*3.1.2. Elevated plus maze measurements*

#### *3.1.2.1. Total distance travelled*

The main effect of the treatment was significant, *F* (3, 39) = 3.06, *p* = .04, η2 = .19. The groups who received diazepam travelled less distance than those who received 100 μg/kg melatonin treatments. No other differences were found to be significant.

The interaction effect between the group and the treatment was also significant on the total distance travelled on elevated plus maze, *F* (3, 39) = 6.52, *p* = 0.001, η2 = .33. This interaction effect reflected the fact that in control condition, the subjects received diazepam travelled less distance than the other treatments, whereas in pinealectomy condition 100 μg/kg melatonin treatments travelled less distance than other treatments, whereas in the pinealectomy condition, the subjects received 100 μg/kg melatonin travelled less distance than the other treatments.

#### *3.1.2.2. Time spent in open arms (Open arm duration)*

The main effect of the treatment was significant, *F* (3, 39) = 6.53, *p* = .001, η2 = .33. The subjects who received 100 μg/kg melatonin treatment spent more time in open arms than those who received other treatments.

An interaction effect between the group and the treatment was also significant, *F* (3, 39) = 6.87, *p* < 0.001, η2 = .35. This effect indicated that in control condition subjects received 100 μg/kg melatonin treatment spent more time than those receiving the other treatments, whereas in pinealectomy condition the subjects who received saline, 100 μg/kg melatonin and diazepam treatments spent more time than those who received 1 μg/kg melatonin treatment.

from Karakas et al.; published by Elsevier, 2011a.)

treatments. No other differences were found to be significant.

*3.1.2.2. Time spent in open arms (Open arm duration)* 

those who received other treatments.

*3.1.2. Elevated plus maze measurements* 

*3.1.2.1. Total distance travelled* 

than the other treatments.

treatment.

**Figure 14.** The velocity. The bar explanations can be seen in Figure 10. (Reproduced with permission

The main effect of the treatment was significant, *F* (3, 39) = 3.06, *p* = .04, η2 = .19. The groups who received diazepam travelled less distance than those who received 100 μg/kg melatonin

The interaction effect between the group and the treatment was also significant on the total distance travelled on elevated plus maze, *F* (3, 39) = 6.52, *p* = 0.001, η2 = .33. This interaction effect reflected the fact that in control condition, the subjects received diazepam travelled less distance than the other treatments, whereas in pinealectomy condition 100 μg/kg melatonin treatments travelled less distance than other treatments, whereas in the pinealectomy condition, the subjects received 100 μg/kg melatonin travelled less distance

The main effect of the treatment was significant, *F* (3, 39) = 6.53, *p* = .001, η2 = .33. The subjects who received 100 μg/kg melatonin treatment spent more time in open arms than

An interaction effect between the group and the treatment was also significant, *F* (3, 39) = 6.87, *p* < 0.001, η2 = .35. This effect indicated that in control condition subjects received 100 μg/kg melatonin treatment spent more time than those receiving the other treatments, whereas in pinealectomy condition the subjects who received saline, 100 μg/kg melatonin and diazepam treatments spent more time than those who received 1 μg/kg melatonin

**Figure 15.** The total distance travelled. Right striated bar represents the saline injection and black bar represents diazepam injections, cross striated bar represents 1 μg/kg melatonin injection and bricks striated bar represents 100 μg/kg melatonin injection for both control and pinealectomy groups. Data are presented as means (± S.E.M.). Different letters indicate the statistically different groups. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

**Figure 16.** The time spent in open arms (TSOA). The bar explanations can be seen in Figure 15. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

#### *3.1.2.3. Time spent in closed arms (Closed arm duration)*

The main effect of the treatment was significant, *F* (3, 39) = 6.56, *p* = .001, η2 = .34. The subjects who received 100 μg/kg melatonin treatment spent less time in closed arms than those who received other treatments.

