacturer’s instructions. After two weeks, a second 1 mg ovalbumin-Alum adjuvant injection was given. Control females were injected with saline at the same two timepoints to control for experimental handling and stress effects. One week later, all females were paired with males for breeding and the day of detection of sperm assigned gestational day 0 (GD0). At GD15, pregnant rats were challenged intranasally with 1% ovalbumin in saline (experimental group) or saline vehicle (control group) (volume: 50 µl per nare), which was placed on each nare under light isoflurane anesthesia and inhaled upon regaining consciousness. At 30 min following challenge, maternal blood was collected to assay for total serum Immunoglobulin E (IgE), using an IgE Rat ELISA kit (Abcam cat#157736) and serum samples run in triplicate. Females were paired in groups of two until GD15 and then housed individually. After birth, animals were sacrificed via perfusion to analyze brain-resident mast cells, microglia or neuronal morphology at specified time points, or were weaned at PN22 into sex-specific groups of three containing both OVA challenged and vehicle exposed offspring for behavioral testing.

Histology and immunohistochemistry

For all in vivo histology experiments, animals were killed via lethal overdose with FatalPlus (Vortech Pharma) followed by transcardial perfusion with saline followed by 4% paraformaldehyde, brains removed, and postfixed for 12 hours. Brains were sectioned coronally at 45 μm thickness on a cryostat (Leica) and mounted onto SuperFrost charged slides (Fisherbrand) for subsequent staining procedures.

Mast cell staining

Mast cells were visualized using staining with acidic Toluidine Blue (Sigma; 0.5% in 60% ethanol; pH = 2.0; 10 minute stain incubation) as detailed in7, and then tissue was cleared with ascending ethanol, defatted with xylenes, and coverslipped using Permount.

Immunohistochemistry (IHC)

Brain sections were rinsed twice with PBS, permeabilized with 0.3% H 2 0 2 in 50% methanol, blocked with 5–10% bovine serum albumin or normal goat serum in PBS + 0.4% Triton X, and incubated with primary antisera against the pan-microglia marker, Iba1 (Wako cat#019-19741 1:1000) for 24 hours at 4 °C. Sections were extensively washed, and processed with biotinylated secondary antibodies (Vector), avidin-biotin complex (Vector), and reacted with Nickel-diaminobenzidine in 0.125 M sodium acetate to visualize chromogen for IHC. Stained sections were coverslipped with DPX mounting medium.

Golgi-Cox staining

Whole brains from P5 pups or P80–90 adults were placed in 15 ml of Golgi-Cox solutions A and B (FD Neurotech) for 10 days, then solution C (sucrose; FD Neurotech) for 1–1.5 weeks, cut into coronal sections 100 µm thick using a Leica vibrotome, and staining embedded using solutions D + E following the FD Neurotech protocol. Tissue was cleared with ascending ethanol, defatted with xylenes, and coverslipped using Permount.

Microscopy and Stereology

Stereology and single cell reconstruction: A Zeiss Axioimager.M2 microscope coupled to a CX9000 Digital Camera and Stereo Investigator software (MBF Bioscience) were used to estimate the total population of mast cells and microglia in the POA, using an average of 4–6 sections per animal encompassing the entire rostrocaudal extent of the POA. At the time of counting, mast cells were categorized as either granulated or degranulated, and microglia were categorized as ameboid or not ameboid. Microglia were considered ameboid if they had an enlarged cell body and either no processes or few, short processes, based on criteria validated in our previously published study4.

For analysis of Golgi-Cox impregnated POA neurons, neurons were chosen for analysis if the cell body was in the middle 50% of the z plane of the tissue section, multiple processes were visible and the cell was easily distinguishable from nearby cells. Four-to-five cells per animal across 3–4 brain sections were reconstructed in three dimensions under a 100x oil objective using Neurolucida software (MBF Bioscience). Morphological parameters for each cell were computed using Neurolucida Explorer, including cell body size, total dendritic length per cell, number of dendritic segments and branch points, and total number of dendritic spines per neuron. Data presented is the average of each of these parameters across multiple neurons for each animal.

