**4.2. Spatial memory performance**

The Morris water maze has been one of widely used tests to measure the spatial memory performance. In this maze, the time passed to find the platform, total distance travelled, the frequency of the entrance to the correct quadrant, the time passed in correct quadrant, mobility and velocity parameters are measured (Morris, 1984).

In this study,( a) diazepam administration increased the total distance travelled more 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 with the high dose of melatonin also travelled more distance than those with the low dose of melatonin and diazepam, (c) the subjects who received 1 μg/kg melatonin spent less time than those who received other treatments, and (d) in the control condition, the subjects with 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 the impairment of learning an association between an auditory cue and food reward (Sutherland and Mc Donalds, 1990), of performance on conditioned place preference task (McIntyre et al., 1998), and working memory (Addy et al., 2003). It is a well known fact that melatonin readily passes all cell membranes, including the blood-brain barrier (Reiter et al., 1993). Melatonin binding sites exist in various brain structures such as the hippocampus and prefrontal cortex are considered to involve in memory function (Brzezinski, 1997; Ekmekçioğlu, 2006; Mazzuchelli et al., 1996; Savaskan et al., 2001; 2005). Moreover, considering that melatonin is a potent sleep inducing enhanced consolidation of hippocampus-dependent memories (Jern et al., 1991; Rasch et al., 2007), 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 (Baydas et al., 2002). Other research has

explanations.

**4.2. Spatial memory performance** 

test to measure the anxiety like behaviors (Dawson and Tricklebank, 1995). 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 traveled 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

The Morris water maze has been one of widely used tests to measure the spatial memory performance. In this maze, the time passed to find the platform, total distance travelled, the frequency of the entrance to the correct quadrant, the time passed in correct quadrant,

In this study,( a) diazepam administration increased the total distance travelled more 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 with the high dose of melatonin also travelled more distance than those with the low dose of melatonin and diazepam, (c) the subjects who received 1 μg/kg melatonin spent less time than those who received other treatments, and (d) in the control condition, the subjects with 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 the impairment of learning an association between an auditory cue and food reward (Sutherland and Mc Donalds, 1990), of performance on conditioned place preference task (McIntyre et al., 1998), and working memory (Addy et al., 2003). It is a well known fact that melatonin readily passes all cell membranes, including the blood-brain barrier (Reiter et al., 1993). Melatonin binding sites exist in various brain structures such as the hippocampus and prefrontal cortex are considered to involve in memory function (Brzezinski, 1997; Ekmekçioğlu, 2006; Mazzuchelli et al., 1996; Savaskan et al., 2001; 2005). Moreover, considering that melatonin is a potent sleep inducing enhanced consolidation of hippocampus-dependent memories (Jern et al., 1991; Rasch et al., 2007), 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

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 (Baydas et al., 2002). Other research has

condition provides melatonin specific outcomes more than the open field condition.

mobility and velocity parameters are measured (Morris, 1984).

also suggested that melatonin may influence memory formation in the hippocampus (El Sherif et al., 2003). 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 (2006) 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 to the regulatory role of amygdala in anxiety, amygdala is of great importance in regulating memory and learning functions. The removal of the temporal lobe in animals leads to an impairment in memory in a way that the subjects experience difficulties in learning new material after the removal of amygdala. Also, damage to amygdala leads to an impairment of learning an association between an auditory cue and food reward. In addition, the muscarinic receptor antagonist administration to amygdala impaired performance on conditioned place preference task (McIntyre et al., 1998). Moreover, the nicotinic receptor antagonist administrations impair working memory (Addy et al., 2003). The results of our study indicate that the administration of melatonin to amygdala with the abolishment of melatonin hormone via pinealectomy produced different effects on anxiety-like and learning behaviors.

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 the darkness (Klein, 1974). However, in pinealectomy the blood melatonin levels drop significantly and the rhythm of melatonin is abolished (Chapman, 1970).

