REVIEW ARTICLE published: 06 March 2013 doi: 10.3389/fendo.2013.00018 The physiological role of /hypocretin neurons in the regulation of sleep/wakefulness and neuroendocrine functions

Ayumu Inutsuka and AkihiroYamanaka* Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

Edited by: The hypothalamus monitors body and regulates various behaviors such as Hubert Vaudry, University of Rouen, feeding, thermogenesis, and sleeping. (also known as hypocretins) were identified France as endogenous ligands for two orphan G-protein-coupled receptors in the lateral hypotha- Reviewed by: lamic area. They were initially recognized as regulators of feeding behavior, but they are Luis De Lecea, Stanford University, USA mainly regarded as key modulators of the sleep/wakefulness cycle. Orexins activate orexin Christelle Peyron, CNRS UMR5292, neurons, monoaminergic and cholinergic neurons in the hypothalamus/brainstem regions, INSERM U1028, France to maintain a long, consolidated awake period. Anatomical studies of neural projections *Correspondence: from/to orexin neurons and phenotypic characterization of transgenic mice revealed various Akihiro Yamanaka, Department of roles for orexin neurons in the coordination of emotion, energy homeostasis, reward Neuroscience II, Research Institute of Environmental Medicine, Nagoya system, and arousal. For example, orexin neurons are regulated by peripheral metabolic University, Furo, Chikusa, Nagoya cues, including , , and glucose concentration. This suggests that they may 464-8601, Japan. provide a link between energy homeostasis and arousal states. A link between the limbic e-mail: [email protected] system and orexin neurons might be important for increasing vigilance during emotional stimuli. Orexins are also involved in reward systems and the mechanisms of drug addiction. These findings suggest that orexin neurons sense the outer and inner environment of the body and maintain the proper wakefulness level of animals for survival. This review discusses the mechanism by which orexins maintain sleep/wakefulness states and how this mechanism relates to other systems that regulate emotion, reward, and energy homeostasis.

Keywords: orexin, hypocretin, sleep, hypothalamus, optogenetics, neuropeptide

INTRODUCTION noradrenergic locus coeruleus (LC), and histaminergic tubero- The hypothalamus plays a critical role in maintaining energy mammillary nucleus (TMN); and all of these nuclei are involved homeostasis by coordinating behavioral, metabolic, and neuroen- in promoting arousal (Saper et al., 2005). Prepro-orexin knockout docrine responses (Bernardis and Bellinger, 1996). Within this mice, orexin receptor knockout mice, and orexin neuron-ablated region, the lateral hypothalamic area (LHA) has been regarded transgenic mice all show severely defective sleep/wakefulness cycles as an important center for feeding and arousal because ani- (Chemelli et al., 1999; Hara et al., 2001; Willie et al., 2003). Con- mal models with LHA lesions exhibit hypophagia and decreased sistently, deficiencies of orexin function were found in human arousal that frequently leads to death. Orexin A and orexin B narcolepsy (Nishino et al., 2000; Peyron et al., 2000; Thannickal (also known as hypocretin 1 and hypocretin 2) are neuropeptides et al., 2000). These findings clearly show the importance of the expressed exclusively by LHA neurons. Orexin-producing neu- orexin system in the regulation of sleep/wakefulness. Past studies rons (orexin neurons) project their axons throughout the brain also revealed roles for orexin neurons beyond feeding and arousal, (Peyron et al., 1998; Nambu et al., 1999), which suggests that their including autonomic nervous system control (Sellayah et al., 2011; functions are varied. Remarkably, dense projections of orexin neu- Tupone et al., 2011) and in reward and stress systems (Boutrel rons are observed in the serotonergic dorsal raphe nucleus (DR), et al., 2005; Harris et al., 2005). In this review, we first discuss the basic biological features of orexins and their receptors, and we then describe the neuronal Abbreviations: AgRP,agouti-related peptide; Arc, arcuate nucleus; BAT, brown adi- pose tissue; BST, bed nucleus of the stria terminalis; CTB, cholera toxin B subunit; inputs and outputs of the orexin neurons. Finally, we discuss the DR, dorsal raphe nucleus; DREADD, designer receptors exclusively activated by various physiological roles of the orexin system, focusing on the designer drugs; FEO, food-entrainable oscillator; GPCRs, G-protein-coupled recep- regulation of sleep and wakefulness. tors; ICV, intracerebroventricular; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; LHA, lateral hypothalamic area; NMDAR, N-methyl-D-aspartate recep- tor; NPY, ; OX1R, orexin receptor 1; OX2R, orexin receptor 2; PPT, OREXIN AND OREXIN RECEPTORS pedunculopontine tegmental nucleus; PTX, pertussis toxin; PVN, paraventricular In 1998, two groups independently found the same new peptides thalamic nucleus; SCN,suprachiasmatic nucleus; TMN,tuberomammillary nucleus; TTC, C-terminal fragment of tetanus toxin; VLPO, ventrolateral preoptic nucleus; by using different strategies. Sakurai et al. (1998) used reverse VTA, ventral tegmental area. pharmacology to identify ligands of orphan G-protein-coupled

