The Neural Circuit of Orexin (Hypocretin): Maintaining Sleep and Wakefulness

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The neural circuit of orexin (hypocretin): maintaining sleep and wakefulness

Takeshi Sakurai Abstract | Sleep and wakefulness are regulated to occur at appropriate times that are in accordance with our internal and external environments. Avoiding danger and finding food, which are life-essential activities that are regulated by emotion, reward and energy balance, require vigilance and therefore, by definition, wakefulness. The orexin (hypocretin) system regulates sleep and wakefulness through interactions with systems that regulate emotion, reward and energy homeostasis.

Narcolepsy The neuropeptides orexin A and orexin B (also known Together, these observations suggest that, broadly A neurological condition as hypocretin 1 and hypocretin 2), produced in speaking, orexin neurons are involved in sensing the mostly characterized by hypothalamic neurons, are crucial regulators of sleep body’s external and internal environments and regulate excessive daytime sleepiness, and wakefulness. These peptides activate wake-active states of sleep and wakefulness accordingly, which is bene- uncontrollable sleep attacks monoaminergic and cholinergic neurons in the hypotha- ficial for survival. This review will discuss the mechanisms and disorder of REM sleep. lamus and brain stem to maintain a long, consolidated by which the orexin system maintains sleep and wakeful- Limbic system awake period, and it is this role in particular that will ness, and how this mechanism relates to other systems A collection of cortical and form the focus of this review. that regulate emotion, reward and energy homeostasis. subcortical structures Orexins were initially identified as endogenous important for processing 1 Narcolepsy and orexins memory and emotional ligands for two orphan G-protein-coupled receptors information. Prominent (BOX 1). They were recognized as regulators of feeding The symptoms and pathophysiology of the sleep dis- structures include the behaviour, firstly because of their exclusive produc- order narcolepsy, caused by an orexin deficiency5–8 hippocampus and amygdala. tion in the lateral hypothalamic area (LHA), a region (BOX 2), provide insight into the physiological roles known as the feeding centre, and secondly owing to their of orexin. Narcolepsy is characterized by the inability Ghrelin Stomach-derived orexigenic pharmacological activity; intracerebroventricular (ICV) to maintain vigilance states, pathological intrusion of peptide. injection of orexins during the light period induces rapid eye movement sleep (REM sleep) and/or non-REM feeding behaviour in rats and mice1–4. Subsequently, the (NREM) sleep into wakefulness, and frequent transitions Leptin finding that an orexin deficiency causes narcolepsy in between states of sleep and wakefulness, which indicates An adipocyte-derived protein hormone that has a key role in humans and animals indicated that these hypothalamic that orexins have important roles in the maintenance regulating energy intake and neuropeptides also have a crucial role in regulating sleep and stabilization of sleep and wakefulness. energy expenditure. and wakefulness5–9. The first clues towards an involvement of the orexins Recent studies of orexin-producing neurons’ efferent in narcolepsy came from animal models; mice lacking the

Department of Pharmacology, and afferent systems, as well as phenotypic characteriza- orexin gene and dogs with null mutations in the orexin Institute of Basic Medical tions of mice with genetic alterations in the orexin system, receptor 2 (OX2R) gene show phenotypes remarkably Science, University of have suggested further roles for orexin in the coordination similar to humans with narcolepsy7,8 (see Supplementary Tsukuba, Ibaraki 305-8575, of emotion, energy homeostasis, reward, drug addiction information S1 (table)). Mice lacking the orexin precur- Japan and the Yanagisawa and arousal10–17. sor prepro-orexin, orexin neuron-ablated (orexin/ataxin 3 Orphan Receptor Project, ERATO, Japan Science and Orexin neurons receive abundant input from the transgenic) mice and OX1R/OX2R double knockout mice 14,15 Technology Agency, limbic system , which might be important for increas- exhibit similar phenotypes that have strong parallels to the Tokyo 135-0064, Japan. ing arousal during emotional stimuli. Orexin neurons are human condition. These are characterized by behavioural e-mail: also regulated by peripheral metabolic cues, including arrests that are similar to a condition called cataplexy [email protected] ghrelin leptin (BOX 2) doi:10.1038/nrn2092 , and glucose, indicating that orexin neurons , occasional direct transitions to REM sleep from 7,9 Published online might provide a link between energy homeostasis and wakefulness, and highly fragmented sleep-wake cycles , 14 February 2007 vigilance states11. all of which are important elements of narcolepsy.

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Rapid eye movement sleep The link between orexin dysfunction and narcolepsy, The orexin system’s role in wakefulness (REM sleep) The stage of sleep especially when accompanied by cataplexy (narcolepsy– How do the orexins physiologically regulate sleep and characterized by rapid cataplexy), has since been supported by findings in human wakefulness, and why does a lack of orexin signalling movements of the eyes. patients. A post-mortem study of human narcolepsy result in narcolepsy? In this section, I will discuss the

