Animals belonging to cohort 1 were checked 6 h after LPS or vehicle injection for a sickness-induced response (feeding and body weight loss, symptoms of sickness, and behavior in the open field test) and 24 h after the immune challenge for depressive-like behaviors. In particular, we evaluated anhedonia using the sucrose preference test and cognitive impairment with the novel object recognition test. The total amount of liquid intake during the sucrose preference test and the open field test were also evaluated at the later time point to exclude bias possibly affecting the evaluation of the depressive-like phenotypes (Biesmans et al., 2016). Each animal underwent behavioral tests subsequently (open field, sucrose preference, and novel object recognition test) during the light cycle phase and was evaluated and analyzed by an observer blinded to the mice treatment.

Exploratory behavior was measured in an open field box constructed of white Plexiglas (45 × 30 × 25 cm) as previously described (Benatti et al., 2011). Tests were recorded using a video camera. Animals were accustomed to the experimental room for 1 h prior to the experiment. The floor and walls of the arena were wiped between trials, first with water and then with 30% v/v alcohol: distilled water solution to prevent mice from following the scent of a previously tested animal. The illumination in the center of the arena was 100 lux. At the beginning of the test, each mouse was placed individually in the center of the arena. In each 5 min session, the following parameters were evaluated using Smart software (version 2.5): total distance traveled and distance traveled in the central part (12 × 12 cm) of the arena (centimeters), time spent in the center, and resting time in the whole arena and the central area (seconds). Total distance traveled is an index of general locomotor (horizontal) behavior, whereas the total number of rearings is an index of locomotor activity (vertical). Time spent within the central area vs. the rest of the open field arena is a measure of anxiety-like behavior.

Behavioral data were collected while mice were in their home cage to avoid stress associated with an unfamiliar environment. Six and 24 h after saline or LPS injection, the presence or absence of the following symptoms was evaluated: 1) curled body posture, 2) ptosis, and 3) piloerection.

Anhedonia, defined as a decreased sensation of pleasure, represents a key symptom of MDD and can be assessed in mice by measuring the preference for a palatable solution (Alboni et al., 2017; Liu et al., 2018). The sucrose preference test consisted of a 3-day training and a test phase performed in the 24 h period following LPS injection, when LPS is known to induce a decrease in sucrose preference. Over the dark phase of the light cycle, single-housed mice were trained with two identical bottles containing either a freshly prepared 1% sucrose solution or water. To control for a side preference in drinking behavior, the position of the two identical bottles was switched every day, and to reduce neophobia in the week before, the test animals were habituated to drink from two identical bottles filled with water in their home cage. Prior to and during testing, mice were not food- and water-deprived. Fluid consumption (grams) was measured by weighing bottles before and after each session. Sucrose preference was calculated as the percentage of sucrose intake volume over the total volume of fluid intake in a 24 h period (Liu et al., 2018).

The novel object recognition (NOR) test has emerged as the most popular test for assessing a rodent’s ability to recognize a previously presented stimulus and evaluate nonspatial memory (Antunes and Biala, 2012; Lueptow, 2017). To test a discrete form of learning and memory that involves the hippocampal formation, animals were tested in boxes from the open field task. After 5 min of open field, we considered animals habituated to the arena. Thus, each animal was exposed for another 5 min to a pair of objects that differed in shape, surface color, contrast, and texture (training session). Three hours and 30 min after the initial exposure, mice were reexposed, in the same arena, for 8 min to one of the original objects and a novel object (test session). The objects were previously shown to induce approximately equal time of exploration in C57BL6/J and were located 10 cm from the side walls. These objects were randomized so that each object could be used either as a familiar or novel object in any given session (Lueptow, 2017). In all tests, the time spent exploring each object was recorded. A mouse was considered to engage in exploratory behavior if it touched the object with its forepaw or nose or sniffed the object within a distance of 1.5 cm. After every exposure, the objects and the cage were wiped with water and then with 30% v/v alcohol: distilled water solution to eliminate odor cues.

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