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receptors (GPCRs). They found a novel family of neuropeptides Orexin A acts on both orexin receptor 1 (OX1R) and 2 (OX2R), that binds to two closely related orphan GPCRs. Because the while orexin B selectively acts on OX2R (Sakurai et al., 1998). injection of the ligands induced feeding behavior, they named While orexin neurons are localized within the LHA, they have the ligands “orexin” after the Greek word orexis, which means widespread projections throughout the brain (Peyron et al., 1998; appetite (Sakurai et al., 1998). At the same time, de Lecea et al. Nambu et al., 1999; Figure 1). Therefore, it is important to (1998) isolated cDNAs selectively expressed within the hypotha- know the distribution pattern of orexin receptors to identify the lamus. Two peptides of the cDNAs showed substantial amino functional neuronal network. Marcus et al. (2001) used in situ acid sequence homology with the gut peptide hormone secretin, hybridization to demonstrate that OX1R and OX2R differ in dis- so they named these peptides “hypocretin.” They suggested that tribution. OX1R mRNA was observed in many brain regions hypocretins function within the central nervous system as neuro- including hippocampus, paraventricular thalamic nucleus (PVN), transmitters. ventromedial hypothalamic nucleus, DR, and LC. OX2R mRNA Prepro-orexin polypeptide is proteolysed to produce two orex- was prominent in a complementary distribution including the ins, orexin A and orexin B. Orexin A is a 33-amino acid peptide of cerebral cortex, hippocampus, DR, and many hypothalamic nuclei 3.5 kDa, with an N-terminal pyroglutamyl residue and C-terminal including PVN, TMN, and the ventral premammillary nucleus. amidation. The four Cys residues of orexin A form two sets Among these regions, DR, LC, and TMN are well known to be of intrachain disulfide bonds. This structure is completely con- involved in maintenance of the awake state. Consistently, orexin- served among several mammalian species (human, rat, mouse, deficient mice display a narcolepsy-like phenotype (Chemelli et al., cow, sheep, dog, and pig). On the other hand, rat orexin B is a 1999), as do dogs with a mutation preventing the expression of 28-amino acid, C-terminally amidated, linear peptide of 2.9 kDa, OX2R (Lin et al., 1999). Note that the regions expressing orexin which is 46% identical in sequence to rat orexin A. The 3.2 kb frag- receptors contain several areas of the hypothalamus, including ment of the 5-upstream region of the human prepro-orexin gene LHA, PVN, and the arcuate nucleus (Arc), which are all strongly is reported to be sufficient to express genes in orexin-containing implicated in the modulation of feeding. neurons (Sakurai et al., 1999; Moriguchi et al., 2002). In situ hybridization of prepro-orexin shows orexin-containing SIGNAL TRANSDUCTION SYSTEM OF OREXIN NEURONS neurons are located in the LHA. Prepro-orexin mRNA was shown OX1R and OX2R are seven-transmembrane GPCRs, which trans- to be upregulated under fasting conditions, indicating that these mit information into the cell by activating heterotrimeric G neurons somehow sense the animal’s energy balance (Sakurai et al., proteins. The signal transduction pathways of orexin receptors 1998). Recently, the forkhead box transcription factor Foxa2, a were examined in cells transfected with OX1R or OX2R. The downstream target of insulin signaling, was reported to be involved inhibitory effect of orexin on forskolin-stimulated cyclic adeno- in this transcriptional regulation (Silva et al., 2009). sine monophosphate (cAMP) accumulation was not observed in

FIGURE 1 | Schematic representation of inputs and outputs of orexin Abbreviations: Arc, arcuate nucleus; DR, dorsal raphe nucleus; LC, locus neurons. Orexin neurons are found only in the lateral hypothalamic area but coeruleus; LDT, laterodorsal tegmental nucleus; PPT, pedunculopontine project throughout the entire central nervous system. Red arrows show tegmental nucleus; TMN, tuberomammillary nucleus; VTA, ventral tegmental excitatory projections, while blue lines show inhibitory projections. area; VLPO, ventrolateral preoptic nucleus.

