Behavioural Brain Research 222 (2011) 141–150 Contents lists available at ScienceDirect Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr Research report The effects of the intraamygdalar melatonin injections on the anxiety like behavior and the spatial memory performance in male Wistar rats Alper Karakaş a,∗ , Hamit Coşkun b , Aliye Kaya a , Ayşegül Kücük c , Bülent Gündüz d a Department of Biology, Faculty of Arts and Sciences, Abant Izzet Baysal University, Bolu, Turkey Department of Psychology, Faculty of Arts and Sciences, Abant Izzet Baysal University, Bolu, Turkey c Department of Physiology, Faculty of Medicine, Dumlupınar University, Kütahya, Turkey d Department of Biology, Faculty of Arts and Sciences, Çanakkale 18 Mart University, Çanakkale, Turkey b a r t i c l e i n f o Article history: Received 22 February 2011 Received in revised form 7 March 2011 Accepted 11 March 2011 Keywords: Pineal gland Melatonin Amygdala Anxiety Memory a b s t r a c t In the present study, the effects of intraamygdalar administrations of melatonin (1 and 100 ␮g/kg), saline and diazepam on the anxiety-like behavior and spatial memory performance in pinealectomized and sham-pinealectomized Wistar rats were investigated. The animals were tested by open field and elevated plus maze tests for anxiety-like behavior, and Morris water maze test for spatial memory. In open field, (a) diazepam was more effective in reducing the anxiety, (b) control subjects were more mobile than pinealectomized subjects and (c) 100 ␮g/kg melatonin administrations reduced the velocity of the animals. In elevated plus maze, (a) 100 ␮g/kg melatonin administrations increased the distance totally travelled and (b) enhanced the time spent in open arms, however, after the pinealectomy, 1 ␮g/kg melatonin administrations decreased it and (c) control animals were less mobile than pinealectomized ones. In Morris water maze, (a) diazepam group travelled more distance than the others in control condition whereas, in pinealectomy condition high dose of melatonin and saline groups travelled more distance than the others, (b) in pinealectomy condition subjects who received 100 ␮g/kg melatonin also travelled more distance than those who received 1 ␮g/kg melatonin and diazepam, (c) the subjects who received 1 ␮g/kg spent less time than those who received other treatments, and (d) in control condition subjects who received 100 ␮g/kg melatonin were slower than those who received the other treatments. In conclusion, melatonin administration to amygdala decreased the anxiety; however, spatial memory performance of the rats was impaired by the pinealectomy and melatonin administrations. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Pineal gland is located in the epithalamia of the brain and the main hormone of this gland is melatonin that informs the body about the environmental light and dark regimen [31]. The production of melatonin follows a circadian fashion; with highest concentrations during the dark phase of the day and lowest during the light phase of the day. This rhythm is similar in all animals whether they are nocturnal or diurnal [5]. The amphilicity of the melatonin is allowing the molecule to enter any cell, compartment or body fluid [45]. The removal of the pineal gland or namely called pinealectomy, abolishes the rhythmic endogenous melatonin release and decreases the plasma levels of melatonin significantly [26]. ∗ Corresponding author. Tel.: +90 374 254 1232; fax: +90 374 253 4642. E-mail address: karakas a@ibu.edu.tr (A. Karakaş). 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.03.029 Melatonin plays a moderator role in the circadian regulation of many physiological functions such as sleep, visual, cerebrovascular, reproductive, neuroendocrine, and neuroimmunological functions [5,10,11,38,58,61]. In addition to its physiological functions, melatonin seems to produce some psychotropic effects in rodents, such as sedative [21], anticonvulsant [21], antidepressant [20,35], and anxiolytic [41,43] effects. Moreover, it affects some behavioral processes [33] such as stress- and anxiety-related behaviors [34]. With regard to behavioral processes, melatonin binding sites have been found in the regions implicated in cognition and memory in the brain [13,60]. Previous studies have shown that passive and active avoidance learning are affected by melatonin [32,37]. Melatonin that decreases recognition time, leads to a facilitation of short-term memory [6]. There is also the research evidence that demonstrate the effect of melatonin implementations on learning performance [30]. Karakaş et al. [30] investigated the effects of pinealectomy, constant release melatonin implants, and timed melatonin injections on spatial memory in male rats by using Morris water maze. They found that spatial memory performance of the 142 A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 rats was impaired by the pinealectomy and melatonin injections since they elongated the latency and shortened the time passed in the correct quadrant. Melatonin implantation did not affect the spatial memory performance of the rats. Their findings suggest that while the removal of the pineal gland and exogenous administration of melatonin via injections are causing impairment, constant release melatonin administration via implantation does not affect the spatial memory in Wistar albino rats. There is also consistent research evidence that a low dose of melatonin given from weaning did lead to learning and memory deficit in rats [12]. Despite this new emerging evidence in the literature, there is more research needed for illuminating the role of the implementations on the various areas of the rat’s brain. For instance, the effect of intraamygdalar melatonin administration on anxiety-like behaviors and spatial learning has not been investigated yet. There are some studies demonstrating that amygdale located in the temporal lobe of the mid brain plays a regulatory role for behaviors related to anxiety and depression [9,16,24,36]. Serotonergic activity is especially high in this brain region [1,2]. Since the percentage of the prevalence of the anxiety disorder is relatively high in general population, researchers tend to focus on some experimental treatments that decrease behavioral incidences of anxiety. For instance, a research has shown that mCPP (a serotonin receptor agonist) microinjections to amygdale increased behavioral indices of anxiety without altering general activity level. In other words, it decreased open arm time and entries, but increased the closed arm ones [16]. Also, Herdade et al. [25] injected locally muscimol (a GABAA receptor agonist) to the medial nucleus of the amygdale and found that such treatment inhibited escape behavior in elevated T maze. The administration of melatonin to amygdale may show either similar or different effects on anxiety like behaviors. An experimental research is needed for illuminating such treatment on anxiety. However, such a plausible effect has not been reported or written in the literature, according to the very best of our knowledge. In addition to the regulatory role of amygdale in anxiety, amygdale is of great importance in regulating memory and learning functions. For instance, the removal of the temporal lobe in animals leads to an impairment in memory and this impairment is global and thus none of the sensory memory is developed. Anterograde amnesia occurs after the removal of amygdale. The subjects experience difficulties in learning new material after the removal of amygdale [4]. One research has shown that amygdale damage enhance new food intake but this damage was suggested to lead to an impairment of learning an association between an auditory cue and food reward [56]. McIntyre et al. [40] also injected scopolamine, the muscarinic receptor antagonist, to amygdala and found that such treatment impaired performance on conditioned place preference task but not a spatial radial maze task. Moreover, Addy et al. [3] have shown that the infusion of nicotinic receptor antagonists methyllycaconitine (MLA) or dihydro-b-erythroidine (DHbE) impaired working memory. Taken together, these studies suggest that amygdala damage influenced the cognitive performance. Despite these endeavors in the literature, the effect of melatonin administration to amygdala is still not known. It is assumed that its administration to amygdala would produce stronger effect than external melatonin administration. The administration of melatonin to amygdala with the abolishment of melatonin hormone via pinealectomy may produce different effects on anxiety-like and learning behaviors. In other words, the endogeneous melatonin concentration and the rhythm of melatonin release may affect the effects of exogeneous melatonin administration on such behaviors. Given the considerations mentioned above, the present study was conducted to investigate the effects of intraamygdalar melatonin injections to amygdale and pinealectomy on the anxiety-like behavior and spatial memory performance of the rats. 2. Materials and methods 2.1. Animal care A total of 47 adult male Wistar rats (200–250 g) were obtained from our laboratory colony maintained at the Abant Izzet Baysal University (AIBU). They were exposed from birth to 12 l (12 h of light, 12 h of darkness, lights off at 18:00 h). Animals were maintained in plastic cages (16 cm × 31 cm × 42 cm) with pine shavings used as bedding. Food pellets and tap water were accessible ad libitum. The procedures in this study were carried out in accordance with the Animal Scientific procedure and approved by the Institutional Animal Care and Use Committee. All lighting was provided by the cool-white fluorescent tubes controlled by automatic programmable timers. The ambient temperatures in the animal facilities were held constant at 22 ± 2 ◦ C in air-ventilated rooms. 2.2. Experimental protocol In the present study, the 47 male adult rats were used and were randomly divided into two groups as control (sham – pinealectomy) and pinealectomy. In the control group animals were exposed to the same surgical procedure with the experimental group except for the removal of the pineal gland. Under the groups, the four subgroups were performed as Melatonin (1 and 100 ␮g/kg) (n:14), Saline (0.9%NaCl) (n:5) and Diazepam (2 mg/kg) (n:5). All pinealectomies and cannulation surgeries were applied before starting the experiment. We started the experiment after a week of the pinealectomies and implantations, when surgery wounds healed up completely. The animals were tested by open field and elevated plus maze tests for anxiety-like behavior, and Morris water maze test for spatial memory. All animals were exposed to these behavioral testings after 30 min of melatonin, saline, and diazepam administrations. 2.3. Anesthesia Before surgery, rats were anesthetized subcutaneously with Ketamine (20 mg/kg BW, Sigma Chemical Company, MO, USA) and intraperiotoneally with pentobarbitol (32.5 mg/kg BW). The depth of anesthesia was monitored by frequent testing for the presence of leg flexion reflexes and active muscle tonus. After awaking from anesthesia, the animals were placed in their cages. 2.4. Cannulation Cannula was implanted into the amygdala. The rats were anesthetized and fixed in a stereotaxic instrument (Stoelting Co., IL, USA) and a hole was opened at the skull by a dental drill; a 22-gauge stainless steel guide cannula 313-G/Spc (Plastics One Inc., VA, USA) was implanted aseptically into amygdala region (coordinates: −2.6 mm posterior to the bregma; +4.3 mm lateral to the midline and −8.4 mm ventral according to the skull). The guide cannula was secured in place by dental cement (Dental Products of Turkey, Istanbul) affixed to two mounting screws. A stainless steel dummy cannula was used to occlude the guide cannula when not in use. Each cannulated rat was then kept individually for a week to recover from surgery. 2.5. Pinealectomy The pinealectomy of Wistar rats was performed according to the method of Hoffmann and Reiter [26]; aspiration was used to control the hemorraging. The anesthetized rats were placed in a stereotaxic apparatus to stabilize the head during surgery. After the head was shaved the surgical area was sterilized with 70% ethanol, an incision was made in the scalp. Muscle attachments were removed from the dorsal skull. After drying the skull, an incomplete circular cut was made with a dental drill burr at the  (lambda) suture and a piece of cranium covering the pineal gland was folded forward anteriorly. The fine-tipped forceps were used to extend into the confluence of the sinuses to grasp and remove the pineal gland. After the removal of the pineal gland, the bone flap was replaced and a small square of absorbable gelatin sponge (Gelfoam, Up John, Kalamazoo, MI) was applied to the skull surface to help promote clotting. The scalp was closed with stainless steel surgical clips. After the surgery, the incision was treated with Newskin adhesive to prevent any contamination. At the end of the experiment, pinealectomized animals were decapitated and checked for the security of the pinealectomy. 