Fear conditioning software

In the last three days of the experiment, the animals underwent a light-dark box test (LDB). The light dark-box test was carried out according to the procedure described by Chocyk et al. [32 (link)] and Chamera et al. [33 (link)] in an apparatus consisting of a cage divided into two sections of equal size (one chamber was brightly illuminated by diodes (100 lux), whereas the other chamber was dark) by a partition with a door (10.6 cm × 10.4 cm). The apparatus was controlled with a computer system (TSE Systems, Bad Homburg, Germany). The whole experiment was carried out in a dark room, and the rats were kept in darkness for an hour before the test. At the beginning of each test session, the animal was placed in one corner of the light compartment, facing away from the gate. Then, the animal was allowed to move freely between the two chambers with the door open for 10 min. The behavioral response of rats during the trials was recorded by Fear Conditioning Software (TSE, Bad Homburg, Germany). The time spent in each compartment was recorded for each animal. After each trial, the chambers were cleaned to prevent bias based on olfactory cues.

The light-dark box test was performed using an apparatus consisting of four cages with a computer-controlled system (TSE Systems, Bad Homburg, Germany) based on the procedure reported by Chocyk et al. [42 (link)]. Each experimental box had two compartments: light (covering ¾ of the cage, brightly lit – 100 lx) and dark (covered with a lid), which were made of clear and black acrylic, respectively. Both sections were permeable to infrared light and were connected by a central gate (10.6 cm × 10.4 cm). Therefore, the two parts of the cage were freely accessible for the animals to explore. The experimental boxes were located in soundproof, ventilated cabinets on base constructions that contained integrated infrared sensors along the horizontal and vertical axes. One hour before the test, male rats (PND88) were kept in total darkness. The entire experiment was also conducted in a dark room. At the beginning of each testing session, which lasted 10 min, an animal was placed in one corner of the light compartment, facing away from the gate. The behavioural response during the trials was recorded by Fear Conditioning Software (TSE, Bad Homburg, Germany). Specifically, the time spent in each compartment, the distance travelled and the average speed were calculated for each animal (n = 8–9 in the kLPS and LPS groups, n = 19–21 in the kPoly and Poly I:C groups).

The fear conditioning was performed in experimental cages under constant white noise conditions for 3 consecutive days. On the first day, the animals were individually placed in the box for 2 min to adapt to the experimental conditions. On the second day, after 5 min of habituation, the animals underwent a fear conditioning procedure that consisted of three footshocks (stimulus 0.7 mA, 1 s, repeated every 59 s). Conditioned fear was tested on the third day by re-exposing the rats to the testing box and recording their freezing response over 10 min (freezing was measured through fear conditioning software, TSE, Bad Homburg, Germany) [31 (link)].