The experiments were designed in such a way that the number of animals used and their Fulvestrant in vivo suffering was minimized. The chemically synthesized NOD1 agonist FK565 was provided by Astellas Pharma Inc. (Ibaraki, Japan) (Watanabe et al., 1985). MDP (N-acetylmuramyl-l-alanyl-d-isoglutamine hydrate, catalogue number A9519, Sigma–Aldrich, Vienna, Austria) was used as synthetic NOD2 agonist and LPS extracted from Escherichiacoli 0127:B8 (purified by gel-filtration chromatography,

catalogue number L3137, Sigma–Aldrich, Vienna, Austria) was used as a TLR4 agonist. The experiments were started after the animals had become accustomed to the institutional animal house over the course of at least 2 weeks. Prior to the behavioral tests, the mice were allowed to adapt to the test room (lights on at 6:00 h, lights off at 18:00 h, set points 22 °C and 50% relative air humidity, maximal light intensity 100 lux) for at least one day. The pattern of locomotion, exploration, feeding as well as sucrose preference (SP) were assessed with the LabMaster system (TSE Systems, Bad Homburg, Germany), allowing

continuous recording of the animals without intervention by any investigator, as described previously ABT-737 mouse (Painsipp et al., 2013). The LabMaster system consisted of test cages (type III, 42.0 × 26.5 × 15.0 cm, length × width × height), surrounded by two external infrared frames and a cage lid equipped with three weight transducers. For recording locomotion and exploration, the two external infrared frames were positioned in a horizontal manner above one another at a distance of 4.3 cm, with the lower frame being fixed 2.0 cm above the bedding floor. The bottom frame was used Mannose-binding protein-associated serine protease to record horizontal locomotion of the mice, whereas the top frame served to record vertical movements (rearing, exploration). The measures of activity (locomotion, exploration) were derived from the light beam interruptions (counts) of the corresponding

infrared frames (Painsipp et al., 2013). The three weight transducers were employed to quantify ingestive behavior. To this end, a feeding bin was filled with standard rodent chow (altromin 1324 FORTI, Altromin, Lage, Germany). In order to assess SP, one drinking bottle was filled with tap water and one with a 1% sucrose solution and the bottles were each attached to a transducer on the cage lid for the total duration of the experiment. SP was calculated using the formula: sucrose intake/(sucrose intake + water intake). In a few cases in which the fluid bottles got obstructed, the data were excluded from analysis. Each test parameter was collected over a 24 h interval and activity scores and food intake recorded during the day before injection were set as 100%, and the daily scores measured post-injection expressed as a percentage of the pre-injection score.