2.6. Melatonin administrations First, melatonin was dissolved in 100% ethanol (1/10 ␮l) and then diluted in saline (0.9% NaCl) (9/10 ␮l) to the desired concentrations. Stock solutions were kept at 4 ◦ C prior to use. The stock was diluted with sterile saline to the desired concentrations in order to make fresh working melatonin solutions. Vehicle solutions were made in the ratio of one part absolute ethanol to 1000 parts sterile saline. Melatonin was injected in a dose of either 1 or 100 ␮g/kg (15:00 p.m.). A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 2.7. Open field Open-field test was taken place in a 80 cm × 80 cm arena with 40 cm high walls. The open field has been the most widely used test in animal psychology. In this test, an animal (usually a rodent) is introduced into a plain and illuminated arena and its behavior is commonly regarded as a fundamental index of general behavior. In this experiment a video camera (Gkb CC-28905S, Commat Ltd. ŞTİ, Ankara, Turkey) was mounted above the arena, recording behavior into the Ethovision videotracking system (Noldus Ethovision, Version 6, Netherland; Commat Ltd. ŞTİ, Ankara, Turkey) that provided a variety of behavioral measures including distance, time in the edge, time in the center, frequency in the edge, frequency in the center, mobility and velocity among the different areas of the arena. All animals were then returned to the breeding and exhibition colonies. 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. 2.9. Morris water maze 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 h 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. 2.10. Statistical analyses Data were analyzed using SPSS (SPSS Statistical Software, SPSS Inc., Los Angeles, CA, USA, Ver. 15.0). Data were analyzed by 2 (pinealectomy and control) × 4 (treatments) ANOVA analysis 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. 3. Results 3.1. Open field measurements 3.1.1. Total distance travelled 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, 2 = .34. This interaction effect reflected the fact that in control condition subjects received 100 ␮g/kg mela- 143 tonin (M = 699.65) and 1 ␮g/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 ␮g/kg melatonin (M = 1070.22), 1 ␮g/kg melatonin (M = 914.38) and saline (M = 902.11) treatments (Fig. 1A). 3.1.2. Time spent at the edge of the open field (edge duration) An interaction effect between the group and the treatment was also significant, F(3, 36) = 5.38, p = .004, 2 = .31. This interaction effect reflected the fact that in control condition subjects received diazepam spent less time than the other treatments whereas, in pinealectomy condition the subjects were not significantly different from each other (Fig. 1B). 3.1.3. Time spent at the center of the open field (center duration) The interaction effect between the group and the treatment was also significant, F(3, 36) = 5.29, p < 0.004, 2 = .31. This interaction effect reflected the fact that in control condition subjects received diazepam spent more time than the other treatments whereas, in pinealectomy condition the subjects were not significantly different from each other (Fig. 1C). 3.1.4. Entrance frequency to the edge of the open field (edge frequency) The interaction effect between the group and the treatment was significant, F(3, 36) = 3.02, p < 0.04, 2 = .20. This interaction effect reflected the fact that in control condition subjects received diazepam entered more frequently to the edge of the open field than the other treatments, whereas in pinealectomy condition the subjects who received saline treatment entered more frequently than the other treatments (Fig. 1D). 3.1.5. Entrance frequency to the center of the open field (center frequency) The interaction effect between the group and the treatment was significant, F(3, 36) = 3.02, p < 0.04, 2 = .20. This interaction effect reflected the fact that in control condition subjects received diazepam entered more frequently to the center of the open field than the other treatments, whereas in pinealectomy condition the subjects who received saline treatment entered more frequently than the other treatments (Fig. 1E). 3.1.6. Mobility The main effect of the group was significant, F(1, 36) = 6.89, p = .01, 2 = .16. Control group was more mobile than pinealectomy group. The main effect of the treatment was also significant, F(3, 36) = 6.73, p = .001, 2 = .36. The subjects who received saline were more mobile on the open field than the other subjects with each being not significantly different from each other (Fig. 1F). In addition, the interaction effect between the group and the treatment was significant, F(3, 36) = 7.08, p < 0.001, 2 = .37. This interaction effect reflected the fact that in control condition subjects received saline was more mobile than the other treatments, whereas in pinealectomy condition the subjects who received 100 ␮g/kg melatonin treatment were more mobile than the other treatments (Fig. 1F). 3.1.7. Velocity The interaction effect between the group and the treatment was significant, F(3, 36) = 6.52, p < 0.001, 2 = .35. This interaction effect reflected the fact that in control condition subjects received saline and diazepam were faster than the other treatments, whereas in pinealectomy condition the subjects who received 100 ␮g/kg melatonin treatment were faster than the other treatments (Fig. 1G). 