For this purpose body temperature and weight were measured immediately before treatment and body temperature was measured again 4 h post-injection. An additional measurement of body weight was taken 21 h post-injection

after the animals had been subjected to the open field (OF) test (n = 8). In a separate experiment (experiment 2.2), mice were euthanized 3 h after injection of PRR agonists ( Fig. 1) and the brains were collected for immunohistochemical visualization of c-Fos expression in select brain regions (n = 3–5). Following euthanasia the brains were removed, put on dry ice and stored at −70 °C ABT-737 chemical structure until use. Protocol 3 was used in 3 separate experiments (Fig. 1) in which the effects of MDP and FK565 in combination with the lower dose of LPS (0.1 mg/kg) were investigated. In experiment 3.1 body temperature and weight were measured before treatment and the body temperature was measured again 4 h post-injection. The OF test was conducted 21 h after the treatment

and body weight was measured after the OF test (Fig. 1). Subsequently the animals were subjected to the tail suspension test (TST) for 6 min (25.5 h post-injection) and euthanized 30 min after start of the TST. Blood was sampled to measure the plasma levels of cytokines, corticosterone, kynurenine and tryptophan (Fig. 1). In addition, the brains were collected, frozen in −70 °C cold 2-methyl butane (Fisher Scientific, Leicestershire, UK) and stored at this temperature until measurement of cytokines (n = 7–8). In experiment 3.2 mice (Fig. 1) were euthanized PTC124 clinical trial 3 h after injection of PRR agonists to record the levels of circulating and brain cytokines and circulating corticosterone without interference by any behavioral test (n = 7–8). In a further experiment (experiment 3.3) singly housed mice were subjected to the forced swim test (FST) 21 h post-injection, since depression-like behavior has been shown to be modified by different housing conditions (Painsipp et al., 2011) (n = 7–8). All compounds were dissolved in pyrogen-free sterile saline (0.9% NaCl) and pyrogen-free

sterile saline injected intraperitoneally (i.p.) at the same volume (50 μL/10 g body weight) was used as vehicle (VEH) control. For the analysis of the interaction between NOD agonists and LPS, two doses of LPS were examined. First, the widely used Avelestat (AZD9668) dose of 0.83 mg/kg LPS inducing the full spectrum of sickness (Frenois et al., 2007 and Painsipp et al., 2011) was used. Since, in combination with the NOD agonists, this dose of LPS led to a marked decrease in body temperature and locomotion, while a ceiling effect was observed with other parameters, a lower dose of 0.1 mg/kg LPS was also tested. The doses of the NOD agonists (see below) were chosen on the basis of their immunological effects in vivo ( Parant et al., 1995 and Shikama et al., 2011) and the results of pilot experiments. Thus, doses of 1 mg/kg (LabMaster studies) and 3 mg/kg (ex LabMaster studies) of MDP and 0.001 mg/kg (LabMaster studies) and 0.