**4. Some basic neurobiological characteristics of sleep-wake cycles**

A study conducted by Qiu and colleagues in 2010 presented the main overall neurobiological activity of basal ganglia neurons associated with the sleep-wake state. The differences in firing patterns across the basal ganglia suggest multiple input sources, such as the cortex, thalamus, and the dopamine system, as well as some other intra basal ganglia inputs, such as the globus pallidus-subtalamic nucleus, and striatum-globus pallidus interactions. The largest nucleus striatum of the basal ganglia is mostly comprised of aminobutiric acid ergic spiny neurons, whose activity is influenced by excitatory glutaminergic projection from the neocortex and thalamus, and dopaminergic projection from the midbrain ventral tegmental area and other known parts. The striatum receiving cortical inputs projects to the globus pallidus, which then projects to the cerebral cortex directly ore by the thalamus (mainly the mediodorsal thalamic nucleus). It was suggested that the lesion of globus pallidus produced a higher increase in wakefulness and frequent sleep-wake transitions, as well as a concomitant decrease in non-REM sleep duration. The results of the study also suggest that the cortico-striato-pallidal loop may be critically involved in the basal ganglia control of arousal.

There are four stages of sleep, which include the brain-active period associated with rapid eye movements called REM sleep (emergent stage 1 EEG), preceded by progressively deeper sleep stages (stages 2, 3, 4) graded on the basis of increasingly slower EEG patterns, called non-REM sleep. Stages 3 and 4 are referred to as slow-wave sleep (SWS) characterized by delta waves (high amplitude and low-frequency). REM sleep and wakefulness are characterized by increased activity in the cerebral cortex with low-amplitude and highfrequency EEG (alpha waves) and in REM by the inhibition of peripheral neurons displayed in the postural muscle atonia. Increased cerebral activity during REM sleep is associated with higher oxygen consumption, blood flow and neural firing (Madsen et al., 1991).

Elemental Mercury Exposure and Sleep Disorder 55

Fig. 1. The rate of glutamate decline during non-REM sleep positively correlated (r = 0.41, p < 0.01) with the amount of SWA. Each data point represents the average SWA and the change in glutamate concentration during non-REM sleep (Adapted from Dash, et al. 2009, The Journal of Neuroscience, Vol. 29, No. 3, pp. 620-629. Copyright 2009, Society for Neuroscience.

originating in SCN, also regulates pineal gland activity. Melatonin can influence the sleeppromoting and sleep-wake rhythm regulating actions through the specific activation of melatonin receptors type 1 and 2, which are highly concentrated in SCN, and are also expressed in the peripheral organs and cells regulating other physiological functions of the so-called circadian 24-hour rhythms. The activation of type 1 occurs by inducing a receptorsuppressed neuronal firing rate in CNS, while type 2 induces a circadian phase shift. The increased secretion of melatonin is also accompanied by other circadian 24-hour rhythms of humans, and in rats studies associated with decreased production of neurotransmitter nitric oxide (NO) (Fig. 2) (Dubocovich et al., 2003; Ebadi, 1992; Geoffriau et al., 1998; Leon et al., 1998; Murphy & Delanty, 2007; Starc, 1998). Some studies do not confirm the influence of melatonin on the duration of sleep (Hughes et al., 1998), while others support the effect of melatonin on the duration and quality of REM sleep because they assume that it either directly influences cholinergic activity in REM sleep, or indirectly influences REM sleep by the elimination of serotonergic or aminergic activity (Jones, 1991; Kunz et al., 2004). It seems that melatonin modulates the release of acethylcholine in the nucleus accumbens and the motor activity of rats (Paredes et al., 1999). Some animal studies suggest that the daily changes in melatonin production may regulate the day-night variation in glutamate and GABA in the neostriatum (Marquez de Prado et al., 2000). It seems that the glutaminergic system negatively regulates norepinephrine-dependent melatonin synthesis in the rat's pineal gland (Yamada et al., 1998). In rats studies melatonin inhibits the glutamate-mediated response of the striatum to motor cortex stimulation and decrease NO content in parietotemporal cortex, striatum and brainstem of rats due to the inhibition of neuronal nitric oxide

Adapted with permission.)