144 A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 Fig. 1. (A) The total distance travelled (TDT), (B) the time spent at the edge of the open field (TSEO), (C) the time spent at the center of the open field (TSCO), (D) the entrance frequency to the edge of the open field (EFEO), (E) the entrance frequency to the center of the open field (EFCO), (F) the mobility time (MT) and (G) the velocity (VEL) are represented for the open field. Right striated bar represents the saline injection and black bar represents diazepam injections, cross striated bar represents 1 ␮g/kg melatonin injection and bricks striated bar represents 100 ␮g/kg melatonin injection for both control and pinealectomy groups. Data are presented as means (±S.E.M.). Different letters indicate the statistically different groups. A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 145 Fig. 2. (A) The total distance travelled (TDT), (B) the time spent in open arms (TSOA), (C) the time spent in closed arms (TSCA), (D) the entrance frequency to the open arms (EFOA), (E) the entrance frequency to the closed arms (EFCA), (F) the mobility time (MT) and (G) the velocity (VEL) are represented for the elevated plus maze. Right striated bar represents the saline injection and black bar represents diazepam injections, cross striated bar represents 1 ␮g/kg melatonin injection and bricks striated bar represents 100 ␮g/kg melatonin injection for both control and pinealectomy groups. Data are presented as means (±S.E.M.). Different letters indicate the statistically different groups. 146 A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 Fig. 3. (A) The total distance travelled (TDT), (B) the time spent to find the platform (TSFP), (C) the time spent in the correct quadrant (TSCQ), (D) the entrance frequency to the correct quadrant (EFCQ), (E) the mobility time (MT) and (F) the velocity (VEL) are represented for the Morris water maze. Right striated bar represents the saline injection and black bar represents diazepam injections, cross striated bar represents 1 ␮g/kg melatonin injection and bricks striated bar represents 100 ␮g/kg melatonin injection for both control and pinealectomy groups. Data are presented as means (±S.E.M.). Different letters indicate the statistically different groups. 3.2. Elevated plus maze measurements 3.2.1. Total distance travelled The main effect of the treatment was significant, F(3, 39) = 3.06, p = .04, 2 = .19. The groups who received diazepam travelled less distance than those who received 100 ␮g/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, 2 = .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 ␮g/kg melatonin treatments travelled less distance than other treatments, whereas in the pinealectomy condition, the subjects received 100 ␮g/kg melatonin travelled less distance than the other treatments (Fig. 2A). 3.2.2. Time spent in open arms (open arm duration) The main effect of the treatment was significant, F(3, 39) = 6.53, p = .001, 2 = .33. The subjects who received 100 ␮g/kg melatonin treatment spent more time in open arms than those who received other treatments (Fig. 2B). An interaction effect between the group and the treatment was also significant, F(3, 39) = 6.87, p < 0.001, 2 = .35. This interaction effect reflected the fact that in control condition subjects received 100 ␮g/kg melatonin treatment spent more time than those receiving the other treatments, whereas in pinealectomy condition the subjects who received saline, 100 ␮g/kg melatonin and diazepam A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 treatments spent more time than those who received 1 ␮g/kg melatonin treatment (Fig. 2B). 3.2.3. Time spent in closed arms (closed arm duration) The main effect of the treatment was significant, F(3, 39) = 6.56, p = .001, 2 = .34. The subjects who received 100 ␮g/kg melatonin treatment spent less time in closed arms than those who received other treatments (Fig. 2C). An interaction effect between the group and the treatment was also significant, F(3, 39) = 7.30, p < 0.001, 2 = .36. This interaction effect reflected the fact that in control condition subjects received 100 ␮g/kg melatonin treatment spent less time than those receiving the other treatments, but there were no significant differences between treatment conditions in pinealectomy (Fig. 2C). 3.2.4. Entrance frequency to open arms The main effect of the group was significant, F(1, 39) = 14.40, p = .001, 2 = .27. The subjects in the control condition entered more frequently to open arm than those in the pinealectomy (Fig. 2D). The main effect of the treatment was also significant, F(3, 39) = 19.39, p = .0001, 2 = .60. The subjects who received 100 ␮g/kg melatonin treatment entered more frequently to the open arm than those who received other treatments (Fig. 2D). In addition, the interaction effect between the group and the treatment was significant, F(3, 39) = 37.65, p = 0.0001, 2 = .74. This interaction effect reflected the fact that in control condition subjects who received 100 ␮g/kg melatonin treatment entered more frequently than those who received the other treatments, whereas in pinealectomy condition the subjects who received saline, 1 ␮g/kg melatonin and diazepam treatments entered more frequently than those who received 100 ␮g/kg melatonin treatment. 147 This interaction effect reflected the fact that there were no significant differences between control and pinealectomy groups in 100 ␮g/kg and 1 ␮g/kg melatonin treatments but was significant differences between these groups in saline and diazepam treatments (Fig. 3B). 3.3.3. Time spent in the correct quadrant The main effect of the treatment was also significant, F(3, 40) = 4.11, p = .01, 2 = .24. The subjects who received 1 ␮g/kg melatonin treatment spent less time than those who received other treatments with each being not significant from each other (Fig. 3C). In addition, the interaction effect between the group and the treatment was also significant, F(3, 40) = 9.29, p = 0.001, 2 = .41. This interaction effect reflected the fact 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. 3.3.4. 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, 2 = .34. This interaction effect reflected the fact 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 (Fig. 3D). 3.2.5. 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 (Fig. 2E). 3.2.6. Mobility The main effect of the group was significant, F(1, 39) = 6.95, p = .01, 2 = .15. The subjects in the pinealectomy condition were more mobile than those in the control condition (Fig. 2F). 3.2.7. Velocity No significant effects were found with regard to the total entrance to the closed arm of the elevated plus maze (Fig. 2G). 3.3. Morris water maze measurements 3.3.1. 