*1.2.1. General information about melatonin hormone*

Melatonin was first identified by Lerner and colleagues in 1958, as the constituent of bovine pineal glands that lightens isolated frog skin. After it's discovery, many studies focused on the physiologic roles of melatonin on pigmentation in lower vertebrates and gonodal maturation in mammals.

Pineal gland functions as a chemical neurotransducer which converts the neural stimuli to a hormonal product as melatonin. This gland regulates many physiological functions by secreting and releasing melatonin. In the secretion of melatonin, the time of the day, age of the animal and in some photoperiodic species, time of year may be important determinant. Melatonin is secreted and released in a circadian fashion, high levels at night and very low levels at day time (Arendt, 1988). The circadian rhythm of melatonin release persists in constant darkness. However, this rhythm can be altered by nighttime light exposure, because light can suppress melatonin production. Many physiological rhythms are synchronized by the normal daily variations of the melatonin secretion. The nighttime sleep initiation and maintenance in diurnal species is also controlled by the melatonin secretion.

Light information is first received by the retina of the eye. This information is transferred to Suprachiasmatic nuclei (SCN) by the retinohypothalamic tract. SCN is capable of measuring the length of the dark/light. The information of light is then transferred to Superior Cervical Ganglia (SCG) of the spinal cord. Pineal gland receives the projections from postganglionic sympathetic nerve endings emerging from the SCG which release norepinephrine (NE). The secretion of melatonin determined by the NE since the release of NE is associated with darkness. In as much as NE release onto the pinealocytes occurs at night, melatonin synthesis likewise occurs primarily during darkness. Therefore, the concentration of melatonin in the blood is greater at night than during the day. Some other factors such as the species and tissues may influence the rate and the pattern of the nocturnal increase in melatonin production (Sugden, 1991; Klein, 1993).

Melatonin has a half life of nearly 20-40 minutes. It does not remain in the blood very long. Unless the pineal gland continues to produce and secrete melatonin, blood levels of the hormone drop quickly. Melatonin is removed from the blood in at least four ways. 1) It is enzymatically degraded primarly to 6-hydroxy melatonin by the liver. 2) Melatonin that is taken up by other cells is non enzymatical degraded when it scavenges hydroxyl radicals. 3) Also, melatonin in the blood rapidly escapes into other body fluids. 4) Finally, melatonin attaches to specific receptors or binding sites located at various locations in the organism (Panke et al, 1979; Steinlechner, 1996).

16 Neuroendocrinology and Behavior

**1.2. Melatonin hormone** 

maturation in mammals.

capillary network in pineal gland (Quay, 1974).

*1.2.1. General information about melatonin hormone* 

melatonin production (Sugden, 1991; Klein, 1993).

The post-ganglionic sympathetic fibers arising from the superior cervical ganglion innervates mainly pineal gland of the mammals. Postganglionic fibers reaching the pineal organ via the nervi conarii release norepinephrine at night. This neurotransmitter then activates adenylate cyclase, stimulating production of the second messenger cyclic adenosine monophosphate (cAMP), which accelerates melatonin synthesis. The vascular supply of the pineal gland is very rich. The arterial supply of the pineal gland is provided by the branches of the posterior choroidal arteries. There is also a well-developed internal

Melatonin was first identified by Lerner and colleagues in 1958, as the constituent of bovine pineal glands that lightens isolated frog skin. After it's discovery, many studies focused on the physiologic roles of melatonin on pigmentation in lower vertebrates and gonodal

Pineal gland functions as a chemical neurotransducer which converts the neural stimuli to a hormonal product as melatonin. This gland regulates many physiological functions by secreting and releasing melatonin. In the secretion of melatonin, the time of the day, age of the animal and in some photoperiodic species, time of year may be important determinant. Melatonin is secreted and released in a circadian fashion, high levels at night and very low levels at day time (Arendt, 1988). The circadian rhythm of melatonin release persists in constant darkness. However, this rhythm can be altered by nighttime light exposure, because light can suppress melatonin production. Many physiological rhythms are synchronized by the normal daily variations of the melatonin secretion. The nighttime sleep initiation and maintenance in diurnal species is also controlled by the melatonin secretion.