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 (Brzezinski, 1997; Borjigin et al., 1999; Arendt, 2000). 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 (Stetson and Tay, 1983; Hong and Stetson, 1987). 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 (Karakas et al., 2011b).

Intraamygdalar Melatonin Administration and Pinealectomy Affect Anxiety Like Behavior and Spatial Memory 43

Addy, NA., Nakijama, A. & Levin, ED., (2003) Nicotinic mechanisms of memory: effects of acute local DH beta E andMLAinfusions in the basolateral amygdale. *Cognit Brain Res*

Almonte,AG., Hamill, CE., Chhatwal, JP., Wingo, TS., Barber, JA., (2007) Learning and memory deficits in mice lacking protease activated receptor-1. *Neurobiol Learn Mem* 88*,* 

Appenrodt, E. & Schwarzberg, H. (1999) Septal vasopressin modulates motility and passive

Appenrodt, E. & Schwarzberg, H. (2000) Central vasopressin administration failed to influence anxiety behavior after pinealectomy in rats. *Physiol Behav* 68*,* 735–9. Appenrodt, E. & Schwarzberg, H. (2003) Pinealectomy blocks modulation of active

Appenrodt, E, Juszczak, M. & Schwarzberg, H. (2002) Septal vasopressininduced preservation of social recognition in rats was abolished by pinealectomy. *Behav Brain* 

Arendt, J. (1995) *Melatonin and the mammalian pineal gland*. Chapman and Hall, London, UK. Arendt, J. (2000) Melatonin, circadian rhythms and sleep. *New England J Med* 343, 1114-1116. Argyriou, A., Prast, H. & Philippu, A. (1998) Melatonin facilitates short-term memory. *Eur J* 

Baydas, G., Nedzvetsky, VS., Nerush, PA., Krchenko, SV., Demchenko, HM., et al.(2002) A novel role for melatonin: regulation of the expression of cell adhesion molecules in the

Benabid, N., Mesfoui, A. & Ouichou, A. (2008). Effects of photoperiod regimen on emotional behaviour in two tests for anxiolytic activity in Wistar rat. *Brain Res Bull* 75,

Binkley, S. (1988) The pineal: endocrine and nonendocrine function. Prentice Hall, New

Blackshear, A., Yamamoto, M., Anderson, B.J., Holmes, P.V., Lundström, L.,et al., (2007) Intracerebroventricular administration of galanin or galanin receptor subtype 1 agonist M617 induces c-Fos activation in central amygdala and dorsomedial hypothalamus.

Borjigin, J., Li, X. & Snyder, S H. (1999) The pineal gland and melatonin: molecular and

Cao, X J., Wang, M., Chen, W H., Zhu, D M., She, J Q., et al., (2009) Effects of chronic administration of melatonin on spatial learning ability and long-term potentiation in

Cardinalli, DP., Vacas, MI. & Boyer, E E. (1979) Specific binding of melatonin in bovine

Chapmann, D I. (1970) Seasonal changes in the gonads and accessory glands of male

hippocampus, cortex and cerebellum. *Neurosci Lett* 326, 109–112.

pharmacologic regulation. *Annu Rev Pharmacol* 39*,* 53-65. Brzezinski, A. (1997) Melatonin in humans. *New Engl J Med* 336,186-195.

lead-exposed and control Rats. *Biomed Environ Sci* 22*,* 70-75.

avoidance by central vasopressin application in rats. *Peptides 24,* 129-136.

avoidance in pinealectomized rats*. Physiol Behav* 66, 757–61.

Arendt, J. (1988) Melatonin. *Clin Endocrinology* 29, 205-29

16, (1): 51-57

*3*, *295-304*

*Res 134,* 67–73.

53-59

Jersey, Usa

*Pharmacol 349,* 159-162.

*Peptides* 28, *5*, *1120-1124*

brain. *Endocrinology* 105, 437-441.

mammals. *Mammal Rev* 1, 231–248.
