3.1.2. Computerized Open Field Test

The cumulative results obtained after 60 min of data collection did not show any significant change in behavior (see Appendix B Figure A3).

However, significant differences were found following the analysis of each 5-min interval. As seen in Figure 2a, the two-factor RM-ANOVA on the distance travelled in the arena revealed a significant main effect for the time factor (F (5.389, 183.2) = 113.8, *p* < 0.0001). Following a peak in the first five minutes the ambulation distance was steeply decreasing until a lower level of basal locomotor activity was reached around 30 min. The distance travelled at 50–55 and 55–60 min was lower in the 1 μg Kp-8 group than in the control group (*p* = 0.0334 and *p* = 0.0410, respectively).

**Figure 1.** Elevated plus maze results: (**a**) percentage of entries into open arms, (**b**) percentage of time spent in the open arms, (**c**) total number of entries into arms, (**d**) total time spent in the arms of the maze, *n* = 7–9, \* *p* < 0.05 vs. control, \*\* *p* < 0.01 vs. control.

**Figure 2.** Open field test results in 5-min intervals: (**a**) total distance travelled in the arena, (**b**) total ambulation time. The color of \* refers to the treatment group which significantly differs from the control group. *n* = 12–13, \* *p* < 0.05 vs. control, \*\* *p* < 0.01 vs. control.

Regarding total ambulation time, there was a significant main effect for the time factor (F (6.138, 208.7) = 98.03, *p* < 0.0001) with a similar pattern of steep then mild decrease (Figure 2b). The 1 μg Kp-8 group spent less time with ambulation than the control group at 50–55 min (*p* = 0.0090) and 55–60 min (*p* = 0.0326), as well.

The two-way ANOVA on immobility yielded a significant main effect for the time factor (F (5.396, 183.5) = 34.51, *p* < 0.0001) and interaction (F (22, 374) = 2.249, *p* = 0.0012). The time spent immobile was increasing during the experiment, showing a tendency reciprocal to that of ambulation time and distance (Figure 3a). Compared to control, the 1 μg dose of Kp-8 significantly increased immobility at 50–55 and 55–60 min (*p* = 0.0202 and *p* = 0.0186, respectively).

**Figure 3.** Open field test results in 5-min intervals: (**a**) total time spent immobile, (**b**) total number of rearings. The color of \* refers to the treatment group which significantly differs from the control group. *n* = 12–13, \* *p* < 0.05 vs. control.

Considering the number of rearing sessions, a significant main effect for the time factor was detected (F (6.756, 229.7) = 7.52, *p* < 0.0001), along with a statistically significant interaction between time and treatment (F (22, 374) = 3.095, *p* < 0.0001). As seen in Figure 3b, a pronounced difference started to appear among treatment groups from 30 min. There was a significant decrease in the number of rearings in the 1 μg Kp-8 group at 30–35 min (*p* = 0.0369), 40–45 min (*p* = 0.0445), 50–55 min (*p* = 0.0182) and 55–60 min (*p* = 0.0108).

Having calculated the average velocity for each timeframe, a significant main effect for the time factor (F (4.044, 129.4) = 12.17, *p* < 0.0001) and interaction (F (22, 352) = 1.940, *p* = 0.0073) could be seen, as shown in Figure 4c. There was no significant difference between treatment groups until 55 min, when the speed of the 1 μg Kp-8 group dropped (*p* = 0.0479).

**Figure 4.** Open field test results in 5-min intervals: (**a**) percentage of distance travelled in the central zone of the arena, (**b**) percentage of time spent in the central zone of the arena, (**c**) average velocity of ambulation. The color of \* refers to the treatment group which significantly differs from the control group. *n* = 12–13, \* *p* < 0.05 vs. control.

Figure 4a shows the percentage of central ambulation distance, calculated by dividing the distance travelled in the central zone of the arena by the total ambulation distance, multiplied by 100. Time factor (F (6.920, 235.3) = 2.207, *p* = 0.0351) and interaction between time and treatment (F (22, 374) = 1.767, *p* = 0.0185) both significantly accounted for the variation, but there was no difference among the groups, except in the first 5 min, when the central ambulation distance of the 1 μg Kp-8 group was higher than that of control (*p* = 0.0429).

The percentage of central ambulation time was calculated by multiplying the ratio of central time and total ambulation time by 100, as shown in Figure 4b. There was a significant main effect for the time factor (F (6.981, 237.3) = 2931, *p* = 0.0059), as well as for the interaction (F (22, 374) = 1.945, *p* = 0.0070). In the first 5 min, the central ambulation time of the 1 μg Kp-8 group significantly exceeded the central time of the control group (*p* = 0.0409), otherwise there was no difference among the groups.

#### 3.1.3. Marble Burying Test

There was no significant difference in the number of buried marbles among the groups (Figure 5a). Two types of goal-oriented interactions with the marbles were distinguished: marble burying and marble moving.

**Figure 5.** Results of marble burying test: (**a**) number of buried marbles (at least 50% covered with bedding material), (**b**) number of marble burying sessions, (**c**) duration of marble burying activity, *n* = 9–10.

Marble burying is an interaction involving digging around the marbles, resulting in marbles covered with bedding material. As seen in Figure 5b,c, neither the number of marble burying sessions, nor the duration of marble burying activity changed significantly with treatment, although a tendency of reduced burying activity was observable.

Marble moving is an interaction that involves rolling, moving the marbles with the forelegs, without successfully covering it with bedding material. Similarly to marble burying, there was no significant difference in the number and duration of marble moving among the groups (Figure 6a,b), although a tendency of suppressed marble moving could be seen in the groups treated with Kp-8.

However, taken the two types of interactions together, the 1 μg Kp-8 group interacted with the marbles fewer times than the control group (Figure 6c, *p* = 0.0499) and they also spent less time with goal-oriented interactions with the marbles (Figure 6d, *p* = 0.0274).

**Figure 6.** Results of marble burying test: (**a**) number of marble moving sessions, (**b**) duration of marble moving activity, (**c**) total number of interactions with marbles, (**d**) total duration of interactions with marbles, \* *p* < 0.05 vs. control. *n* = 9–10.

#### *3.2. Serum Corticosterone, LH and Total Protein*

The results of serum corticosterone and LH measurement can be seen in Figure 7. One-way ANOVA showed a significant effect of Kp-8 treatment both on corticosterone (F (2, 10) = 12.02, *p* = 0.0022) and LH concentration (F (2, 15) = 41.31, *p* < 0.0001). A robust increase in serum corticosterone concentration was detected 30 min after icv. treatment with 1 μg of Kp-8 (*p* = 0.001 vs. control). The 0.1 μg dose had a tendency to elevate corticosterone concentration, but the change was not significant (*p* = 0.306 vs. control). The 1 μg dose of Kp-8 also raised serum LH concentration 15 min after icv. treatment (*p* = 0.0001 vs. control), but the 0.1 μg dose had no effect on LH (*p* = 0.961 vs. control). There was no difference in serum protein concentration among the groups (F (2, 17) = 2.365, *p* = 0.124, *n* = 5–8): The mean serum protein concentrations with SD were 45.74 ± 4.898, 40.44 ± 3.115 and 45.13 ± 7.045 g/L in the control, 0.1 μg and 1 μg groups, respectively.

**Figure 7.** ELISA results: (**a**) serum corticosterone concentration (pg/mL), *n* = 4–5, \*\* *p* < 0.01 vs. control, (**b**) serum LH concentration (mIU/mL), *n* = 4–9, \*\* *p* < 0.01 vs. control.
