Behavioral testing

The behavioral experiments were conducted in the order presented below, and the interval between subsequent tests was 2–3 days during the light phase. The experimenter was blind to the animal genotype and/or treatment. At the beginning of behavioral testing, the animals were 2–3 months (young adult) or 12 months (middle-aged) old.

4.3.1 Exploratory activity/Open field (I, II, III)

The spontaneous exploratory activity of mice was automatically monitored in a transparent, well-illuminated (~ 300 lx) cage equipped with two stacked frames of infrared photo detectors enabling separate monitoring of horizontal (XY-move time) and vertical activity (rearing). The young adult mice were tested using an activity monitor produced by MED Associates (cage dimensions, 28.5 cm × 28.5 cm x 20 cm; St. Albans, VT, USA), while middle-aged mice were tested with the TruScan®

activity monitor (cage dimensions, 26 cm x 26 cm x 39 cm; Coulbourn Instruments, CO, USA). The test sessions for young adult mice were 30 min, while the sessions for middle-aged mice were 10 min. To avoid odor traces, the test cage was cleaned with 70% ethanol before each mouse.

For analysis of results, the cages were divided into two compartments: a compartment near the walls (7 cm from the walls) and a central area compartment. Interruptions of infrared photo beams were used to calculate the following parameters: the distance traveled (cm), time spent in compartments, immobility time and rearing.

4.3.2 Light/dark-box test (III)

The LD test was performed for 10 min in an acrylic cage (28.5 × 28.5 × 20 cm; MED Associates, St. Albans, VT, USA) divided into two equal size compartments: one with transparent walls that was open topped and brightly illuminated (~ 450 lx from a 40-W light bulb fixed 55 cm above the floor) and another compartment that was constructed from black plastic (passing infrared light) and covered by a lid. The two compartments were separated by a partition containing an opening (7 × 5 cm) in its center at floor level. The mouse was placed in the center of the light compartment facing away from the opening. The latency to enter the dark area, the time spent in the compartments, the total distance traveled, the immobility time and the number of entries into the dark compartment were measured over 10 min. Rearing time was also calculated. The testing apparatus was thoroughly cleaned after each animal using 70% ethanol.

4.3.3 Elevated Plus-maze test (I, III)

This test was performed in an elevated maze (40 cm above the floor) consisting of two open arms (30 cm x 5 cm), two enclosed arms (30 cm x 5 cm with 15-cm-high transparent or black-painted side and end walls) and a connecting central platform (5 cm x 5 cm). The mouse was placed on the central platform facing one of the enclosed arms, and the time spent in the open and closed arms and the number of total arm entries was observed for 5 minutes. A video camera positioned above the maze recorded the experiments (Ethovision XT 7, Noldus Information Technology, Wageningen, Netherlands). The total number of arm visits was taken as a measure of general activity, while the % time spent in the open arms was used as a measure of anxiety. Only mice with at least 5 arm entries were considered when calculating the latter parameter. Testing occurred in a dimly lit room. To avoid odor traces, the test cage was cleaned with 70% ethanol before each mouse.

4.3.4 Forced swimming test (I, II, III)

Mice were placed individually in glass cylinders (19 cm in diameter, 24 cm high) filled with water at 21 ± 1 °C to a height of 14 cm. If used, the test compounds and vehicle were administered 45 minutes prior to testing. The time spent immobile (passive floating, during which the animal was motionless or moving the tail or one hind limb only slightly) was measured during the 6 min test. The latency to the first bout of immobility was also recorded. The results were normalized to the respective controls and expressed as a percentage of the control.

4.3.5 Novel object recognition (I)

A pair of identical objects was left overnight in the home cage and removed the next morning.

After 4 hours, a new pair of objects, one identical to the previous pair and a novel object, was placed side by side at the back of the cage (the side of the novel object was randomized between days). The behavior of the animal was monitored for 5 minutes, and the approaches to the objects were counted. Four pairs of objects were used [three of them were Duplo^R toy building blocks (Lego, Denmark)] that were similar in size to the mouse; the last pair of objects was a drinking glass and a metal tea jar (height ~ 9 cm). The total number of approaches to the objects and novelty preference was counted over the four days. The formula for novelty preference was as follows:

[(visits to novel – visits to familial)/total visits] * 100%

4.3.6 Marble Burying (I)

The test was conducted in the home cage (27 cm x 45 x cm 14.5 cm). In the afternoon, 1 l of extra bedding was added to the cage bottom, and 9 glass marbles (1 cm in diameter) were left on the top of the new bedding in a 3 x 3 array. The next morning, the number of uncovered marbles was counted.

4.3.7 Fear conditioning, extinction, renewal and reinstatement (IV)

Freezing behavior was measured with an automatic infrared beam detection system, which was placed on the sides of the chamber of the fear conditioning apparatus (TSE Systems GmbH, Bad Homburg, Germany). The mouse was considered to be frozen only if it was not moving for at least 3 s, and the measurement was expressed as the percentage of the time spent freezing. Fear conditioning and extinction occurred in two different contexts unless otherwise stated. Fear conditioning context (A) was a transparent Plexiglas chamber with metal grids on the floor, whereas extinction context (B) was a black nontransparent Plexiglas chamber with a planar floor. Both context A and context B were cleaned before each session with 70% ethanol or 70% 2-propanol, respectively.

Fear conditioning. During the fear conditioning, the mice were conditioned with 5 pairings of the CS (total CS duration 30 s, 1 Hz, white noise, 80 dB) with the US (1 s foot shock of 0.6 mA, inter-trial interval: 20-120 s). The US co-terminated with the CS. The freezing level during the first CS, preceding the first US, was taken as the baseline freezing during CS.

Fear extinction. After fear conditioning (Days 2 and 3), mice were submitted to extinction training in context B. During this training, the mice received 12 presentations of the CS on each day (inter-trial interval: 20-60 s). Spontaneous recovery and context-dependent fear renewal were tested 7 days later in context B and context A, respectively, using 4 presentations of the CS (inter-trial interval: 20-60 s).

Fear renewal. After fear conditioning, the mice were assigned to 2 groups with equal levels of freezing: one received fluoxetine in the drinking water until the end of the experiment, while the other received tap water. On days 14 and 15, the conditioned mice were submitted to extinction training in context B, during which they received 12 daily presentations of the CS (inter-trial interval:

20-60 s). Spontaneous recovery and context-dependent fear renewal were tested 7 days later in context B and context A, respectively, using 4 presentations of the CS (inter-trial interval: 20-60 s).

Fear reinstatement. All experimental procedures were conducted in context A. After fear conditioning, mice were assigned to 2 groups with equal levels of freezing: one received fluoxetine in the drinking water until the end of the experiment, while the other received tap water. On days 14 and 15, the conditioned mice were submitted to extinction training, during which they received 12

daily presentations of the CS (inter-trial interval: 20-60 s). 7 days later, the mice received 5 unsignaled US; 24 hours later, fear reinstatement was tested using 4 presentations of the CS (inter-trial interval: 20-60 s). To control for the context specificity of the fear reinstatement test, mice were additionally tested in the new context B 2 hours later using 4 presentations of the CS (inter-trial interval: 20-60 s), during which the mice did not exhibit elevated freezing behavior.