**2. Orexinergic neurons, their receptors, and physio-pharmacological aspects of orexinergic system related to the sleep-wake cycle**

Prepro-orexin protein is the precursor protein, generating the excitatory neuropeptides orexins A and B (hypocretins 1 and 2). Orexin A (hypocretin 1), with a structure of 33 amino acids and 3.5 kDa, is completely conserved among different mammals which reflects its physiological relevance. Orexin B (hypocretin 2) is a 28-amino acid peptide with 2.9 kDa with 46% similarity to orexin A [14]. Their neurons, located on the LH, project widely throughout the brain and spinal cord [15]. Orexin excites target neurons through two types of expressed G-proteincoupled receptors. Orexin 1 receptor (OX1R) is dominantly expressed in the locus coeruleus (LC) and orexin 2 receptor (OX2R) is dominantly expressed in the arcuate nucleus (Arc), ventrolateral hypothalamus (VMH), LH, and TMN. Both OX1R and OX2R are expressed in the raphe nucleus and ventral tegmental area (VTA).

Similar to other wake-promoting neurons, orexin neurons fire mainly during active wakefulness when orexin levels are highest and are silenced during NREM and REM sleep, concurring with the lowest levels of orexin [16].

Different neuronal pathways involving orexin and neurotransmitters affecting its activity were identified. Neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the Arc project to orexin neurons [17]. Also, serotoninergic neurons in the median/paramedian raphe nucleus and GABAergic neurons in the ventrolateral preoptic (VLPO) nucleus send axons to orexin neurons [18]. VLPO is of major importance on initiating and maintaining NREM sleep as their neurons are activated by the somnogens adenosine [19] and prostaglandin D2 [20], and VLPO damage reduces NREM and REM sleep [21]. Orexinergic neurons are also targeted by neuronal projections from the bed nucleus of the stria terminalis (BST), supraventricular zone, and dorsomedial hypothalamus (DMH) [18] and receive neuronal projections from the suprachiasmatic nucleus—the human master circadian clock [22]. A direct neuronal pathway between SCN and orexinergic neurons was not identified until now.

**3. Melanin-concentrating hormone (MCH) and MCH neurons**

physiology.

sleep.

on REM sleep duration [41].

of sleep-wake physiological cycle [51–53].

The melanin-concentrating hormone is a 19-amino acid peptide predominant in specific neurons with the cell body located in the lateral hypothalamus and incerto-hypothalamic area of mammals. Apart from the sleep-active neurons in the preoptic area, these groups of neurons are also active during sleep, especially in REM sleep [32]. MCH neurons project throughout the brain with a dense innervation of the cholinergic and monoaminergic arousal centers [33]. MCH decreases cAMP levels in the cell through the MCH receptor 1(MCHR1), a G-protein-coupled receptor linked to Gq, Gi, and Go subunits which are expressed widely in the brain [34], and cellular electrophysiological studies showed that MCH has both presynaptic and postsynaptic strong inhibitory effects [35, 36]. The evidence that MCHR1 is expressed in several areas of the brain including those which are part of physiological pathways within sleep-wake control mechanisms (hippocampus, subiculum, basolateral amygdala, shell of the nucleus accumbens, ventromedial nucleus, arcuate nucleus, tuberomammillary nucleus, dorsolateral pons including dorsal raphe, and locus coeruleus) [37] supports that MCH neurons must play an essential role on sleep-wake

Hypothalamic Control of Sleep-Wake Circadian Cycle http://dx.doi.org/10.5772/intechopen.79899 35

Furthermore, while intracerebroventricular infusion of MCH peptide facilitates REM and NREM sleep [38], knockout of MCH is associated to a more active wakefulness state [39] and to a reduction on either REM or NREM sleep. Optogenetically selectively activated MCH neurons generally increase REM sleep duration [40–42]. Consistent results have shown that MCH neurons are strongly activated on REM sleep and de-activated during NREM, suggesting that MCH neurons promote REM sleep [32]. However, studies with timing-controlled ablation of MCH neurons revealed an increase in wakefulness and a reduction in NREM sleep, showing that MCH is also involved in the regulation of NREM

MCH neurons seem to inhibit some awake center neurons through GABAergic-inhibitory synapses onto histaminergic neurons of tuberomammillary nucleus. Recent work showed that the acute activation of MCH neurons, at the onset of REM sleep, extended the duration of this sleep stage but not that of the NREM sleep [42]. The inhibition of MCH neurons on the other hand reduces the frequency of theta rhythms from the hippocampus without interfering

MCH neurons are excited by orexin, AMPA agonists, NMDA, and cannabinoid type-1 receptor agonists [43–45] and inhibit orexinergic and adjacent GABAergic neurons [46]. It is clear, however, that orexin may also inhibit MCH neurons via GABAa receptors [47]. Dopamine is also an MCH neuronal inhibitor either via alpha-2 receptor [48] or via D1- and D2-like receptors [49]. Furthermore, MCH neurons are inhibited by MCH itself and by GABA, noradrenaline, serotonin, acetylcholine, neuropeptide Y, and histamine [50]. This mutual inhibitory interaction between orexin neurons and MCH neurons in the LH is crucial for the regulation

Since orexinergic neurons in LH are scarce and difficult to distinguish from other neurons just by morphology, a slice-path clamp technique, an electrophysiological method based on the expression of enhanced green fluorescent protein (EGFP) under the control of orexin promoter in transgenic mice, has been used in order to identify substances affecting orexinergic neuron activity [23, 24]. For instance, this allowed to assume the effects of distinct neurotransmitters on orexin neurons: glutamate receptor agonists AMPA and NMDA depolarize orexin neurons, while GABAA and GABAB receptor agonists muscimol and baclofen hyperpolarize those cells. Serotonin and noradrenaline hyperpolarize all orexin neurons through two receptors coupled to inhibitory Gi proteins (5HT1A and alpha 2A receptors, respectively) and subsequently activate protein-coupled inwardly rectifying potassium channels. Recent optogenetic methods allowed to confirm that the activation of serotoninergic neuron terminal inhibits orexin neurons either directly (via 5-HT1A receptor) or indirectly (via facilitation of GABAergic-inhibitory inputs) [25]. Dopamine also hyperpolarizes orexin neurons possibly by an indirect action through alpha 2A receptor [26], and glycine inhibits the activity of orexin neurons either directly and indirectly [27].

One complementary method to study the function of orexinergic neurons is to look for the physiological consequences of its ablation. Hara and coworkers generated transgenic mice, in which orexin neurons are ablated, and showed a phenotype similar to human narcolepsy [11], which also occurred in OX1r and OX2r knockout mice [28]. In transgenic mice, experimentally induced gradual ablation of orexin neurons using a specific "timecontrolled death" technique was associated to a fragmentation of the usual sleep-wake cycle [29]. The anatomical proximity and the genetic co-localization of the orexin neurons regulating sleep-wake state have recently benefitted from optogenetics. Using this kind of approach, Adamantidis and collaborators showed that by increasing the activity of orexin neurons, there was also an increased probability of transition to wakefulness from either NREM or REM sleep [30]. On the other hand, results from Zhang group using the same kind of approach indicate that the acute inhibition of orexinergic neurons leads to a time-ofday-dependent induction of NREM sleep [31]. To overcome some difficulties related to the study of neuronal networks located deeper in the brain, several new-generation optogenetic tools are being developed with an expected great impact on the near future in the areas of chronobiology and sleep physiology.