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, 2 = .27. This interaction effect reflected the fact that in control condition, the subjects received diazepam travelled more distance than the other treatments, whereas in pinealectomy condition subjects who received 100 ␮g/kg melatonin and the saline treatments travelled more distance than those who received 1 ␮g/kg melatonin and diazepam treatments (Fig. 3A). 3.3.2. Time spent to find the platform (latency) The main effect of the treatment was significant, F(3, 40) = 3.02, p = .04, 2 = .19. The subjects who received diazepam treatment spent more time than those who received 1 ␮g/kg melatonin treatment (Fig. 3B). In addition, the interaction effect between the group and the treatment was also significant, F(3, 40) = 4.90, p = 0.005, 2 = .41. Fig. 4. The figure represents the amydala region of the brain where the injections were applied. Blue colored region of the brain represents the amygdala region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) 148 A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 3.3.5. Mobility No significant effects were found with regard to the mobility in the Morris water maze (Fig. 3E). 3.3.6. Velocity The main effect of the group was significant, F(1, 40) = 11.31, p = .002, 2 = .22. The subjects in the control condition were faster than those in the pinealectomy condition (Fig. 3F). The main effect of the treatment was also significant, F(3, 40) = 4.16, p = .01, 2 = .24. The subjects who received diazepam treatment were slower than those who received other treatments (Fig. 3F). In addition, the interaction effect between the group and the treatment was significant, F(3, 40) = 4.13, p = 0.01, 2 = .24. This interaction effect reflected the fact that in control condition subjects who received 100 ␮g/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. 3.4. Evaluation of the placement of the cannula After all experiments finished, animals were decapitated and the brains were removed. The placement of the cannulas was checked histologically if they were placed to the amygdala of the brain or not. Fig. 4 represents a histological section of a brain which the cannula was placed correctly. 4. Discussion The results of the present study can be classified under two main headings: anxiety-like behavior (in open field apparatus and elevated plus maze) and spatial memory performance (in Morris water maze). 4.1. Anxiety-like behaviors In open field, (a) diazepam was more effective in reducing the anxiety since the time passed at the center of the open field was longer especially than those the 0.1 melatonin administration treatments, (b) the biggest difference in between the controls and the pinealectomies was apparent in mobility. The control subjects were more mobile than the pinealectomized subjects and (c) 100 ␮g/kg melatonin administration reduced the velocity of the animals. The finding of the present research showed that diazepam was more effective in reducing the anxiety. This effect was expected since the diazepam inhibits the serotonergic activity via GABAergic system. Benzodiazepines are widely used in reducing the anxietylike behaviors. They are preferred because of their effectiveness and wide therapeutic index. They make their effect by binding their receptors which are found near the GABA receptors and by making an allosteric effect. By this way, they increase the affinity of these GABA receptors to benzodiazepines [52]. The second finding was that the biggest difference in between the controls and the pinealectomies was apparent in mobility. This suggests that mobility measurement is more sensitive to the removal of pineal gland. It should be kept in mind that such effect was not observed in terms of other indices of the anxiety-like behaviors in the present study. This finding also suggests that the amount and the rhythm of the endogeneous melatonin release in the pinealectomized animals is abolished; however, this endogeneous rhythm in the sham pinealectomized animals is intact. Therefore, the plausible effect of external high dose of melatonin administration may not become evident. Our results, which showed that the anxiety like behavior was not significantly affected by the pinealectomy in rats, are in good agreement with the findings of the previous studies indicating that pinealectomy alone did not have a significant effect on anxiety behavior [29,32]. This suggests that the pineal gland is partially involved in the anxiety-like behaviors. The third finding was that the high dose of melatonin (100 ␮g/kg) administrations reduced the velocity of the animals. This effect of melatonin might be due to the direct inhibition of locomotor activity, rather than an effect on the circadian clock. In elevated plus maze, (a) the high dose of melatonin administrations increased the distance totally travelled. However, the high dose of melatonin administration after pinealectomy decreased the distance totally travelled, (b) the high dose of melatonin administration enhanced the time spent in open arms, however, after the pinealectomy, the low dose of melatonin (1 ␮g/kg) administration decreased it and (c) control animals were less mobile than pinealectomized ones. The present finding that the high dose of melatonin administration induced increase in travelled distance was reversed by the pinealectomy suggest that internal melatonin concentrations and rhythm may be more likely to change the effects of exogeneous melatonin administration in the anxiety like behaviors. It is well known fact that pinealectomy abolishes the rhythmic endogenous melatonin release and decreases the plasma levels of melatonin significantly [26]. Thus, after the removal of the pineal gland a high dose of melatonin could show its effect on anxiety-like behavior. The second finding in the elevated plus maze was that the high dose of melatonin administration enhanced the time spent in open arms, while, after the pinealectomy, the low dose of melatonin (1 ␮g/kg) administration decreased it. There is evidence in the literature that suggesting the interaction of melatonin with central gama aminobutyric acid (GABA) neurotransmission. Melatonin has been shown to enhance the GABA in rat brain tissue in vitro [15,42]. When melatonin was applied in vivo, it increased the GABA levels in several brain regions in rats [49,62]. In conclusion, our findings can be attributed the fact that high dose of melatonin administration increased the GABA levels, which in turns reduce