An interaction effect between the group and the treatment was also significant, *F* (3, 39) = 7.30, *p* < 0.001, η2 = .36. This interaction effect reflected the fact that in control condition subjects received 100 μg/kg melatonin treatment spent less time than those receiving the other treatments, but there were no significant differences between treatment conditions in pinealectomy.

Intraamygdalar Melatonin Administration and Pinealectomy Affect Anxiety Like Behavior and Spatial Memory 33

**Figure 18.** The entrance frequency to the open arms (EFOA). The bar explanations can be seen in Figure

No significant effects were found with regard to the total entrance to the closed arm of the

The main effect of the group was significant, *F* (1, 39) = 6.95, *p* = .01, η2 = .15. The subjects in

**Figure 19.** The mobility time (MT). The bar explanations can be seen in Figure 15. (Reproduced with

permission from Karakas et al.; published by Elsevier, 2011a.)

the pinealectomy condition were more mobile than those in the control condition.

15. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

*3.1.2.5. Entrance frequency to closed arms* 

elevated plus maze.

*3.1.2.6. Mobility* 

**Figure 17.** The time spent in closed arms (TSCA). The bar explanations can be seen in Figure 15. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

#### *3.1.2.4. Entrance frequency to open arms*

The main effect of the group was significant, *F* (1, 39) = 14.40, *p* = .001, η2 = .27. The subjects in the control condition entered more frequently to open arm than those in the pinealectomy.

The main effect of the treatment was also significant, *F* (3, 39) = 19.39, *p* = .0001, η2 = .60. The subjects who received 100 μg/kg melatonin treatment entered more frequently to the open arm than those who received other treatments.

In addition, the interaction effect between the group and the treatment was significant, *F* (3, 39) = 37.65, *p* = 0.0001, η2 = .74. This reflected the fact that in control condition subjects who received 100 μg/kg melatonin treatment entered more frequently than those who received the other treatments, whereas in pinealectomy condition the subjects who received saline, 1 μg/kg melatonin and diazepam treatments entered more frequently than those who received 100 μg/kg melatonin treatment.

**Figure 18.** The entrance frequency to the open arms (EFOA). The bar explanations can be seen in Figure 15. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

#### *3.1.2.5. Entrance frequency to closed arms*

No significant effects were found with regard to the total entrance to the closed arm of the elevated plus maze.

#### *3.1.2.6. Mobility*

32 Neuroendocrinology and Behavior

pinealectomy.

An interaction effect between the group and the treatment was also significant, *F* (3, 39) = 7.30, *p* < 0.001, η2 = .36. This interaction effect reflected the fact that in control condition subjects received 100 μg/kg melatonin treatment spent less time than those receiving the other treatments, but there were no significant differences between treatment conditions in

**Figure 17.** The time spent in closed arms (TSCA). The bar explanations can be seen in Figure 15.

The main effect of the group was significant, *F* (1, 39) = 14.40, *p* = .001, η2 = .27. The subjects in the control condition entered more frequently to open arm than those in the

The main effect of the treatment was also significant, *F* (3, 39) = 19.39, *p* = .0001, η2 = .60. The subjects who received 100 μg/kg melatonin treatment entered more frequently to the open

In addition, the interaction effect between the group and the treatment was significant, *F* (3, 39) = 37.65, *p* = 0.0001, η2 = .74. This reflected the fact that in control condition subjects who received 100 μg/kg melatonin treatment entered more frequently than those who received the other treatments, whereas in pinealectomy condition the subjects who received saline, 1 μg/kg melatonin and diazepam treatments entered more frequently than those who

(Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

*3.1.2.4. Entrance frequency to open arms* 

arm than those who received other treatments.

received 100 μg/kg melatonin treatment.

pinealectomy.

The main effect of the group was significant, *F* (1, 39) = 6.95, *p* = .01, η2 = .15. The subjects in the pinealectomy condition were more mobile than those in the control condition.