Acetylcholine, norepinephrine, serotonin, histamine and hypocretin levels are increased in wakefulness and low in non-REM sleep, whereas during REM sleep the noradrenergic, serotonergic and histaminergic cells become silent (Jones, 2005). A high cholinergic tone in the pontine reticular formation combined with a low GABAergic tone contributes to the generation of REM sleep (Vanini et al., 2011). Animal studies showed that the neurotransmitter glutamate enhances REM sleep by activation of the kainite receptor within the cholinergic cell compartment of the brainstem pedunculo pontine tegmentum of cat and rat (Datta, 2002). During REM sleep and waking, the release of acetylcholine activated dopamine in the ventral tegmental neurons, which were higher in the prefrontal cortex and nucleus accumbens. It was also suggested that glutamate and asparate release can reciprocally affect dopamine release (Forster and Blaha, 2000; Morari et al., 1998). The animal study of Lena and colleagues in 2005 also showed elevated levels of dopamine during waking and REM sleep in the medial prefrontal cortex and nucleus accumbens.

The impairment of the subcortical dopaminergic system may cause disinhibition of the GABAergic inhibitory circuitry at the motor cortex level (Entazry-Taher et al., 1999; Ziemann et al., 1996). It is suggested that the diencephalon-spinal dopaminergic tract could be important as a potential anatomic site of dopaminergic dysfunction in restless leg syndrome, and of periodic leg contractile movements in sleep. The diencephalon-spinal dopaminergic tract projects to the limbic system, sensory cortex and spinal cord (Ondo et al., 2000). The periodic leg contractile movements occur mainly during non-REM sleep. The results of the study of Rijsman et al. in 2005 indicate diminished inhibition at spinal level in subjects with periodic leg movements disorder, probably because of the altered function of the descending spinal tracts and peripheral changes in the inter-neural circuitry at the spinal level. Dreams and nightmares occur usually at the end of the night, when REM sleep is longer. On the other side, sleep terrors occur more often in children than in adults, while children have more delta sleep (Pinel, 2009; Lee & Douglass, 2010).

Another recent animal study (John et al., 2008) showed a rapid increase in the glutamate level during REM sleep and awakening in the histamine-containing posterior hypothalamic region and the perifornical-lateral hypothalamus, and its reduction shortly after the termination of REM sleep and awakening. In the animal study of Dash and colleagues conducted in 2009, which employed a very sensitive method (in vivo amperometry) to measure cortical extracellular glutamate, a progressive increase was observed in the cortical extracellular glutamate concentration during REM sleep and waking. It was suggested that extrasynaptic glutamate is released from astrocytes and neurons in extracellular space, where it is accumulated, and then declines during non-REM sleep due to the intracellular reuptake mediated by glutamate/asparate transporters. The rate of glutamate decline during non-REM sleep positively correlated with the levels of slow wave activity (SWA) (Fig. 1). The authors of the study concluded that perhaps the glutamate-decreasing effect of non-REM sleep is especially relevant in a pathological condition.

It is thought that the pineal gland itself takes part in regulating the rhythm of sleep and wakefulness, entrained by the light/dark cycle. Neural impulses from the retina enter the pineal gland, which coordinates the formation and secretion of serotonin and melatonin, through the suprachiasmic nuclei (SCN) of the hypothalamus. Light induces serotonin secretion, while melatonin is produced at night directly from serotonin by acetylation. However, melatonin production can be acutely interrupted by light exposure during the night. Norepinephrine, which is released at night in response to stimulatory signals

Acetylcholine, norepinephrine, serotonin, histamine and hypocretin levels are increased in wakefulness and low in non-REM sleep, whereas during REM sleep the noradrenergic, serotonergic and histaminergic cells become silent (Jones, 2005). A high cholinergic tone in the pontine reticular formation combined with a low GABAergic tone contributes to the generation of REM sleep (Vanini et al., 2011). Animal studies showed that the neurotransmitter glutamate enhances REM sleep by activation of the kainite receptor within the cholinergic cell compartment of the brainstem pedunculo pontine tegmentum of cat and rat (Datta, 2002). During REM sleep and waking, the release of acetylcholine activated dopamine in the ventral tegmental neurons, which were higher in the prefrontal cortex and nucleus accumbens. It was also suggested that glutamate and asparate release can reciprocally affect dopamine release (Forster and Blaha, 2000; Morari et al., 1998). The animal study of Lena and colleagues in 2005 also showed elevated levels of dopamine during waking and REM sleep in the medial prefrontal cortex and nucleus accumbens. The impairment of the subcortical dopaminergic system may cause disinhibition of the GABAergic inhibitory circuitry at the motor cortex level (Entazry-Taher et al., 1999; Ziemann et al., 1996). It is suggested that the diencephalon-spinal dopaminergic tract could be important as a potential anatomic site of dopaminergic dysfunction in restless leg syndrome, and of periodic leg contractile movements in sleep. The diencephalon-spinal dopaminergic tract projects to the limbic system, sensory cortex and spinal cord (Ondo et al., 2000). The periodic leg contractile movements occur mainly during non-REM sleep. The results of the study of Rijsman et al. in 2005 indicate diminished inhibition at spinal level in subjects with periodic leg movements disorder, probably because of the altered function of the descending spinal tracts and peripheral changes in the inter-neural circuitry at the spinal level. Dreams and nightmares occur usually at the end of the night, when REM sleep is longer. On the other side, sleep terrors occur more often in children than in adults, while