anxiety like behaviors. Through this mechanism, the high dose of melatonin administered subjects spent more time in open arms than the others. The third finding in the elevated plus maze was that pinealectomy increased the mobility time in compared to controls suggest that mobility measurement is more sensitive to the removal of pineal gland. One can see that, this effect was opposite of what was found in open arms. This difference may be due to the task difference between open field and elevated plus maze. Motor functions such as spontaneous activity is measured by the open field. Open field test is also used to measure the anxiety like behavior in rodents [8]. The total distance travelled, 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 [46]. In this maze, if the anxiety of the animal is high, the number of the entries to the edge of the open field is increasing and the total distance travelled is decreasing. The total number of the entries into the center and the edges provides a built-in control measure for general hyperactivity or sedation. On the other hand, the elevated plus maze has been one of popular or widely used test to measure the anxiety like behaviors [17]. In this maze, if the anxiety of the animal is high, the number of the entries to closed arms is increasing and the total distance travelled is decreasing. The total number of the entries into all arms provides a built-in control measure for general hyperactivity or sedation. Regarding elevated plus maze and open field tests, the present study represent a difference in mobility, which needs a further investigation. Our findings also suggest that the elevated plus maze condition A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 provides melatonin specific outcomes more than the open field condition. 4.2. Spatial memory performance In the Morris water maze, the present study has shown that (a) the diazepam group did travel more distance than the others in the control condition whereas, in the pinealectomy condition the high dose of melatonin and saline groups travelled more distance than the others, (b) in the pinealectomy condition the subjects who received the high dose of melatonin treated animals also travelled more distance than those who received the low dose of melatonin and diazepam, (c) the subjects who received 1 ␮g/kg melatonin spent less time than those who received other treatments, and (d) in the control condition the subjects who received the high dose of melatonin treatment were slower than those who received the other treatments. Longer distance travelled and less time spent in the correct quadrant indicates less spatial learning in this maze. It should be especially noted that the high doses of melatonin decreased some behavioral indices of spatial memory. In line with this finding, other studies have consistently shown that amygdala damage through various implementations leads to impairment of learning an association between an auditory cue and food reward [56], of performance on conditioned place preference task [40], and working memory [3]. It is a well known fact that melatonin readily passes all cell membranes, including the bloodbrain barrier [48]. Melatonin binding sites exist in various brain structures such as the hippocampus and prefrontal cortex are considered to involve in memory function [11,18,39,50,51]. Moreover, considering that melatonin is a potent sleep inducing enhanced consolidation of hippocampus-dependent memories [28,47], it is possible that ‘sleep-like’ melatonin effects on consolidation in the aftermath of encoding added to its effects on encoding. Despite this evidence, exact mechanism of melatonin concerning cognitive performance is still not known and there are some plausible explanations. One explanation deals with its pathway. Melatonin could have direct or indirect effect on memory. Some studies have provided evidence for its direct effect. For instance, a research has suggested that melatonin could be involved in structural remodeling of synaptic connections during memory and learning processes [7]. Other research has also suggested that melatonin may influence memory formation in the hippocampus [19]. In addition to its direct action, indirectly, melatonin may act as an antioxidant to reduce oxidative damage to the synapses in hippocampus and therefore improves learning and memory deficits. Tuzcu and Baydas [57] have found evidence indicating that melatonin significantly ameliorated the cognitive impairment, reduced lipid per oxidation, and increased glutathione levels in diabetic rats. In conclusion, the effect of melatonin on learning performance could be in both ways. Even though the present study was not aimed to directly test this explanation, its results suggest that melatonin injection seems to have direct effect on spatial memory that has been related to limbic system of rat brain. Melatonin may also have an indirect effect on learning performance via some neurotransmitter such as gama amino butyric acid (GABA). An increase in melatonin level via injection may also affect the GABA, an inhibitory neurotransmitter, which in turn may decrease the neural transmission in the limbic system. Through this way, melatonin microinjection to amygdala may show its impairing effect on learning and memory processes. In addition, melatonin may also show its effects through its reciprocal relationship with some parts of rat brain such as suprachiasmatic nucleus (SCN). While SCN is generating and controlling the circadian rhythm of melatonin, melatonin hormone is also acting on SCN as a negative feedback agent in order to control the activity of the SCN. It is well known fact that the release of the melatonin hormone in rats shows a circadian pattern which is high throughout 149 the darkness [44]. However, in pinealectomy the blood melatonin levels drop significantly and the rhythm of melatonin is abolished [14]. The other explanation for the effects of melatonin on learning performance is related with the circadian effects of melatonin. Several studies have demonstrated the regulatory roles of melatonin in circadian rhythms [5,10,11]. For instance, our recent experiment has shown that daily injections of melatonin can entrain the activity rhythms of the pinealectomized Mongolian gerbils (Meriones unguiculatus) (unpublished data). This effect of melatonin