**Figure 19.** The mobility time (MT). The bar explanations can be seen in Figure 15. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

#### *3.1.2.7. Velocity*

No significant effects were found with regard to the total entrance to the closed arm of the elevated plus maze.

Intraamygdalar Melatonin Administration and Pinealectomy Affect Anxiety Like Behavior and Spatial Memory 35

**Figure 21.** The time spent to find the platform (TSFP) The bar explanations can be seen in Figure 20.

The main effect of the treatment was also significant, *F* (3, 40) = 4.11, *p* = .01, η2 = .24. The subjects who received 1 μg/kg melatonin treatment spent less time than those who received

**Figure 22.** The time spent in the correct quadrant (TSCQ). The bar explanations can be seen in Figure

In addition, the interaction effect between the group and the treatment was also significant, *F* (3, 40) = 9.29, *p* = 0.001, η2 = .41, indicating that in control condition subjects who received diazepam treatment spent more time than those who received the other treatments, whereas

20. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

(Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

other treatments with each being not significant from each other.

*3.1.3.3. Time spent in the correct quadrant* 

#### *3.1.3. Spatial memory measures (Morris water maze measures)*

#### *3.1.3.1. Total distance travelled*

The interaction effect between the group and the treatment was significant on the total distance travelled on elevated plus maze, *F*(3, 40) = 4.84, *p* = 0.006, η2 = .27. This reflected the fact that in control condition, the subjects received diazepam travelled more distance than the other treatments, whereas in pinealectomy condition subjects who reveived 100 μg/kg melatonin and the saline treatments travelled more distance than those who received 1 μg/kg melatonin and diazepam treatments.

**Figure 20.** The total distance travelled (TDT). Right striated bar represents the saline injection and black bar represents diazepam injections, cross striated bar represents 1 μg/kg melatonin injection and bricks striated bar represents 100 μg/kg melatonin injection for both control and pinealectomy groups. Data are presented as means (± S.E.M.). Different letters indicate the statistically different groups. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

#### *3.1.3.2. Time spent to find the platform (Latency)*

The main effect of the treatment was significant, *F* (3, 40) = 3.02, *p* = .04, η2 = .19. The subjects who received diazepam treatment spent more time than those who received 1 μg/kg melatonin treatment.

In addition, the interaction effect between the group and the treatment was also significant, *F* (3, 40) = 4.90, *p* = 0.005, η2 = .41. This interaction effect reflected the fact that there were no significant differences between control and pinealectomy groups in 100 μg/kg and 1 μg/kg melatonin treatments but was significant differences between these groups in saline and diazepam treatments.

**Figure 21.** The time spent to find the platform (TSFP) The bar explanations can be seen in Figure 20. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

#### *3.1.3.3. Time spent in the correct quadrant*

34 Neuroendocrinology and Behavior

No significant effects were found with regard to the total entrance to the closed arm of the

The interaction effect between the group and the treatment was significant on the total distance travelled on elevated plus maze, *F*(3, 40) = 4.84, *p* = 0.006, η2 = .27. This reflected the fact that in control condition, the subjects received diazepam travelled more distance than the other treatments, whereas in pinealectomy condition subjects who reveived 100 μg/kg melatonin and the saline treatments travelled more distance than those who received 1

**Figure 20.** The total distance travelled (TDT). Right striated bar represents the saline injection and black bar represents diazepam injections, cross striated bar represents 1 μg/kg melatonin injection and bricks striated bar represents 100 μg/kg melatonin injection for both control and pinealectomy groups. Data

The main effect of the treatment was significant, *F* (3, 40) = 3.02, *p* = .04, η2 = .19. The subjects who received diazepam treatment spent more time than those who received 1 μg/kg

In addition, the interaction effect between the group and the treatment was also significant, *F* (3, 40) = 4.90, *p* = 0.005, η2 = .41. This interaction effect reflected the fact that there were no significant differences between control and pinealectomy groups in 100 μg/kg and 1 μg/kg melatonin treatments but was significant differences between these groups in saline and

are presented as means (± S.E.M.). Different letters indicate the statistically different groups.

(Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

*3.1.3. Spatial memory measures (Morris water maze measures)* 

*3.1.2.7. Velocity* 

elevated plus maze.

*3.1.3.1. Total distance travelled* 

μg/kg melatonin and diazepam treatments.

*3.1.3.2. Time spent to find the platform (Latency)* 

melatonin treatment.

diazepam treatments.

The main effect of the treatment was also significant, *F* (3, 40) = 4.11, *p* = .01, η2 = .24. The subjects who received 1 μg/kg melatonin treatment spent less time than those who received other treatments with each being not significant from each other.

**Figure 22.** The time spent in the correct quadrant (TSCQ). The bar explanations can be seen in Figure 20. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

In addition, the interaction effect between the group and the treatment was also significant, *F* (3, 40) = 9.29, *p* = 0.001, η2 = .41, indicating that in control condition subjects who received diazepam treatment spent more time than those who received the other treatments, whereas

in pinealectomy condition the subjects who received saline treatments spent more time than those who received other treatments.

Intraamygdalar Melatonin Administration and Pinealectomy Affect Anxiety Like Behavior and Spatial Memory 37

After all experiments finished, animals were decapitated and the brains were removed. We checked the placement of the cannulas histologically whether they were placed to the amygdala of the brain or not. Figure 24 represents a histological section of a brain which the

**Figure 24.** The figure represents the amydala region of the brain where the injections were applied. Blue colored region of the brain represents the amygdala region. (Reproduced with permission from

The results of the present study can be described under the two main headings: anxiety-like behaviour (in open field apparatus and elevated plus maze) and spatial memory

Open field test is used to measure the anxiety like behaviors in rodents (Benabid et al. 2008). The total distance traveled, the total number of entries to the center and the edge of the open field, the time spent in the center of the open field versus time spent at the edge of the open field and the mobility are frequently used parameters measured in open field test in the literature (Pyter and Nelson, 2006). In this maze, if the anxiety of the animal is high, (a) the number of the entries to the edge of the open field tends to increase, whereas that of entries to the center of the open field decrease, (b) the time passed at the edge of the open field increases, while the time passed at the center of the open field decreases, and (c) the total

**3.2. Evaluation of the correct placement of the cannula** 

cannula was placed correctly.

Karakas et al.; published by Elsevier, 2011a.)

performance (in Morris water maze).

**4.1. Anxiety-like behaviours** 

**4. Discussion** 

#### *3.1.3.4. The entrance frequency to the correct quadrant*

The interaction effect between the group and the treatment was significant, *F*(3, 40) = 6.72, *p* = 0.001, η2 = .34, indicating that in control condition subjects who received diazepam entered more frequently those who received the other treatments, whereas in pinealectomy condition the subjects who received saline treatments entered more frequently than those who received other treatments.

#### *3.1.3.5. Mobility*

No significant effects were found with regard to the mobility in the Morris water maze.

#### *3.1.3.6. Velocity*

The main effect of the group was significant, *F* (1, 40) = 11.31, *p* = .002, η2 = .22. The subjects in the control condition were faster than those in the pinealectomy condition.

**Figure 23.** The velocity (VEL) are represented for the Morris water maze. The bar explanations can be seen in Figure 20. (Reproduced with permission from Karakas et al.; published by Elsevier, 2011a.)

The main effect of the treatment was also significant, *F* (3, 40) = 4.16, *p* = .01, η2 = .24. The subjects who received diazepam treatment were slower than those who received other treatments.

In addition, the interaction effect between the group and the treatment was significant, *F* (3, 40) = 4.13, *p* = 0.01, η2 = .24. This interaction effect reflected the fact that in control condition subjects who received 100 μg/kg melatonin treatment were slower than those who received the other treatments, whereas in pinealectomy condition, the subjects who received diazepam were slower than those who received other treatments.
