Deep isoflurane anesthesia

Rats were sacrificed by rapid decapitation under deep isoflurane anesthesia (Baxter, Deerfield, IL). Brains were flash-frozen in isopentane chilled on dry ice and stored at −80°C until tissue punching. Frozen brains were sect ioned coronally by cryostat and bilateral 1.2 mm diameter punches were taken of central amygdala (CeA; AP −1.8 to −2.8). One tissue punch was processed for western blotting and one was processed for qPCR as follows. Western blot samples were homogenized by sonication (Branson SLPe; Emerson Industrial, St. Louis, MO) in homogenization buffer (10mM Tris-HCl, 1% (w/v) SDS, 1% (v/v) HALT protease inhibitor, 5mM EDTA). Protein concentration from lysates were determined using a BCA assay (Thermo-Fisher; Rockford, IL). RNA was extracted from qPCR samples using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to manufacturer’s instructions and eluted in 30uL of nuclease free water (Life Technologies Corp., TX, USA). RNA concentration and purity for each sample were determined using a spectrophometer (Nanodrop 2000, Thermo-Fisher).

Immediately following the acoustic startle response and approximately 25 hours after injection, rats were euthanized via rapid decapitation under deep isoflurane anesthesia (Baxter, Deerfield, IL). Spleens were collected from all rats, and brain tissue from the high dose (3 mg/kg) cohort was used to assess neuroimmune signaling. Brains were flash-frozen in isopentane and placed on dry ice immediately after extraction. The brains were then stored at −80 °C until tissue punching. Whole spleens were weighed immediately.