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OX1R-expressing cells. In addition, orexin-stimulated elevation to the orexin field such as the medial septum possibly because of in [Ca2+]i in OX1R- or OX2R-expressing cells was not affected transport to second-order neurons or ectopic expression of the by pertussis toxin (PTX) pretreatment. These results suggest transgene. In addition, the TTC::GFP technique also appears to be that OX1R does not couple to Gi proteins. On the other hand, less sensitive than conventional retrograde tracers, as it failed to forskolin-stimulated cAMP accumulation in OX2R-expressing label neurons in the lateral septum or VTA – regions that probably cells was inhibited by orexin in a dose-dependent manner, and this innervate orexin neurons as indicated by anterograde tracing and effect was abolished by pretreatment with PTX. These results indi- other retrograde tracing studies (Yoshida et al., 2006; Richardson cate that OX2R couples to both PTX-sensitive and PTX-insensitive and Aston-Jones, 2012). proteins (Zhu et al., 2003). Note that orexin has two independent Given that inputs to orexin neurons are so anatomically actions on neuronal activity: activation of noisy cation channels varied and associated with multiple functions, it might be rea- that generate depolarization and activation of a protein kinase C sonable to hypothesize the existence of subgroups of orexin (PKC)-dependent enhancement of Ca2+ transients mediated by neurons. Indeed, anterograde tracers injected into the DR L-type Ca2+ channels (Kohlmeier et al., 2008). marked the lateral LHA preferentially, while injections into Orexin neurons innervate monoaminergic neurons. In partic- the VMH preferentially stained neurons in the medial LHA ular, noradrenergic neurons of the LC, dopaminergic neurons of (Yoshida et al., 2006). With current technology we cannot only the ventral tegmental area (VTA), and histaminergic neurons of trace neuronal projections but also analyze functional connecti- the TMN are activated by orexins (Hagan et al., 1999; Horvath vity by utilizing optogenetic and pharmacogenetic tools (Lammel et al., 1999b; Nakamura et al., 2000; Yamanaka et al., 2002). LC et al., 2012). neurons exclusively express OX1R, while TMN neurons express OX2R, suggesting that both OX1R and OX2R signaling are excita- INPUT FROM HYPOTHALAMUS tory on neurons. Orexin colocalizes with dynorphin (Chou et al., Previous studies indicated that the LHA is innervated by several 2001) and glutamate (Abrahamson et al., 2001). It has also been hypothalamic regions, and some of these innervations project demonstrated that orexin increases local glutamate signaling by toward orexin neurons in the LHA. The LHA has long been con- facilitation of glutamate release from presynaptic terminals (Li sidered a brain region regulating food intake and body weight. et al., 2002). The localization of orexin neurons to the LHA and the func- tions ascribed to orexin neurons suggest that they may constitute INPUT TO OREXIN NEURONS components of a central circuitry controlling energy metabolism. ANATOMICAL ANALYSIS OF NEURONAL INPUT TO OREXIN NEURONS Therefore, it is reasonable to assess their connectivity to other It has been challenging to study the neuronal afferents to orexin neuronal populations involved in ingestive behaviors. Neuropep- neurons because they are scattered sparsely within the LHA. To tide Y (NPY), produced by specific neurons in the hypothalamic address this point, retrograde tracing studies were performed. The Arc (Allen et al., 1983; Chronwall et al., 1985), was demonstrated non-toxic C-terminal fragment of tetanus toxin (TTC) can be uti- to be a prominent inducer of food intake upon central administra- lized to retrogradely transfer the fused protein to interconnected tion (Clark et al., 1984; Stanley and Leibowitz, 1985). Projections neurons and transport toward the cell bodies of higher-order to orexin neurons from NPY/agouti-related peptide (AgRP) neu- neurons (Maskos et al., 2002). Sakurai et al. (2005) generated rons in the Arc were identified by anatomical studies (Broberger transgenic mouse lines expressing a fused protein of TTC and et al.,1998; Elias et al.,1998). Furthermore, orexin neurons express green fluorescent protein (GFP) exclusively in orexin neurons NPY receptors, and direct administration of NPY agonists into by using the promoter of human prepro-orexin. They identi- the LHA increases Fos-like immunoreactivity in orexin neu- fied several brain regions including the basal forebrain cholinergic rons (Campbell et al., 2003). These findings suggest NPY excites neurons, gamma-aminobutyric acid (GABA)ergic neurons in the orexin neurons; however, electrophysiological analyses showed ventrolateral preoptic nucleus (VLPO), and serotonergic neurons that direct application of NPY instead reduces spike frequency and in the median raphe and paramedian raphe nucleus. Moreover, hyperpolarizes the membrane