*1.3.1. General features of amygdala*

18 Neuroendocrinology and Behavior

affect anxiety of animals.

*1.2.3. Pinealectomy* 

Reiter, 1965; Hoffmann, 1974).

amino butyric acid (GABA) neurotransmission (Golombek et al., 1996). The literature findings have provided evidence for an interaction between melatonin and central GABA neurotransmission. GABA release is augmented by melatonin in rat brain tissue *in vitro* (Niles et al., 1987; Coloma and Niles, 1988). Also, when melatonin was applied *in vivo*, GABA levels increased in several brain regions in rats (Rosenstein and Cardinali, 1986; Xu et al., 1995). These findings mean that melatonin increases GABA levels, which in turn may

It has been shown that melatonin affects passive and active avoidance learning. Melatonin that decreases recognition time, leads to a facilitation of short-term memory. We have previously shown that melatonin implementations have some effects on learning performance depending on treatment. We investigated the effects of pinealectomy, constant release melatonin implants, and timed melatonin injections on spatial memory in male rats by using Morris water maze. Our findings showed that spatial memory performance of the 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 change significantly the spatial memory performance of the rats. This outcome suggests that while the removal of the pineal gland and exogenous administration of melatonin via injections did impair learning performance, constant release melatonin administration via implantation did not affect the spatial memory in Wistar albino rats. There is also consistent research evidence that melatonin given from weaning did lead to learning and memory deficit in rats (Cao et al., 2009). 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.

Pinealectomy is one of the methods to investigate the effect of melatonin in animals. It eliminates the melatonin hormone from blood circulation. It is well-recognized that the removal of the pineal gland abolishes the rhythmic endogenous melatonin release and decreases the plasma levels of melatonin significantly (Hoffman and Reiter, 1965). It prevents the animal from responding against the changes in day length (Hoffmann and

The effects of pinealectomy have been mostly studied on the reproductive system. The reproductive cycle desynchronizes from the environmental photoperiodic cycle by the pinealectomy. The effects of pinealectomy on reproductive system have been well documented in some hamster species. Pinealectomy prevents the regression effect of short photoperiods while gonadal maintenance on long photoperiods is not affected in Syrian hamster (Hoffmann and Reiter, 1965). Pinealectomy blocks short photoperiod induced gonadal regression of hamsters previously housed on long photoperiod (Hoffmann, 1974).

In addition to studies on the effects of regulatory function of pinealectomy on the reproductive system, it has been received a research attention in the behavioral studies. The amygdala, a complex mass of gray matter, is located within the anterior-medial portion of the temporal lobe, just rostral to the hippocampus. The subnuclei and cortical regions of the amygdala are connected to other nearby cortical areas on the ventral and medial aspect of the hemispheric surface. The amygdala has three major functional and anatomical subdivisions, each of which are connected to the other parts of the brain. The first subdivision, namely the medial group of subnuclei, is connected to the olfactory bulb and the olfactory cortex. The second one, the basal-lateral group, has major projections with the cerebral cortex. The third one, the central and anterior group of nuclei, makes connections with the hypothalamus and brainstem which process sensory information with hypothalamic and brainstem effector systems. The visual, somatic sensory, visceral sensory, and auditory stimuli information are provided by the cortical inputs. The amygdala and the hypothalamus are separated from each other by the pathways from sensory cortical areas (Gilman and Newman, 1992.

The amygdala receives some projections directly from thalamic nuclei, the olfactory bulb, and visceral sensory relays in the brainstem. There is evidence for this convergence of sensory information. For instance, many neurons in the amygdala are sensitive to visual, auditory, somatic, sensory, visceral sensory, gustatory, and olfactory stimuli. In addition to sensory inputs, the prefrontal and temporal cortical connections of the amygdala also make connections with cognitive neocortical circuits or integrative areas, especially for integration of the emotional significance of sensory stimuli with guide complex behavior, or vice versa. Moreover, projections from the amygdala to the hypothalamus and brainstem involve in the processing of emotions such as fear, anger, and pleasure (Gilman and Newman, 1992).

#### *1.3.2. The role of amygdala on anxiety like behavior and learning performance*

It has been demonstrated that amygdala plays a regulatory role for behaviors related to anxiety and depression (Hale et al., 2006; Blackshear et al., 2007; Martinez et al., 2007). Serotonergic activity is especially high in amygdala (Abrams et al., 2004 a, 2004b). For

instance, a research has indicated that mCPP (a serotonin receptor agonist) microinjections to amygdala 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 (Cornelio and Nunes de Souza, 2007). In another study, Herdade et al. (2006) injected locally muscimol (a GABAA receptor agonist) to the medial nucleus of the amygdala and found that such treatment inhibited escape behavior in elevated T maze.

Intraamygdalar Melatonin Administration and Pinealectomy Affect Anxiety Like Behavior and Spatial Memory 21

group, animals were exposed to the same surgical procedure with the experimental group except for the removal of the pineal gland. We performed the four subgroups as Melatonin (1 and 100 μg/kg) (n:14), Saline (0.9%NaCl) (n:5) and Diazepam (2mg/kg) (n:5) under control and pinealectomy groups. All pinealectomies and cannulation surgeries were applied before starting the experiment. The experiments were started after a week of the pinealectomies and implantations, when surgery wounds healed up completely. The anxiety-like behaviour of animals were tested by open field and elevated plus maze tests, and spatial memory was tested by means of the Morris water maze test. All animals were exposed to these behavioral

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

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

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

tests after 30 minutes of melatonin, saline, and diazepam administrations.

**2.3. Anesthesia** 

their cages.

**2.4. Cannulation** 

to recover from surgery.

**2.5. Pinealectomy** 

In addition to the regulatory role of amygdala in anxiety, amygdala is of great importance in regulating memory and learning functions. The amygdala is responsible for determining what memories are stored and where the memories are stored in the brain. 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. For instance, the subjects experience difficulties in learning new material (i.e., anterograde amnesia) after the removal of amygdala (Almonte et al., 2007). One research has shown that amygdala damage leads to an impairment of learning an association between an auditory cue and food reward. When scopolamine, the muscarinic receptor antagonist, was injected to amygdala, it impaired performance on conditioned place preference task but not a spatial radial maze task (McIntyre et al., 1998). Moreover, the infusion of nicotinic receptor antagonists methyllycaconitine (MLA) or dihydro-b-erythroidine (DHbE) impaired working memory (Addy et al., 2003). Taken together, these findings suggest that amygdala damage has detrimental effect on the cognitive performance. However, the effect of melatonin administration to amygdala was not well known prior to the research mentioned below. The administration of melatonin to amygdala with the abolishment of melatonin hormone via pinealectomy might produce different effects on anxiety-like and learning behaviors. In other words, the endogeneous melatonin concentration and the rhythm of melatonin release might affect the effects of exogeneous melatonin administration on such behaviors.