Light information is first received by the retina of the eye. This information is transferred to Suprachiasmatic nuclei (SCN) by the retinohypothalamic tract. SCN is capable of measuring the length of the dark/light. The information of light is then transferred to Superior Cervical Ganglia (SCG) of the spinal cord. Pineal gland receives the projections from postganglionic sympathetic nerve endings emerging from the SCG which release norepinephrine (NE). The secretion of melatonin determined by the NE since the release of NE is associated with darkness. In as much as NE release onto the pinealocytes occurs at night, melatonin synthesis likewise occurs primarily during darkness. Therefore, the concentration of melatonin in the blood is greater at night than during the day. Some other factors such as the species and tissues may influence the rate and the pattern of the nocturnal increase in

Melatonin has a half life of nearly 20-40 minutes. It does not remain in the blood very long. Unless the pineal gland continues to produce and secrete melatonin, blood levels of the hormone drop quickly. Melatonin is removed from the blood in at least four ways. 1) It is enzymatically degraded primarly to 6-hydroxy melatonin by the liver. 2) Melatonin that is

The melatonin receptors involved in mediating the effects of melatonin on the reproductive and endocrine systems are presumed to be those located in the pars tuberalis of the anterior pituitary gland (Stankov et al, 1991). These cells are in close proximity to the primary portal plexus and the terminals of the hypothalamic releasing hormone neurosecretory cells in the median eminence. Melatonin theoretically controls the release of substances, e.g., gonadotropins or other factors, that act in a paracrine manner in the nearby median eminence thereby regulating the release of the hypothalamic releasing hormones, e.g., gonadotropin releasing hormone (GnRH). In this manner melatonin can obviously regulate the functional status of the gonads and control the reproductive capability of an animal on a seasonal basis.

Melatonin modulates many physiological functions such as sleep, circadian, visual, cerebrovascular, reproductive, neuroendocrine, and neuroimmunological functions (Arendt, 2000; Wirz-Justice, 2001; Borjigin et al., 1999; Brzezinzki, 1997; Masana and Dubocovich, 2001; Vanecek, 1999; Hardeland et al., 2006). The amphilicity of the melatonin is allowing the molecule to enter any cell, compartment or body fluid (Poeggeler et al., 1994). In addition to physiological functions, melatonin influences the behavioural processes such as learning, stress, anxiety like behaviors, and depression (Krause and Dubocovich, 1990; Mantovani et al, 2003; Naranjo-Rodriguez et al., 2000; Loiseau et al., 2006). With regard to behavioural processes, melatonin binding sites have been found in the regions implicated in cognition and memory in the brain (Cardinalli et al., 1979; Weaver et al., 1989). The previous studies have shown that passive and active avoidance learning are affected by melatonin (Martini, 1971; Kovács et al., 1974). Melatonin that decreases recognition time, leads to a facilitation of short-term memory (Argyriou et al, 1998]. Taken together, these findings suggest the beneficial effect of melatonin on cognition and memory.

Melatonin receptors represent saturation by the melatonin concentrations, which are close to physiologic nighttime melatonin levels. Because of this reason, these receptors show a dosage dependent activity. The sleep-promoting and activity-inhibiting effects of melatonin are provided by its low levels (e.g.,50 pg/mL in blood plasma) at the beginning of the night. However, the high levels of melatonin (e.g.,150 pg/mL in blood plasma) do not enhance these behavioral parameters. Some diurnal variations are also evident in the sensitivity of the melatonin receptors since melatonin receptors are more sensitive during the daytime when the time endogenous melatonin is not secreted. The circadian phase shifting effect of melatonin may be due to the enhanced sensitivity of melatonin receptors to melatonin in the morning or in the evening hours in response to small increases in melatonin secretion (Reppert, 1997).

### *1.2.2. The role of melatonin hormone on anxiety and learning performance*

Melatonin seems to produce anxiolytic (Naranjo-Rodriguez et al., 2000; Papp et al., 2000) effects. The effect of melatonin on anxiety is suggested to be mediated by central gamma

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 affect anxiety of animals.

Intraamygdalar Melatonin Administration and Pinealectomy Affect Anxiety Like Behavior and Spatial Memory 19

However, these studies have provided rather inconsistent findings. For instance, while the pinealectomy itself did not have a detrimental effect on cognitive performance in rats, the interaction of it with the other lesion (i.,e, lesion on habenula) impaired such performance (Lecourtier et al., 2005). Many studies have shown that pinealectomy did not have a significant effect on the acquisition and extinction of the active avoidance behavior (Appenrodt and Schwarzberg, 2003), anxiety behavior (Appenrodt and Schwarzberg, 2000), passive avoidance learning (Appenrodt and Schwarzberg, 1999), open field exploratory

activity (Kovács et al., 1974), and social recognition (Appenrodt et al., 2002).

and learning performance will be examined.

*1.3.1. General features of amygdala* 

(Gilman and Newman, 1992.

**1.3. The amygdala regulate the behaviours related to the anxiety and memory** 

In this section, the general features of the amygdala and its role on anxiety like behaviors

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

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).

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

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

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.
