**4.1. Anxiety-like behaviours**

Open field test is used to measure the anxiety like behaviors in rodents (Benabid et al. 2008). The total distance traveled, 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 (Pyter and Nelson, 2006). In this maze, if the anxiety of the animal is high, (a) the number of the entries to the edge of the open field tends to increase, whereas that of entries to the center of the open field decrease, (b) the time passed at the edge of the open field increases, while the time passed at the center of the open field decreases, and (c) the total

distance traveled, mobility and velocity in the open field decrease. The total number of the entries into the center and the edges provides a built-in control measure for general hyperactivity or sedation.

Intraamygdalar Melatonin Administration and Pinealectomy Affect Anxiety Like Behavior and Spatial Memory 39

animal is high, (a) the number of the entries to closed arms increases whereas those to open arms decreases, (b) the time passed in closed arm increases whereas that passed in open arms decreases and (c) the total distance traveled, mobility and velocity decrease. The total number of the entries into all arms provides a built-in control measure for general

In sum, our findings were in elevated plus maze that, a) the high dose (100 μg/kg) of melatonin increased the distance totally travelled, whereas such dose after pinealectomy decreased it, b) 100 μg/kg melatonin increased the time spent in open arms; however, after the pinealectomy, the low dose of melatonin (1 μg/kg) decreased it and c) pinealectomized

The increase in travelled distance induced by high dose of melatonin administration was reversed by the pinealectomy. This suggests 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 (Hoffman and Reiter, 1965). 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 increased the time spent in open arms, while, after the pinealectomy, the low dose of melatonin decreased it. In the literature, there is evidence for the interaction of melatonin with central gama aminobutyric acid (GABA) neurotransmission. Melatonin has been shown to increase the GABA levels in rat brain tissue *in vitro* (Niles et al., 1987; Coloma and Niles, 1988). When melatonin was applied *in vivo*, it increased the GABA levels in several brain regions in rats (Rosenstein and Cardinali, 1986; Xu et al., 1995). In conclusion, our findings can be attributed the fact that high dose of melatonin increased the GABA levels, which in turns reduce anxiety like behaviors. Through this mechanism, the high dose of melatonin administered subjects spent more time

The third finding in the elevated plus maze was that pinealectomy increased the mobility time in compared to controls. This finding suggests 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 (Benabid et al., 2008). The total distance traveled, 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 (Pyter and Nelson, 2006). 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 traveled 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

hyperactivity or sedation.

in open arms than the others.

animals were more mobile than control ones.

In sum, our findings were in open field that 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 control subjects were more mobile than the pinealectomized ones, and c) 100 μg/kg melatonin administration in contrast to other treatment conditions reduced the velocity of the animals.

Our findings reflected the fact 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 anxiety-like behaviours. 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 (Sinclair and Nutt, 2007). There is also possibility that amygdala also plays an important role on anxiety. The high serotonergic activity in amygdala may be one plausible explanation for this important role. This role of amygdala is supported by some research evidence that (a) a serotonin receptor agonist increased behavioral indices of anxiety without altering general activity level, and (b) a GABAA receptor agonist treatment to the medial nucleus of the amygdala inhibited escape behavior in elevated T maze.

The second finding was that there was the biggest difference in between the controls and the pinealectomies in mobility. This means 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 behaviours in this 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 behaviour 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 [Kovacs et al., 1974; Juszcak et al., 1996). This suggests that the pineal gland is partially involved in the anxiety-like behaviours. The third finding was that the high dose of melatonin (100 μg/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.

The elevated plus maze has been one of widely used tests to measure the anxiety like behaviours (Dawson and Tricklebank, 1995). In this test, the total distance traveled, the total number of entries to the closed and open arms, the time spent in closed and open arms, the mobility and the velocity are used parameters measured. In this maze, if the anxiety of the

hyperactivity or sedation.

treatment conditions reduced the velocity of the animals.

melatonin administration may not become evident.

on the circadian clock.

distance traveled, mobility and velocity in the open field decrease. The total number of the entries into the center and the edges provides a built-in control measure for general

In sum, our findings were in open field that 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 control subjects were more mobile than the pinealectomized ones, and c) 100 μg/kg melatonin administration in contrast to other

Our findings reflected the fact 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 anxiety-like behaviours. 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 (Sinclair and Nutt, 2007). There is also possibility that amygdala also plays an important role on anxiety. The high serotonergic activity in amygdala may be one plausible explanation for this important role. This role of amygdala is supported by some research evidence that (a) a serotonin receptor agonist increased behavioral indices of anxiety without altering general activity level, and (b) a GABAA receptor agonist treatment

to the medial nucleus of the amygdala inhibited escape behavior in elevated T maze.

The second finding was that there was the biggest difference in between the controls and the pinealectomies in mobility. This means 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 behaviours in this 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

Our results, which showed that the anxiety like behaviour 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 [Kovacs et al., 1974; Juszcak et al., 1996). This suggests that the pineal gland is partially involved in the anxiety-like behaviours. The third finding was that the high dose of melatonin (100 μg/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

The elevated plus maze has been one of widely used tests to measure the anxiety like behaviours (Dawson and Tricklebank, 1995). In this test, the total distance traveled, the total number of entries to the closed and open arms, the time spent in closed and open arms, the mobility and the velocity are used parameters measured. In this maze, if the anxiety of the animal is high, (a) the number of the entries to closed arms increases whereas those to open arms decreases, (b) the time passed in closed arm increases whereas that passed in open arms decreases and (c) the total distance traveled, mobility and velocity decrease. The total number of the entries into all arms provides a built-in control measure for general hyperactivity or sedation.

In sum, our findings were in elevated plus maze that, a) the high dose (100 μg/kg) of melatonin increased the distance totally travelled, whereas such dose after pinealectomy decreased it, b) 100 μg/kg melatonin increased the time spent in open arms; however, after the pinealectomy, the low dose of melatonin (1 μg/kg) decreased it and c) pinealectomized animals were more mobile than control ones.

The increase in travelled distance induced by high dose of melatonin administration was reversed by the pinealectomy. This suggests 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 (Hoffman and Reiter, 1965). 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 increased the time spent in open arms, while, after the pinealectomy, the low dose of melatonin decreased it. In the literature, there is evidence for the interaction of melatonin with central gama aminobutyric acid (GABA) neurotransmission. Melatonin has been shown to increase the GABA levels in rat brain tissue *in vitro* (Niles et al., 1987; Coloma and Niles, 1988). When melatonin was applied *in vivo*, it increased the GABA levels in several brain regions in rats (Rosenstein and Cardinali, 1986; Xu et al., 1995). In conclusion, our findings can be attributed the fact that high dose of melatonin 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. This finding suggests 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 (Benabid et al., 2008). The total distance traveled, 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 (Pyter and Nelson, 2006). 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 traveled 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 (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 condition provides melatonin specific outcomes more than the open field condition.

Intraamygdalar Melatonin Administration and Pinealectomy Affect Anxiety Like Behavior and Spatial Memory 41

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

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

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

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

drop significantly and the rhythm of melatonin is abolished (Chapman, 1970).

locomotor activity, rather than an effect on the circadian clock.

effects on anxiety-like and learning behaviors.