potential of orexin neurons (Fu regions associated with emotion including the amygdala, infral- et al., 2004). imbic cortex, shell region of the nucleus accumbens, and the bed nucleus of the stria terminalis (BST) were found to innervate INPUT FROM LIMBIC SYSTEM orexin neurons. Given that depletion of orexin neurons induces the sleep disor- In addition to TTC, the cholera toxin B subunit (CTB) is der narcolepsy, the limbic system might also provide important also used to retrogradely trace neuronal projections. Yoshida et al. projections to orexin neurons. Narcolepsy patients often suffer (2006) injected CTB into the LHA and counted every labeled cell from an attack called“cataplexy,”which is characterized by sudden in rats. Interestingly, they found strong projections from the lat- weakening of postural muscle tone. Cataplexy is often triggered eral septum, preoptic area, BST, and posterior hypothalamus. In by strong, generally positive emotion while consciousness is pre- addition, they also found that hypothalamic regions preferentially served during the attack (Honda et al., 1986). This fact implies that innervate orexin neurons in the medial and perifornical parts of orexin neurons may play a role in the physiological responses asso- the field, but most projections from the brainstem target the lateral ciated with emotions. Consistently, local injection of orexin into part of the field. the pedunculopontine tegmental nucleus (PPT) strongly inhibited The results of these two papers present slight distinctions. rapid eye movement (REM)-related atonia in the cat (Takakusaki TTC::GFP sometimes labeled regions with no known projections et al., 2005). Therefore, it is hypothesized that emotional stimuli

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increase orexin release in the PPT to prevent muscle atonia in FACTORS THAT INFLUENCE ACTIVITY OF OREXIN NEURONS wild-type animals. Electrophysiological studies have identified several modulators The innervations from limbic system may mediate emotional that regulate activity of orexin neurons. Recordings from trans- arousal and fear-related responses. Prepro-orexin knockout mice genic mice expressing GFP in orexin neurons demonstrated that showed weaker cardiovascular and locomotor responses to emo- agonists of ionotropic glutamate receptors activated orexin neu- tional stress in an awake and freely moving condition (Kayaba rons, while glutamate antagonists reduced their activity (Li et al., et al., 2003). Consistently, air jet stress-induced elevations of 2002; Yamanaka et al., 2003b). These results indicate that orexin blood pressure and heart rate were attenuated in conscious neurons are tonically activated by glutamate. orexin/ataxin-3 mice, in which orexin neurons were specifically Dopamine, noradrenaline, and serotonin (5-HT) hyperpolar- ablated by expressing neurotoxic protein (Zhang et al., 2006). ize and inhibit orexin neurons via alpha2 and 5-HT1A receptors, respectively (Yamanaka et al., 2003b; Muraki et al., 2004; Li and INPUT FROM PREOPTIC AREAS van den Pol, 2005). Dopamine-induced hyperpolarization is most The preoptic area, especially the VLPO, plays a critical role in non- likely mediated by alpha2-adrenergic receptors since a very high REM sleep initiation and maintenance (Lu et al., 2002). The VLPO concentration of dopamine is necessary to induce hyperpolar- has multiple inhibitory projections to neurons that release wake- ization and also because dopamine-induced hyperpolarization is promoting neurotransmitters, including histamine neurons in the inhibited by the alpha2-adrenergic receptor antagonist, idazoxan TMN, noradrenergic neurons in the LC, serotonergic neurons in (Yamanaka et al., 2006). However, it is noteworthy that dopamine the DR, and acetylcholinergic neurons (Sherin et al., 1996, 1998; potentially affects both dopamine receptors and adrenergic recep- Steininger et al., 2001; Lu et al., 2002). tors, while the dopamine D2 receptor antagonist eticlopride blocks Neurons in the VLPO fire at a rapid rate during sleep, with the actions of dopamine on spike frequency and membrane attenuation of firing during the awake period. Likewise, neurons potential (Li and van den Pol, 2005). Thus, dopamine might in wake-promoting centers fire rapidly during wakefulness and are act through both alpha2-adrenergic receptor and dopamine D2 relatively quiescent during sleep, with the exception of cholinergic receptor. neurons, which are divided into two classes of neurons: one is Recently, it was found that orexin itself excites orexin neurons active in both the awake and REM sleep period, and the other is via OX2R (Yamanaka et al., 2010). This suggests that orexin neu- active only in the REM sleep period. rons form a positive-feedback circuit through indirect and direct Orexin neurons are strongly inhibited by both a GABAA ago- pathways, which results in the preservation of the orexin neuron nist, muscimol (Yamanaka et al., 