might be due to the direct inhibition of locomotor activity, rather than an effect on the circadian clock. It should be kept in mind that we implemented microinjections in the afternoon when the melatonin receptors are re-sensitive to the melatonin hormone. According to the internal coincidence hypothesis, melatonin exerts an effect only when its circadian secretion is coincident with target tissue sensitivity. This hypothesis supposes that the time of presence of melatonin is important [22,23,27,53–55,59]. In line with this explanation, we found in our another study that pinealectomy and only admistration of melatonin via timed injections caused impairment of the learning performance of the rats [30]. In conclusion, the results of the present experiment have indicated that the data coming from the elevated plus maze and the open field are consistent to each other. However, our results have suggested that elevated plus maze measurements were more sensitive to the melatonin microinjections to amygdala than open field measurements, since the differences were more evident in this maze. This suggests that pinealectomy treatment interacts with anxiety provoking test situations. In open field, it was assumed that the anxiety level experience by animals may be greater in elevated plus maze than open field. In open field the mobility was smaller in pinealectomized rats than controls since the anxiety level may be low compared to the elevated plus maze. This explanation requires further experimental research that illuminates differential effects of pinealectomy on testing conditions. Taken together, these results are unique contribution to the field of anxiety like behavior and spatial learning in the literature. Further research should take multiple measures of anxiety and learning in the given consideration that melatonin injection produce different outcomes in the investigated parameters in open field, elevated plus maze and Morris water maze. Acknowledgement This study was supported by the AIBU Scientific Research Project 2009.03.01.310. References [1] Abrams JK, Johnson PL, Shekhar A, Lowry CA. S.07.01 Anxiogenic drugs act selectively ontopographically distinct midbrain, pontine, and medullary serotonergic neurons. Eur Neuropsychopharm 2004;14(Suppl. 3):S124. [2] Abrams JK, Johnson PL, Shekhar A, Lowry CA. P.2.01 Anxiogenic drugs act selectively on topographically distinct midbrain, pontine, and medullary serotonergic neurons. Eur Neuropsychopharm 2004;14(Suppl. 1):S21. [3] Addy NA, Nakijama A, Levin ED. Nicotinic mechanisms of memory: effects of acute local DH beta E and MLA infusions in the basolateral amygdale. Cognit Brain Res 2003;16(1):51–7. [4] Almonte AG, Hamill CE, Chhatwal JP, Wingo TS, Barber JA, Lyuboslavsky PN, et al. Learning and memory deficits in mice lacking protease activated receptor1. Neurobiol Learn Mem 2007;88(3):295–304. [5] Arendt J. Melatonin, circadian rhythms and sleep. N Engl J Med 2000;343:1114–6. [6] Argyriou A, Prast H, Philippu A. Melatonin facilitates short-term memory. Eur J Pharmacol 1998;349:159–62. [7] Baydas G, Nedzvetsky VS, Nerush PA, Kırıchenko SV, Demchenko HM, Reiter RJ. A novel role for melatonin: regulation of the expression of cell adhesion molecules in the hippocampus, cortex and cerebellum. Neurosci Lett 2002;326:109–12. 150 A. Karakaş et al. / Behavioural Brain Research 222 (2011) 141–150 [8] Benabid N, Mesfıoui A, Ouichou A. Effects of photoperiod regimen on emotional behaviour in two tests for anxiolytic activity in Wistar rat. Brain Res Bull 2008. [9] Blackshear A, Yamamoto M, Anderson BJ, Holmes PV, Lundström L, Langel U, et al. Intracerebroventricular administration of galanin or galanin receptor subtype 1 agonist M617 induces c-Fos activation in central amygdala and dorsomedial hypothalamus. Peptides 2007;28(5):1120–4. [10] Borjigin J, Li X, Snyder SH. The pineal gland and melatonin: molecular and pharmacologic regulation. Annu Rev Pharmacol 1999;39:53–65. [11] Brzezinski A. Melatonin in humans. New Engl J Med 1997;336:186–95. [12] Cao XJ, Wang M, Chen WH, Zhu DM, She JQ, Ruan DY. Effects of chronic administration of melatonin on spatial learning ability and long-term potentiation in lead-exposed and control rats. Biomed Environ Sci 2009;22:70–5. [13] Cardinalli DP, Vacas MI, Boyer EE. Specific binding of melatonin in bovine brain. Endocrinology 1979;105:437–41. [14] Chapmann DI. Seasonal changes in the gonads and accessory glands of male mammals. Mammal Rev 1970;1:231–48. [15] Coloma FM, Niles LP. Melatonin enhancement of [3 H]-gamma-aminobutyric acid and [3 H] muscimol binding in rat brain. Biochem Pharmacol 1988;37:1271–4. [16] Cornélio AM, Luiz Nunes-de-Souza R. Anxiogenic-like effects of mCPP microinfusions into the amygdala (but not dorsal or ventral hippocampus) in mice exposed to elevated plus-maze. Behav Brain Res 2007;178(1):82–9. [17] Dawson GR, Tricklebank MD. Use of the elevated plus maze in the search for novel anxiolytic agents. Trends Pharmacol Sci 1995;16(2):33–6. [18] Ekmekcioglu C. Melatonin receptors in humans: biological role and clinical relevance. Biomed Pharmacother 2006;60:97–108. [19] El-Sherif Y, Tesoriero J, Hogan MV, Wieraszko A. Melatonin regulates neuronal plasticity in the hippocampus. J Neurosci Res 2003;72:454–60. [20] Ergun Y, Ergun UG, Orhan FO, Kucuk E. Co-administration of a nitric oxide synthase inhibitor and melatonin exerts an additive antidepressant-like effect in the mouse forced swim test. Med Sci Monit 2006;12:307–12. [21] Golombek DA, Pevet P, Cardinalli DP. Melatonin effects on behavior: possible mediation by the central GABAergic system. Neurosci Biobehav Rev 1996;20:403–12. [22] Gündüz B, Stetson MH. A test of the coincidence and duration models of melatonin action in Siberian hamsters: the effects of one hour melatonin infusions on testicular development of intact and pinealectomized juvenile Siberian hamsters, (Phodopus sungorus). J Pineal Res 2001;30:97–107. [23] Gündüz B, Stetson MH. A test of the coincidence and duration models of melatonin action in Siberian hamsters: II: The effects of 4- and 8-hr melatonin infusions on testicular development of pinealectomized juvenile Siberian hamsters, (Phodopus sungorus). J Pineal Res 2001;30:56–64. [24] Hale MW, Bouwknecht JA, Spiga F, Shekhar A, Lowry CA. Exposure to high- and low-light conditions in an open-field test of anxiety