For adult analyses, dendritic spines were analyzed, but single cells were not reconstructed in three dimensions. Dendritic segments chosen for analysis were unobstructed by other Golgi-stained material, and were at least 25 μm in length without a bifurcation. Only one segment was analyzed per given cell. Four-to-five dendritic segments per animal across multiple brain sections were reconstructed, and dendritic spine density was analyzed using Neurolucida Explorer software. Data are presented and were analyzed using the average dendritic spine density for each animal.

Western blot

Tissue was homogenized in RIPA buffer containing 1% Igepal CA630, 0.25% deoxycholic acid, 1 mM EDTA, 154 mM NaCl, and 65 mM Trizma Base, with added protease and phosphatase inhibitors (1:1000). All chemicals were obtained from Sigma unless otherwise specified. Protein supernatant was extracted after 20 minutes of centrifugation at 3000 rpm at 4 °C, and total protein concentration determined via Bradford assay (BioRad). Fifteen μg of protein was electrophoresed on an 8–16% precast SDS polyacrylamide gel (Life Technologies) and transferred onto a single polyvinyl difluoride membrane (Bio-Rad). Membranes were blocked in 50% Odyssey blocking buffer (LI-COR) in TBS or 10% nonfat milk in 0.1% Tween in Tris-buffered saline (TTBS) and subsequently incubated with spinophilin primary anti-serum (Millipore cat#06–842, 1:1000) in 5% milk in TTBS overnight at 4 °C. Membranes were rinsed and incubated with HRP-conjugated secondary antibody (1:200) for two hours. A Phototype chemilluminescence system (New England Biolabs) was used to detect the immunoblots by exposing the membrane to Hyperfield ECL (GE Healthcare). Integrative grayscale pixel area densitometry captured with a CCD camera was quantified with NIH Image software. Ponceau S staining appearing at 45 kDa was used as a loading control, and immunoblot densitometry values for each lane expressed as a percentage of Ponceau staining for the same lane.

Behavior

Sexual behavior testing

Between PN50–54, animals were gonadectomized under isoflurane anesthesia and implanted subcutaneously with a 30-mm silastic capsule (1.57 mm inner diameter, 3.18 mm outer diameter) filled with crystalline testosterone (Sigma) placed between the scapula. This capsule length releases testosterone in a manner that mimics physiological levels of testosterone circulating in adult males and allows appropriate activational hormones for developmentally-masculinized females to perform male-typical copulatory behavior5. Two weeks following surgery, animals were video recorded for at least 20 min during the dark phase of the light cycle, in a Plexiglass behavioral arena in the presence of a hormonally-primed receptive stimulus female under red light illumination. Behavioral data was collected and analyzed by an observer blind to the experimental treatment of each animal. Measures included number of mounts, latency to mount, frequency of ejaculation, time of each ejaculation and latency to resume mounting after ejaculation (ejaculation measures in males only). In order to record the full post-ejaculatory interval for situations during which a male started a post-ejaculatory interval with only a portion of the 20 min testing period remaining, the male was observed beyond the 20 min period until he started mounting again. These mounts outside of the 20 min period were not tallied in total mount measures. Mount rate was calculated from the total mounts divided by the 20 minutes less the total time during that 20 min the animal was in a refractory post-ejaculatory state.

Olfactory preference test

Olfactory preference test was performed on a separate cohort of adult animals (~PN60) from those used for sexual behavior testing. The test was performed using a modified protocol based on13. Animals were placed in the same Plexiglas arena used for sexual behavior testing, with two ceramic dishes in opposite corners of the arena (counterbalanced across groups and animals). Dishes contained soiled bedding from adult male or female non-littermate conspecifics’ cages, freshly collected 5 days after bedding change. Animals were placed in the center of the arena and allowed to openly explore the arena for 5 min under red light illumination during the dark phase of the light cycle, while being videotaped. The number of seconds spent actively investigating (e.g., sniffing, digging in, or climbing on/in) each dish of soiled bedding was tallied and a female bedding preference score tabulated ([Time spent investigating female bedding- time spent investigating male bedding]/total investigation time).

Data analysis