2003b), and a GABAB receptor network at a high activity level and/or for a longer period. agonist, baclofen (Xie et al., 2006). Orexin neurons are also inner- Calcium imaging using transgenic mice in which orexin vated by cells in the VLPO that also contain GABA (Sakurai neurons specifically express yellow cameleon 2.1 showed that et al., 2005; Yoshida et al., 2006). These observations suggest that neurotensin, sulfated octapeptide form of , oxy- VLPO neurons send GABAergic inhibitory projections to wake- tocin, and vasopressin activate orexin neurons, while 5-HT, promoting neurons including orexin neurons. This pathway might noradrenaline, dopamine, and muscimol, a GABAA receptor ago- be important to initiate and maintain sleep. nist inhibit these cells (Tsujino et al., 2005). Recently, it was also reported that orexin neurons express glycine receptors through- INPUT FROM SUPRACHIASMATIC NUCLEUS out adulthood and that glycine inhibits the electric activity of Given that sleep/wakefulness is a circadian phenomenon, it is orexin neurons directly and indirectly (Hondo et al.,2011; Karnani reasonable to consider that orexin neurons receive information et al., 2011b). from the suprachiasmatic nucleus (SCN), which is the center Other factors that reportedly influence the activity of of the circadian rhythm according the environmental light–dark orexin neurons include corticotrophin-releasing factor (Winsky- information. Indeed, the circadian fluctuation of orexin levels Sommerer et al., 2004), ATP (Wollmann et al., 2005), NPY (Fu in the cerebrospinal fluid (CSF) disappears when the SCN is et al., 2004), and physiological fluctuations in acid and CO2 lev- removed (Deboer et al., 2004). Although direct input to orexin els (Williams et al., 2007). It is noteworthy that the factors that neurons from the SCN appears to be sparse, orexin neurons receive are supposed to be influenced by feeding (such as glucose, ghre- abundant innervations from the BST, supraventricular zone, and lin, and leptin) inhibit the activity of orexin neurons (Yamanaka dorsomedial hypothalamus (DMH; Sakurai et al., 2005; Yoshida et al., 2003a). The large variety of factors regulating orexin neu- et al., 2006), which receive input from the SCN (Leak and Moore, ronal activities demonstrates the integral role of orexin neurons 2001). This suggests the possibility that orexin neurons receive in monitoring circadian rhythms, energy balance, and vigilance circadian influences from the SCN via these regions. Note that level. excitotoxic lesions of the DMH reduce the circadian rhythmicity of wakefulness. The DMH projects to orexin neurons (Chou et al., REGULATION OF OREXIN NEURONS BY HUMORAL FACTORS 2003), although the DMH also projects to multiple brain areas Motivated behaviors such as food-seeking are deeply involved in such as the LC and the VLPO that are involved in sleep and wake- maintenance of arousal. Orexin neurons are thought to function as fulness. In addition, considering that the orexin system is involved the sensor of energy balance. Electrophysiological studies revealed in food-entrainable oscillator (FEO; Mieda et al., 2004), the circa- that increasing extracellular glucose concentrations induce strik- dian change in the activity of orexin neurons might be regulated ing hyperpolarizations, while decreasing the glucose concentration by other elements, such as energy balance. induces depolarization and increases the frequency of action

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potentials of orexin neurons (Yamanaka et al., 2003a; Burdakov FUNCTIONS IN SLEEP AND WAKEFULNESS et al., 2005). Importantly, this mechanism is sufficiently sensitive The roles of orexin neurons in the regulation of sleep and to respond to physiological fluctuations of glucose concentration arousal have been reported repeatedly. ICV injection of orexin induced by normal feeding. Note that other dietary nutrients, A or orexin B during the light period increased awake time and amino acids, also activate orexin neurons, and they can sup- reciprocally decreased REM and non-REM sleep time (Hagan press the glucose response of orexin neurons at physiological et al., 1999; Bourgin et al., 2000; Piper et al., 2000). Sleep frag- concentration (Karnani et al., 2011a). mentation observed in orexin knockout mice (Chemelli et al., In addition, the orexigenic peptide ghrelin activated isolated 1999), orexin receptor knockout mice (Willie et al., 2003), and orexin neurons with depolarization and an increase in action orexin neuron-ablated transgenic mice (Hara et al., 2001) shows potential frequency (Yamanaka et al., 2003a). In contrast, the us the importance of their physiological functions. Narcolepsy strong anorectic factor leptin robustly inhibited orexin neu- is a sleep disorder characterized by primary disorganization of rons, causing hyperpolarization and decreasing the firing rate sleep/wakefulness cycles. It has also been reported that the