83 mg/kg dose of LPS. Lower doses of the PRR agonists were administered in the LabMaster system because pilot experiments had shown that mice kept singly in the LabMaster system were more SB203580 clinical trial sensitive to the treatments than group-housed animals. Mice receiving just one of the compounds (MDP, FK565 or LPS) were first injected with saline followed by the respective compound 4 h later. The first injection was given 3 h after start of the light phase. In experiments involving combination treatments, MDP + LPS and FK565 + LPS were given with a time
lag of 4 h between injection of the NOD and TLR agonist, since this timing has been shown to have the strongest priming effect on the immune system (Takada et al., 1990 and Takada and Galanos, 1987). Sickness responses were examined 3–4 h after injection and depression-like behavior 21–26 h post-treatment. The time points for the recording of the sickness responses were chosen according to the known time course of the sickness response to MDP or LPS (Frenois et al., 2007 and Engeland
et al., 2003). Sickness behavior has been shown to pass into depression-like behavior 1 day after injection of LPS (0.83 mg/kg), which Galunisertib nmr was the reason for choosing the second time point (Frenois et al., 2007). Since MDP or FK565 alone did not induce behavioral changes in experiment 2.1 and only induced a modest cytokine response 3 h post-treatment, the single treatment with MDP or KF565 was not investigated in experiments 3.1 and 3.2. Mice were deeply anesthetized with selleck chemicals pentobarbital (150 mg/kg i.p.). Blood was sampled by cardiac puncture using citrate (3.8%) as an anticoagulant. Following centrifugation for 10 min at 4 °C and 1000×g, blood plasma was collected and stored at −70 °C until assay. The plasma levels of corticosterone were determined with an enzyme immunoassay kit (Assay Designs, Ann Arbor, Michigan,
USA). According to the manufacturer’s specifications, the sensitivity of the assay is 27 pg/mL, and the intra- and inter-assay coefficient of variation amounts to 7.7% and 9.7%, respectively. Kynurenine and tryptophan were measured in plasma samples by high-performance liquid chromatography (HPLC) with ultraviolet detection (Herve et al., 1996). In brief, 100 μL plasma samples were deproteinized by adding of 100 μL of 5% (v/v) perchloric acid. After vortexing and 5 min centrifugation at 11,000×g, 20 μL of the clear supernatant was injected in the chromatographic system. Separations were achieved on a Chromolith RP18e column (100 × 4.6 mm, 5 μm, Merck Darmstadt, Germany) at 30 °C by isocratic elution with a mobile phase consisting of 50 mmol/L ammonium acetate, 250 mol/L zinc acetate and 3% (v/v) acetonitrile (pH 4.9) at a flow rate of 0.8 mL/min. Kynurenine and tryptophan were detected on a LaChrom UV-Detector Merck HITACHI L-7400 at 235 nm.