children have more delta sleep (Pinel, 2009; Lee & Douglass, 2010).

REM sleep is especially relevant in a pathological condition.

Another recent animal study (John et al., 2008) showed a rapid increase in the glutamate level during REM sleep and awakening in the histamine-containing posterior hypothalamic region and the perifornical-lateral hypothalamus, and its reduction shortly after the termination of REM sleep and awakening. In the animal study of Dash and colleagues conducted in 2009, which employed a very sensitive method (in vivo amperometry) to measure cortical extracellular glutamate, a progressive increase was observed in the cortical extracellular glutamate concentration during REM sleep and waking. It was suggested that extrasynaptic glutamate is released from astrocytes and neurons in extracellular space, where it is accumulated, and then declines during non-REM sleep due to the intracellular reuptake mediated by glutamate/asparate transporters. The rate of glutamate decline during non-REM sleep positively correlated with the levels of slow wave activity (SWA) (Fig. 1). The authors of the study concluded that perhaps the glutamate-decreasing effect of non-

It is thought that the pineal gland itself takes part in regulating the rhythm of sleep and wakefulness, entrained by the light/dark cycle. Neural impulses from the retina enter the pineal gland, which coordinates the formation and secretion of serotonin and melatonin, through the suprachiasmic nuclei (SCN) of the hypothalamus. Light induces serotonin secretion, while melatonin is produced at night directly from serotonin by acetylation. However, melatonin production can be acutely interrupted by light exposure during the night. Norepinephrine, which is released at night in response to stimulatory signals

Fig. 1. The rate of glutamate decline during non-REM sleep positively correlated (r = 0.41, p < 0.01) with the amount of SWA. Each data point represents the average SWA and the change in glutamate concentration during non-REM sleep (Adapted from Dash, et al. 2009, The Journal of Neuroscience, Vol. 29, No. 3, pp. 620-629. Copyright 2009, Society for Neuroscience. Adapted with permission.)

originating in SCN, also regulates pineal gland activity. Melatonin can influence the sleeppromoting and sleep-wake rhythm regulating actions through the specific activation of melatonin receptors type 1 and 2, which are highly concentrated in SCN, and are also expressed in the peripheral organs and cells regulating other physiological functions of the so-called circadian 24-hour rhythms. The activation of type 1 occurs by inducing a receptorsuppressed neuronal firing rate in CNS, while type 2 induces a circadian phase shift. The increased secretion of melatonin is also accompanied by other circadian 24-hour rhythms of humans, and in rats studies associated with decreased production of neurotransmitter nitric oxide (NO) (Fig. 2) (Dubocovich et al., 2003; Ebadi, 1992; Geoffriau et al., 1998; Leon et al., 1998; Murphy & Delanty, 2007; Starc, 1998). Some studies do not confirm the influence of melatonin on the duration of sleep (Hughes et al., 1998), while others support the effect of melatonin on the duration and quality of REM sleep because they assume that it either directly influences cholinergic activity in REM sleep, or indirectly influences REM sleep by the elimination of serotonergic or aminergic activity (Jones, 1991; Kunz et al., 2004). It seems that melatonin modulates the release of acethylcholine in the nucleus accumbens and the motor activity of rats (Paredes et al., 1999). Some animal studies suggest that the daily changes in melatonin production may regulate the day-night variation in glutamate and GABA in the neostriatum (Marquez de Prado et al., 2000). It seems that the glutaminergic system negatively regulates norepinephrine-dependent melatonin synthesis in the rat's pineal gland (Yamada et al., 1998). In rats studies melatonin inhibits the glutamate-mediated response of the striatum to motor cortex stimulation and decrease NO content in parietotemporal cortex, striatum and brainstem of rats due to the inhibition of neuronal nitric oxide