num- (Yamanaka et al., 2003a). Notably, insulin exerted no direct effects ber of orexin neurons is greatly reduced, and orexin peptide levels on orexin neurons. These findings are consistent with the idea in the cerebrospinal fluid are decreased to undetectable levels that orexin neurons act as a sensor of nutritional status (Sakurai in narcoleptic patients (Nishino et al., 2000; Peyron et al., 2000; et al., 1998). Indeed, transgenic mice without orexin neurons fail Thannickal et al., 2000). Orexin-ataxin-3 mice are a well known to show fasting-induced arousal (Yamanaka et al., 2003a). mouse model of narcolepsy. However, in orexin-ataxin-3 mice, orexin neurons are absent from birth, and therefore other neu- PHYSIOLOGICAL FUNCTIONS OF OREXIN NEURONS ronal mechanisms might compensate for the function of orexin FUNCTIONS IN FEEDING BEHAVIORS AND ENERGY HOMEOSTASIS neurons during development. Indeed, the frequency of cataplexy Orexin neuron-ablated transgenic mice show hypophagia and late- is not high in these mice. Timing-controlled neuronal ablation onset (Hara et al., 2001), although the severity of the obese models using the tTA-TetO system might overcome this problem. phenotype critically depends on genetic background (Hara et al., The activities of monoaminergic neurons in the brainstem and 2005). These findings imply a role for orexin in the regulation of hypothalamus are known to be associated with sleep and awake energy homeostasis. Although orexins stimulate feeding behav- states. Furthermore, the DR, LC, and TMN monoaminergic neu- ior, they do not slow metabolic rate, which might be expected in rons express orexin receptors and are densely innervated by orexin a system geared for weight gain. Instead, orexins increase both neurons. These findings suggest that these regions mediate the food intake and metabolic rate (Lubkin and Stricker-Krongrad, effects of orexins. Consistently, noradrenergic neurons of the LC 1998). Because animals must be aware and active when they (Hagan et al., 1999), serotonergic neurons of the DR (Brown et al., seek and eat food, this function might be important for feeding 2002; Liu et al., 2002), and histaminergic neurons of the TMN behavior. (Huang et al., 2001; Yamanaka et al., 2002)havebeenshownto The Arc is attributed to the regulation of feeding behav- be activated by orexins. These observations suggest that the activ- iors. Orexin neurons densely project to this region (Date ity of these monoaminergic neurons is at least partly regulated et al., 1999; Horvath et al., 1999a). Principal components of by orexins. Orexins also have a strong direct excitatory effect the regulating system of feeding behaviors include antagonis- on cholinergic neurons of the basal forebrain (Eggermann et al., tic and complementary appetite-stimulating (orexigenic) and 2001), which is hypothesized to play an important role in arousal. appetite-suppressing (anorectic) pathways: NPY/AgRP neurons The PPT and the laterodorsal tegmental nucleus (LDT) pro- and proopiomelanocortin (POMC) neurons. It was reported vide cholinergic afferents to several brain regions and play a that intracerebroventricular (ICV) injection of orexin induced pivotal role in the regulation of REM sleep and wakefulness. These c-Fos expression in NPY neurons of the Arc (Yamanaka et al., regions are also strongly innervated by orexin neurons (Peyron 2000). Therefore, orexin-stimulated feeding may occur at least et al., 1998; Nambu et al., 1999). Electrophysiological experiments partly through NPY pathways. However, because NPY antag- revealed that the firing rate of cholinergic neurons is increased onist (which completely abolished NPY-induced feeding) only by orexin A (Burlet et al., 2002). Microinjection of orexin A into partially abolished orexin-induced feeding in rats, other path- the LDT increases awake time and decreases REM sleep time in ways by which orexin induces feeding might exist. POMC neurons cats (Xi et al., 2001), and when orexin A is injected into the PPT of the Arc are known to suppress appetite, and lack of POMC- in cats, an increased stimulus at the PPT is required to induce derived peptides or electrical silencing of POMC neurons causes muscle atonia (Takakusaki et al., 2005). However, it is noteworthy obesity. Orexin neurons might affect feeding behavior by inhibit- that the LDT/PPT contains other neuronal types beside choliner- ing POMC-expressing neurons (Muroya et al., 2004). Indeed, gic neurons that show activity associated with sleep/wake cycles orexin suppresses action potential firing and hyperpolarizes the (Sakai, 2012). membrane potential of POMC neurons in theArc (Ma et al.,2007). Emerging new studies using optogenetics have revealed the Intracerebroventricular injection of orexin induces water intake physiological roles of orexin neurons in vivo. Direct selective pho- as well as food intake (Kunii et al., 1999). Additionally arginine- tostimulation of orexin neurons expressing channelrhodopsin2 vasopressin, also known as antidiuretic hormone, activates orexin increases the probability of transition from non-REM or REM neurons via the V1a receptor (Tsunematsu et al., 2008). These sleep to wakefulness (Adamantidis et al., 2007) and activates results suggest a role for orexin neurons in fluid homeosta- downstream wake-promoting nuclei such as LC and TMN (Carter sis too. et al., 2009). Consistently, it was also shown that direct selective