increases c-Fos expression in specific subdivisions of the rat basolateral amygdaloid complex. Brain Res Bull 2006;71(1–3):174–82. [25] Hardeland R, Pandi-Perumal SR, Cardinali DP. Melatonin. Int J Biochem Cell Biol 2006;38(3):313–6. [26] Hoffman RA, Reiter RJ. Rapid pinealectomy in hamsters and other small rodents. Anat Record 1965;24:83–9. [27] Hong SM, Stetson MH. Detailed diurnal rhythm of sensitivity to melatonin injections in Turkish hamsters. Mesocricews brandsi. J Pineal Res 1987;4:69–78. [28] Jern C, Manhem K, Eriksson E, Tengborn L, Risberg B, Jern S. Hemostatic responses to mental stress during the menstrual cycle. Thromb Haemostasis 1991;66:614–8. [29] Juszcak M, Drobnik J, Guzek JW, Schwarzberg H. Effect of pinealectomy and melatonin on vasopressin-potentiated passive avoidance in rats. J Physiol Pharmacol 1996;47:621–7. [30] Karakaş A, Coşkun H, Kaya A. The effects of pinealectomy, melatonin injections and implants on the spatial memory performance of male Wistar rats. Biol Rhythm Res; doi:10.1080/09291016.2010.537443, in press. [31] Klein DC. The mammalian melatonin rhythm generating system. In: Watterberg L, editor. Light and biological rhythms in man. New York: Pergamon Pres; 1993. p. 55–70. [32] Kovács GL, Gajari I, Telegdy G, Lissak K. Effects of melatonin and pinealectomy on avoidance and exploratory activity in the rat. Physiol Behav 1974;13:349–55. [33] Krause DN, Dubocovich ML. Regulatory sites in the melatonin system of mammals. Trends Neurosci 1990;13:464–70. [34] Loiseau F, Bihan CL, Hamon M, Thiebot MH. Effects of melatonin and agomelatine in anxiety-related procedures in rats: interaction with diazepam. Eur Neuropsychopharm 2006;16:417–28. [35] Mantovani M, Pertile R, Calixto JB, Santos AR, Rodrigues AL. Melatonin exerts an antidepressant-like effect in the tail suspension test in mice: evidence for [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] involvement of N-methyl-d-aspartate receptors and the l-arginine-nitric oxide pathway. Neurosci Lett 2003;343:1–4. Martinez LA, Klann E, Tejada-Simon MV. Translocation and activation of Rac in the hippocampus during associative contextual fear learning. Neurobiol Learn Mem 2007;88(1):104–13. Martini L. Behavioral effects of pineal principles. In: Wolsten-holme GEW, Knight J, editors. The pineal gland. Edinburgh: Livingstone; 1971. p. 368–72. Masana MI, Dubocovich ML. Melatonin receptor signaling: finding the path through the dark. Sci STKE 2001:pe39. Mazzucchelli C, Pannacci M, Nonno R, Lucini V, Franchini F, Stankov BM. The melatonin receptor in the human brain: cloning experiments and distribution studies. Brain Res Mol Brain Res 1996;39:117–26. McIntyre CK, Ragozzino ME, Gold PE. Intra-amygdala infusions of scopolamine impair performance on a conditioned place preference task but not a spatial radial maze task. Behav Brain Res 1998;95(2):219–26. Naranjo-Rodriguez EB, Ortiz Orsornio A, Hernandez-Avitia E, MendozaFernandez V, Escobar A. Anxiolytic-like actions of melatonin, 5-methoxytryptophan, 5-hydroxytryptophol and benzodiazepines on a conflict procedure. Prog Neuropsychopharmacol Biol Psychiatry 2000;24:117–29. Niles LP, Pickering DS, Arciszewski MA. Effects of chronic melatonin administration on GABA and diazepam binding in rat brain. J Neural Transm 1987;70:117–24. Papp M, Litwa E, Gruca P, Mocaer E. Anxiolytic-like activity of agomelatine and melatonin in three animal models of anxiety. Behav Pharmacol 2006;17: 9–18. Pickavance L, Tadayyon M, Williams G, Vernon RG. Lactation suppresses diurnal rhythm of serum leptin. Biochem Biophys Res Commun 1998;248: 196–9. Poeggeler B, Saarela S, Reiter RJ, Tan DX, Chen LD, Manchester LC, et al. Melatonin, a highly potent endogeneous radical scavenger and electron donor, new aspects of the antioxidant chemistry of this indole accessed in vitro. Ann Ny Acad Sci 1994;738, xi–xii, 1–467. Pyter LM, Nelson RJ. Enduring effects of photoperiod on affective behaviors in Siberian hamsters (Phodopus sungorus). Behav Neurosci 2006;120(1): 125–34. Rasch B, Buchel C, Gais S, Born J. Odor cues during slowwave sleep prompt declarative memory consolidation. Science 2007;315:1426–9. Reiter RJ, Poeggeler B, Tan DX, Chen LD, Manchester LC, Guerro JM. Antioxidant capacity of melatonin: a novel action not requiring a receptor. Neuroendocrinol Lett 1993;15:103–16. Rosenstein RE, Cardinali DP. Melatonin increases in vivo Gaba accumulation in rat hypothalamus, cerebellum, cerebral cortex and pineal gland. Brain Res 1986;398:403–6. Savaskan E, Olivieri G, Brydon L, Jockers R, Krauchi K, Wirz-Justice A, et al. Cerebrovascular melatonin MT1-receptor alterations in patients with Alzheimer’s disease. Neurosci Lett 2001;308:9–12. Savaskan E, Ayoub MA, Ravid R, Angeloni D, Franchini F, Meier F, et al. Reduced hippocampal MT2 melatonin receptor expression in Alzheimer’s disease. J Pineal Res 2005;38:10–6. Sinclair L, Nutt D. Anxiolytics. Psychiatry 2007;6(7):284–8. Stetson MN, Tay DE. Time course of sensitivity of golden hamsters to melatonin injections throughout the day. Biol Reprod 1983;29:432–8. Stetson MN, Sarafidis E, Rollag MD. Sensitivity of adult male Djungarian hamsters (Phodopus sungorus) to melatonin injections throughout the day: effects on the reproductive system and the pineal. Biol Reprod 1986;35:618–32. Stetson MN, Watson-Whitmyre M. Effect of exogenous and endogenous melatonin on gonadal function in hamsters. J Neural Transm 1986;21:55–80. Sutherland RJ, Mc Donalds RJ. Hippocampus, amygdale and memory deficits. Behav Brain Res 1990;34:57–79. Tuzcu M, Baydas G. Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. Eur J Pharmacol 2006;537:106–10. Vanecek J. Inhibitory effect of melatonin on GnRH induced LH release. Rev Reprod 1999;4:67–72. Watson-Whitmyre M, Stetson MH. A mathematical method for estimating paired testes weight from in situ testicular measurements in three species of hamster. Anat Record 1985;213:473–6. Weaver DR, Rivkees SA, Reppert SM. Localization and characterization of melatonin receptors in rodent brain by in vitro aotoradiography. J Neurosci 1989;9:2581–90. Wirz-Justice A. Treatment tools in chronobiology. Rev Med Interne 2001;22(Suppl. 1):37–8. Xu F, Li JC, Ma KC, Wang M. Effects of melatonin on hypothalamic gammaaminobutyric acid, aspartic acid, glutamic acid, beta-endorphin and serotonin levels in male mice. Biol Signals 1995;4:225–31.