**2.8. Elevated plus maze**

The elevated plus maze consisted of the two open and two closed 10 cm wide arms in a plus-sign configuration 55 cm off the floor. The closed arms were enclosed by 41 cm tall black Plexiglas. All arms were covered with contact paper to prevent the animals from sliding off, and all surfaces were wiped with 70% alcohol between animals. Each animal was released into one of the closed arms and allowed to move freely on the maze for a 5-min testing period that was videotaped from above the maze. Animals that fell off the maze into compartments below were placed back on the maze for the remainder of the testing period. An observer uninformed about experimental conditions scored the videotapes with the Observer Software (EthoVision XT ) (Noldus Ethovision, Version 6, Netherland; Commat LTD.ŞTİ. Ankara/Turkey) for distance, duration in the open arm, frequency in the open arm, duration in the closed arms, frequency in the closed arms, mobility, and velocity. Animals were considered to have entered an arm when all four paws crossed onto the arm.

For the spatial memory, the performance in the Morris water maze was evaluated. The experiments were carried out in a circular, galvanized steel maze (1,5 m in diameter and 60 cm in depth), which was filled with 40 cm deep water kept at 28 °C and rendered opaque by the addition of a non-toxic, water soluble dye. The maze was located in a large quiet test room, surrounded by many visual cues external to the maze (e.g. the experimenter, ceiling lights, rack, pictures, etc.), which were visible from within the pool and could be used by the rats for spatial orientation. The locations of the cues were unchanged throughout the period of testing. A video camera fixed to the ceiling over the center of the maze was used for recording and monitoring movements of the animals. There were the four equally divided quadrants in the pool. In one of the quadrants, a platform (1.0 cm below water surface, 10 cm in diameter) was submerged centrally and fixed in position which was kept constant throughout the acquisition or probe trials. The rats performed the five trials per day for the four consecutive days (20 trials). In the swimming trials each individual rat was released gently into the water at a randomly chosen quadrant except for the one that contained the hidden platform for facing an extra maze cue. The rat swam and learned how to find the hidden platform within 60 s. After reaching, the rat was allowed to stay on the platform for 15 s and was then taken back into the cage. During the inter-trial intervals, the rats were kept in a dry

In order to assess the spatial memory, the platform was kept away from the maze for 24 hours in the final trial. Each rat was placed into the water as in the training trials and the time in seconds spent in the quadrant formerly occupied by the platform (correct quadrant) was recorded. The platform remained in the same quadrant during the entire experiment. The rats were required to find the platform using only the distal spatial cues available in the

testing room. The cues were kept constant throughout the testing.

**Figure 7.** The quarters of the water maze and the hidden platform are seen.

**2.9. Morris water maze** 

home cage for 60 s.

**Figure 4.** Open and closed arms of the elevated plus maze are seen.

**Figure 5.** Rat is seen in the closed arm of the elevated plus maze.

**Figure 6.** Time spent in open arm is high. The anxiety is low.

## **2.9. Morris water maze**

24 Neuroendocrinology and Behavior

**Figure 4.** Open and closed arms of the elevated plus maze are seen.

**Figure 5.** Rat is seen in the closed arm of the elevated plus maze.

**Figure 6.** Time spent in open arm is high. The anxiety is low.

For the spatial memory, the performance in the Morris water maze was evaluated. The experiments were carried out in a circular, galvanized steel maze (1,5 m in diameter and 60 cm in depth), which was filled with 40 cm deep water kept at 28 °C and rendered opaque by the addition of a non-toxic, water soluble dye. The maze was located in a large quiet test room, surrounded by many visual cues external to the maze (e.g. the experimenter, ceiling lights, rack, pictures, etc.), which were visible from within the pool and could be used by the rats for spatial orientation. The locations of the cues were unchanged throughout the period of testing. A video camera fixed to the ceiling over the center of the maze was used for recording and monitoring movements of the animals. There were the four equally divided quadrants in the pool. In one of the quadrants, a platform (1.0 cm below water surface, 10 cm in diameter) was submerged centrally and fixed in position which was kept constant throughout the acquisition or probe trials. The rats performed the five trials per day for the four consecutive days (20 trials). In the swimming trials each individual rat was released gently into the water at a randomly chosen quadrant except for the one that contained the hidden platform for facing an extra maze cue. The rat swam and learned how to find the hidden platform within 60 s. After reaching, the rat was allowed to stay on the platform for 15 s and was then taken back into the cage. During the inter-trial intervals, the rats were kept in a dry home cage for 60 s.

In order to assess the spatial memory, the platform was kept away from the maze for 24 hours in the final trial. Each rat was placed into the water as in the training trials and the time in seconds spent in the quadrant formerly occupied by the platform (correct quadrant) was recorded. The platform remained in the same quadrant during the entire experiment. The rats were required to find the platform using only the distal spatial cues available in the testing room. The cues were kept constant throughout the testing.

**Figure 7.** The quarters of the water maze and the hidden platform are seen.

An interaction effect between the group and the treatment was significant on the total distance travelled on the open field, *F*(3, 36) = 6.15, *p* < 0.002, η2 = .34. This effect reflected the fact that in control condition subjects received 100 μg/kg melatonin (M = 699.65) and 1 μg/kg melatonin (M = 690.46) treatments travelled less distance than those received diazepam (M = 1400.04) and saline (M = 1214.95), whereas in the pinealectomy condition, the subjects received diazem (M = 643.75) travelled less distance than those received 100 μg/kg melatonin (M = 1070.22), 1 μg/kg melatonin (M = 914.38) and saline (M = 902.11) treatments.

**Figure 10.** 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

An interaction effect between the group and the treatment was also significant, *F* (3, 36) = 5.38, *p* = .004, η2 = .31. Indicating that in control condition subjects received diazepam spent less time than the other treatments whereas, in pinealectomy condition the subjects were not

The interaction effect between the group and the treatment was also significant, *F*(3, 36) = 5.29, *p* < 0.004, η2 = .31. Indicating that in control condition subjects received diazepam spent

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.)

*3.1.1.2. Time spent at the edge of the open field (Edge duration)* 

*3.1.1.3. Time spent at the center of the open field (Center duration)* 

significantly different from each other.

**3. Results** 

**3.1. Anxiety measures** 

*3.1.1. Open field measurements* 

*3.1.1.1. Total distance travelled* 

**Figure 8.** Rat is on the platform after it found the hidden platform.

**Figure 9.** Total distance travelled is high. Time to find the platform is long.

### **2.10. Statistical analyses**

Data were analyzed using SPSS (SPSS Statistical Software, SPSS Inc., Los Angeles, CA, USA, Ver. 15.0). A 2 (pinealectomy and control) X 4 (treatments: saline, diazepam, 1 μg/kg melatonin and 100 μg/kg melatonin) ANOVA analyses on data were performed with the last factor as a within subject or repeated design. Significant ANOVA results were also tested by the post test, namely the Tukey test which is assumed to be a strong test for comparison of groups that has equal variance and sample size. Values were considered statistically significant at p ≤ 0.05. Data are presented as MEAN SEM after back transforming from ANOVA results.