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inhibition of orexin neurons expressing halorhodopsin induces increased non-REM sleep time (Sasaki et al., 2011). Additionally, non-REM sleep (Tsunematsu et al., 2011). Furthermore, optoge- melanopsin, a photosensitive G-protein-coupled photopigment, netic stimulation of the LC produces immediate sleep-to-wake makes it possible to control wakefulness by blue light in a way transitions, whereas the inhibition causes a decrease in wakefulness similar to channelrhodopsin (Tsunematsu et al., 2012). (Carter et al.,2010). Recently it was also reported that photoinhibi- tion of LC neurons during the photostimulation of orexin neurons FUNCTIONS IN AUTONOMIC NERVOUS SYSTEM cancels these sleep-to-wake transitions (Carter et al., 2012). These Orexin-deficient mice show lower blood pressure than wild-type findings indicate that the LC is a major effector of orexin neurons littermates (Kayaba et al., 2003; Zhang et al., 2006). Consistently, in the regulation of sleep and wakefulness. However, it is notewor- ICV injection of orexins increases blood pressure and heart rate thy that LC noradrenergic neurons express only OX1R, and that (Shirasaka et al., 1999), and these effects are abolished by admin- OX1R knockout mice show only weak fragmentation of sleep and istration of alpha1-adrenergic receptor antagonist, prazosin, or no cataplexy (Mieda et al., 2011). beta-adrenergic receptor antagonist, propranolol. These results Recently, not only photo-activated ion channels or ion pumps suggest that orexins physiologically stimulate the sympathetic but also other natural and modified proteins have been used to nervous system and regulate energy expenditure. regulate the activity of specific neuronal circuits in vivo. One Heat production in brown (BAT) also con- interesting example is “Designer Receptors Exclusively Activated tributes to body weight regulation through the maintenance of by Designer Drugs (DREADD).” This method employs modified body temperature. Recently Tupone et al. (2011) reported that muscarinic receptors (hM3Dq for excitation and hM4Di for inhi- orexinergic projections to raphe pallidus increase BAT thermoge- bition) that have lost their affinity for endogenous acetylcholine nesis in rat. This finding provides a new mechanism contributing but can be activated by a synthetic ligand, clozapine-N-oxide, to the disrupted regulation of body temperature and energy which can cross the blood–brain barrier (Armbruster et al., 2007; metabolism in the absence of orexin. Orexin neuron-ablated Alexander et al., 2009). Because stimulation of GPCRs with a spe- transgenic mice show late-onset obesity, although they also show cific ligand has a longer effect on cellular signaling than optical hypophagia (Hara et al., 2001). The regulation of BAT thermogen- stimulation, the DREADD system can facilitate the examination of esis by orexin neurons might account for this phenotype of energy the chronic effects of modulating the activity of specific neurons. metabolism (Sellayah et al., 2011). Using this technique, it was reported that the excitation of orexin neurons significantly increased the amount of time spent in wake- FUNCTIONS IN REWARD AND STRESS SYSTEMS fulness and decreased both non-REM and REM sleep times and Toattenuate the symptoms of the sleep disorder, psychostimulants that inhibition of orexin neurons decreased wakefulness time and such as amphetamine or methylphenidate are often given to

FIGURE 2 | A schematic diagram to illustrate the integrative tissue; BST, bed nucleus of the stria terminalis; DA, dopamine; DR, physiological roles of orexin neurons. Orexin neurons regulate various dorsal raphe nucleus; GABA, gamma-aminobutyric acid; HA, histamine; physiological phenomena such as wakefulness, feeding, reward, and LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; LHA, lateral thermogenesis. The body energy level influences orexin neuronal activity hypothalamic area; NA, noradrenalin; NAc, nucleus accumbens; NPY, to coordinate arousal and energy homeostasis. Inputs from the limbic neuropeptide Y; POA, preoptic area; POMC, proopiomelanocortin; system may be important to regulate the activity of orexin neurons to PPT, pedunculopontine tegmental nucleus; SCN, suprachiasmatic evoke emotional arousal or fear-related responses. Abbreviations: 5-HT, nucleus; TMN, tuberomammillary nucleus; VTA, ventral tegmen- serotonin; ACh, acetylcholine; Arc, arcuate nucleus; BAT, brown adipose tal area.