Elemental Mercury Exposure and Sleep Disorder 57

In vitro and in vivo studies have shown that due to the increased production of free radicals as well as blocked sodium and calcium channels, Hg inhibits the uptake of some neurotransmitters, especially glutamate, into astrocytes, which increases their extracellular concentration, thus increasing the sensitivity of neighbouring neurons for stimulating excitotoxic effects (Aschner et al., 2007; Castoldi et al., 2001). The increased production of the neurotransmitter nitrogen oxide (NO) mediated by Hg (Ikeda et al., 1999) is also indirectly included in the excitotoxic effects of glutamate (Dawson et al., 1991). Hgº thus additionally increases the physiological level of extracellular glutamate and its glutaminergic activity during REM sleep and awakening. It is not expected that Hg++-mediated glutamate accumulation in extracellular space can decline during non-REM-SWA sleep through intracellular-astrocyte uptake by glutamate/asparate transporters and its degradation into glutamine, whose capacity is satisfactory in physiological conditions (Dash et al., 2009), but probably not in Hg++-enhanced glutaminergic activity. It seems that the decrease of melatonin mediated by interaction with Hg++ can also decrease the uptake of glutamate in astrocytes, which additionally contributes to pathological glutaminergic overactivity at

Given the results of some animal studies (Lena et al., 2005; Morari et al., 1998) and human data (Burbure et al., 2006; Entezari-Taher et al., 1999; Lucchini et al., 2003; Missale et al., 1998), it is expected that Hg++ enhances the dopaminergic effect in CNS, otherwise associated with cortical hyperexcitability and changes in the control of locomotor function. The impaired subcortical dopaminergic system, which may cause disinhibition at motor cortex level, could be associated with periodic contractile movements of the legs in the sleep (Entezari-Taher et al., 1999; Ondo et al., 2000; Rijsman et al., 2005; Ziemann et al., 1996) observed in miners during increased Hgº absorption and intoxication. We can not completely exclude the potential additive effect of sub-clinical peripheral neuropathy observed in miners, which can trigger and modify the appearance of periodic leg contractile

Melatonin is decreased in the night-time, either because of decreased synthesis under the influence of Hg-mediated, increased NO production, which in SCN operates similarly to a light signal (Ding et al., 1994; Ikeda et al., 1999), or by lowering its precursor tryptophan through its increased metabolizing, and because of its consumption in interaction with free radicals (McNally et al., 2008; Sener et al., 2003; Tan et al., 2000). A lower melatonin level is, at the same time, associated with the increased production of NO and its free radicals, peroxyntrites, which also increase the excitotoxic effects of glutamate (Acuna-Castroviejo et al., 1995; Leon et al., 1998). However, it has been established in many studies that melatonin plays a role in mediation between the circardian pacemaker and sleep-wake behaviour, and may have soporiphic properties and induce sedation, as well as the decreased nocturnal melatonin level labilised circadian rhythm function (Rodenbeck & Hajak, 2001; Stone et al.,

Increased extracellular glutamate and its decreased uptake in astrocytes (Dash et al., 2009) could hypothetically lead to longer REM periods and more frequent awakening associated with more frequent dreaming during increased Hgº absorption or intoxication. Hypothetically, persistent glutaminergic activity can also disrupt delta wave sleep, which could be associated with the sleep terrors observed in intoxicated miners. Further animal studies would be very helpful in elucidating the potential effects of Hg on the uptake of

increased Hg++ concentrations in CNS.

movements in sleep.

2000; Turek & Gillette, 2004).

synthase activity. On the other site the administration of high doses of melatonin have paradoxal effect and can decrease GABA and increase glutamate levels (Bikjdaouene et al., 2003; Leon et al., 1998). The synaptically released glutamate is taken up into astrocytes, where it is degraded into glutamine by the glutamate-metabolizing enzyme, glutamate synthetase. It is suggested that astrocytes are primarily responsible for controlling the extracellular level of glutamate, and melatonin seems to have a direct effect on astrocytes (Marquez de Prado et al., 2000; Segovia et al., 1999).

Fig. 2. Melatonin rhythm acts as an endogenous synchroniser adjusted to the 24-hour light/dark cycle, which (rats studies) regulates also the NO production (Adapted from Geoffrieau et al., 1998 ; Leon et al., 1998, *Hormone Research*, Vol. 49, pp. 136-141. Copyright 1998, S. Karger AG, Medical and Scientific Publishers. Adapted with permission.)
