4.5. Behavioral Testing

Behavioral testing was performed in the latent (from Days 7 to 10 following pilocarpine-induced SE) and chronic (from Days 45 to 52) phases of the lithium–pilocarpine model (Figure 14).

An open-field test (OFT) [59] was used to assess motor and explorative activity. The open-field arena had a diameter of 1 m, wall height of 30 cm, illumination of 8 Lx, and 4 cm round holes in the floor. The rat was placed in the center of the arena. Each rat’s movement was recorded for 3 min. The recordings were analyzed offline using the Round and Cross and Field4W software (Institute of Experimental Medicine, St. Petersburg, Russia). We defined the form of the tracks and locomotor characteristics in the different field zones. The time spent in the center of the arena (1/4 of arena radius) and near the wall (less than 20 cm from the wall, thigmotaxis) were measured to estimate anxiety level. Total distance, average speed, time of locomotion, and immobility were calculated to determine locomotor activity. The number and total duration of the following behavioral patterns were measured: hole exploration, sniffing, climbing, and rearing (explorative activity) as well as locomotion and actions in a place (locomotor activity).

The Y maze [60] was used to measure spatial working memory. The maze consisted of three arms (each 50 × 10 cm) with opaque, 30 cm high walls. The rat was placed in the center, and the sequence of entries into arms was analyzed for 8 min. Entry was considered correct if it differed from two previous entries, for instance, 1-2-3, 2-3-1, or 1-3-2. Repeated numbers, such as 1-1, were considered two entries. The coefficient of alternation (C_A) was used as an index of operative spatial memory. It was calculated as follows: C_A = N_right/(N_total − 2), where N_right is the number of correct entries into a new arm, and N_total is the total number of entries.

The fear conditioning test of short-term and long-term fear-associated memory [61] was performed over 3 days. Two Plexiglas cages were used in this test. Cage A (45 × 30 cm, height = 20 cm) had an electroconductive floor. Cage B was larger (60 × 30 cm, height = 40 cm) and had no electroconductive floor. On Day 0 (habituation day), the rat habituated to the conditioning of Cage A for 3 min. On Day 1 (conditioning day), the rat was placed into Cage A. Five steps of conditioning were performed: 120 s of habituation (Step 1-1), 20 s of an auditory cue (80 dB sound) followed by 2 s of mild (0.6 mA) foot shock through the electroconductive floor (Step 1-2), a 120 s break (Step 1-3), repetition of Step 1-2 (Step 1-4), and 60 s of rest (Step 1-5). On day 2 (testing day), rats were placed in Cage A for 3 min (Step 2-1) without a cue or current to estimate condition-associated fear (contextual conditioning testing). The rat was then moved to Cage B (Steps 2-2 to 2-4), which was a different size and had pictures of geometrical figures on the walls and a vanillin drop on the floor to create a new odor that made Cage B unfamiliar to the rat. Thus, Cage B was not associated with Cage A. After 3 min of habituation (Step 2-2), the same current-associated sound was given for 3 min (Step 2-3) to estimate cue-associated fear (cued conditional testing). No stimuli were given during the last 1 min step (Step 2-4).

During each step, the total time of freezing was measured to estimate fear response to an aversive stimulus. Since the length of steps was different, the relative time of freezing (% of step duration) was used to measure fear-associated memory. Freezing during Steps 1-3 and 1-5 (immediately after the foot shock) reflected short-term fear-related memory.

A social interaction test was used to estimate social behavior [62]. Rats were placed into a Plexiglas cage (60 × 30 cm, height = 40 cm) for 30 min before the test to decrease anxiety from the new environment [62]. Then, an unfamiliar, adult, intact male Wistar rat was placed into the same cage for 5 min. The following patterns were measured: communication (sniffing and grooming the intruder’s body and sniffing intruder’s tail and genitalia), aggression, defense, sexual-like behavior to a male intruder (mounting and genitalia licking after mounting), and noncommunicative behavior (autogrooming).

The sucrose preference test [63] was performed for 2 consecutive days to estimate depressive-like behavior (anhedonia). On Day 1, a bottle with a 1% sucrose solution was placed in the home cage so that the rats could grow accustomed to the sweet taste. On Day 2, the rats were put into individual cages (30 × 30 cm, height = 40 cm, one rat per cage) with two bottles, the first one containing plain drinking water and the other containing 1% sucrose solution. After 18 h (6 p.m. to 12 p.m.), the water and sucrose solution intakes were measured by weighing. The sucrose preference was calculated as the ratio of sucrose solution consumption to total liquid consumption.