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narcolepsy patients. Interestingly, drug addiction hardly occurs exist, orexin neurons themselves monitor various physiolog- in these patients. This finding suggests that the orexin system ical conditions and coordinate various behaviors to respond mediates the establishment of drug addiction. The LHA, where to environmental change adequately (Figure 2). For example, orexin neurons exist, is a brain region historically implicated feeding behaviors affect the activity of orexin neurons through in reward and motivation, and orexin neurons project to many changes in concentration of glucose or amino acids, and these brain areas including the LC, nucleus accumbens, and VTA (Fadel changes modulate the vigilance state, regulating aspects of the and Deutch, 2002) that are implicated in behavioral responses to autonomic nervous system such as blood pressure, heart rate, drugs of abuse. Orexin directly activates VTA dopaminergic neu- and thermogenesis at the same time. These findings indicate rons (Nakamura et al., 2000; Korotkova et al., 2003). ICV or local a critical role for orexin neurons in the regulation of vigi- VTA infusion of orexin drives behavior motivated by either food lance states, according to internal and external environments, for or drug rewards (Sakurai et al., 1998; Boutrel et al., 2005; Harris survival. et al., 2005). By combining viral-mediated tracing, electrophysiology, and It was demonstrated that orexin A input to the VTA potentiates optogenetic manipulations, it might be determined that there are N-methyl-D-aspartate receptor (NMDAR)-mediated neurotrans- several subpopulations of orexin neurons that project to different mission via a PLC/PKC-dependent insertion of NMDARs in VTA target areas. For example, the distribution pattern of orexin neu- dopamine neuron synapses (Borgland et al., 2006). Furthermore, rons appears to be divided into two groups: medial and lateral. intra-VTA microinjection of an OX1R antagonist abolished a con- Some of the input projections to orexin neurons demonstrate a ditioned place preference for morphine (Narita et al., 2006) and preference between these two areas as well. With new tools to locomotor sensitization to cocaine (Borgland et al., 2006). These manipulate specific neuronal projections, we can now study phys- data indicate that orexin signaling plays an important role in neural iological differences within the orexin system. These upcoming plasticity relevant to addiction in the VTA. findings may reveal that discrete functional units underlie the integral role of the orexin system. CONCLUDING REMARKS Although the name orexin is derived from the word orex- ACKNOWLEDGMENTS igenic after its function in feeding, mounting evidence has This study was supported by JST PRESTO program and Grant-in- revealed various physiological roles for orexin other than feed- Aid for Scientific Research (B) (23300142), Grant-in-Aid for Sci- ing, such as maintenance of sleep, autonomous regulation, and entific Research on Innovative Area “Mesoscopic Neurocircuitry” reward processing. Orexin neurons in the LHA are anatomi- (23115103) from the Ministry of Education, Culture, Sports, cally well placed to provide a link between the limbic system, Science and Technology (MEXT) of Japan (Akihiro Yamanaka), energy homeostasis, and brain stem monoaminergic or cholin- and Grant-in-Aid for Young Scientists (B) (24790192) (Ayumu ergic neurons. Like the hypothalamus where orexin neurons Inutsuka).

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