Cataplexy patients found that orexin peptides were undetectable in mechanisms of action of these peptides at both An episodic condition featuring the cortex and pons, in which orexinergic projections are the cellular and the systems level. loss of muscle function, ranging normally found (FIG. 1), and that there was an 80–100% from slight weakness (such as reduction in the number of neurons containing detect- Orexins stabilize wakefulness. It has been shown that limpness at the neck or knees, able prepro-orexin mRNA or orexin-like immunoreac- ICV injection of orexin A or orexin B in rats during sagging facial muscles or 5,6 inability to speak clearly) to tivity in the hypothalamus . This supports earlier reports the light (rest) period, the equivalent of night-time complete body collapse. that orexin A was undetectable in the cerebro spinal fluid in humans, increases awake time and decreases REM of narcolepsy patients18. Approximately 90% of patients and NREM sleep time23. By what mechanisms does the with narcolepsy, especially those with narcolepsy– orexin system evoke this pharmacological effect? cataplexy, show decreased orexin A levels in the cerebro- Orexin neurons originate in the hypothalamus and spinal fluid19. Therefore, a low cerebrospinal fluid level are almost exclusively localized in the LHA and pos- of orexin A is now one of the diagnostic criteria for terior hypothalamus24–26. These neurons are variable in narcolepsy–cataplexy, according to the 2nd edition of the size (the cell body diameter ranges from 15–40 μm) and international classification of sleep disorders20. shape (spherical, fusiform or multipolar), and have been A recent finding showing concomitant loss of dynor- assumed to number around 3000 in the rat brain, or phin, neuronal activity-regulated pentraxin and orexin, 7000 in the human brain25,26. From these regions, orexin which colocalize in orexin neurons, further indicates neurons project widely to the entire neuroaxis, a loss of orexin neurons in narcolepsy–cataplexy21. The excluding the cerebellum24–26 (FIG. 1). The densest cause of the specific loss or degradation of orexin neurons staining of orexin-immunoreactive nerve endings in narcolepsy has been unknown so far, but because of its in the brain is found in the paraventricular nucleus strong association with certain human leukocyte antigen of the thalamus, the arcuate nucleus and, most notably, the (HLA) alleles22 it is possible that narcolepsy could result locus coeruleus (LC, containing noradrenergic from selective immune-mediated degeneration of orexin neurons), dorsal raphe (DR, which contains sero- neurons, although no specific antibody against tonergic neurons) and tuberomammillary nucleus orexin neurons has been found in the serum of affected (TMN, containing histaminergic neurons)5,24,25. individuals. Regardless of the cause of the neuron loss, The distribution of mRNA for the orexin receptors the orexin signalling deficiency in narcolepsy–cataplexy is consistent with these projection sites; within the

shows that this neuropeptide system has an important role brain, OX1R mRNA is most abundantly expressed in

in the regulation of sleep and wakefulness, especially in the LC, whereas OX2R mRNA is highly expressed in the the maintenance of long, consolidated awake periods. TMN27. Both regions are important for the maintenance of arousal27. The DR and ventral tegmental area (VTA) 27 contain both OX1R and OX2R mRNA . These observa- Box 1 | Overview of the orexin/hypocretin system tions indicate that these monoaminergic regions are important effector sites of orexins. Orexin A and orexin B were identified by our group as endogenous ligands for two Consistent with this hypothesis, electrophysiological orphan G-protein-coupled receptors1. Orexin A and B are derived from a common experiments using brain slice preparations or isolated precursor peptide, prepro-orexin. An mRNA encoding the same precursor peptide was independently isolated as a hypothalamus-specific transcript98. It was predicted that the cells have shown that cells of these nuclei are activated by 23,28 transcript encoded a polypeptide precursor that is cleaved to form two neuropeptides, orexins in vitro. Indeed, noradrenergic cells of the LC , 29 termed hypocretin 1 and hypocretin 2. To avoid confusion, the orexin nomenclature is dopaminergic cells of the VTA , serotonergic cells of used throughout this review, but it should be noted that the names ‘orexin’ and the DR30,31 and histaminergic cells of the TMN32 have ‘hypocretin’ are currently used synonymously in many papers. all been shown to be activated by orexins. Many of these Orexins constitute a novel peptide family, with no significant homology with any monoaminergic neurons are implicated in increasing previously described peptides41. Orexin A is a 33 amino acid peptide with an amino arousal and promoting wakefulness. The activity of mono- (N)-terminal pyroglutamyl residue, two intra-chain disulphide bonds and carboxy aminergic neurons in the TMN, LC and DR is known to (C)-terminal amidation. This structure is completely conserved among several be synchronized and strongly associated with sleep and mammalian species (human, rat, mouse, cow, sheep, dog and pig). Orexin B is a 28 amino wakefulness: the neurons fire tonically during wakeful- acid, C-terminally amidated linear peptide. The C-terminal half of orexin B is very similar to that of orexin A, whereas the N-terminal half is more variable. ness, less during NREM sleep, and not at all during REM 33 The actions of orexins are mediated by two receptors, named orexin 1 (OX R) and sleep . These observations indicate that orexin-mediated 1 arousal results from the activation of these wake-active orexin 2 (OX2R) receptors (also known as HCRTR1 and HCRTR2). OX1R has one-order-of- magnitude greater affinity for orexin A than orexin B. By contrast, orexin A and orexin B monoaminergic neurons. Specifically, orexin neurons, 1 α bind OX2R with similar affinities . OX1R is thought to transmit signals through the G 11 activated during wakefulness, exert an excitatory influ- class of G protein, which results in the activation of phospholipase C with subsequent ence on these wake-active neurons, thereby sustaining triggering of the phosphatidylinositol cascade and influx of extracellular Ca2+, probably their activity. through transient receptor potential (TRP) channels. OX R is thought to be coupled to 2 Additional evidence for a role of orexin in wakeful- both Gα and inhibitory G G proteins99. OX R and OX R mRNAs exhibit a markedly 11 i 1 2 ness is provided by the strong, direct excitatory effect of different and basically complementary distribution, indicating that these receptors have orexins on cholinergic neurons in the basal forebrain34, distinct physiological roles27. which are important for maintaining arousal35.

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Box 2 | What is narcolepsy (narcolepsy–cataplexy)? Narcolepsy is a serious neurological disorder that affects approximately 1 in 2000 individuals in the United States100. Onset of the condition is usually during adolescence (around 12–14 years old). A cardinal symptom of the disorder is excessive daytime sleepiness (an insurmountable urge to sleep), which manifests itself primarily as the subject falling asleep at inappropriate times (‘sleep attacks’). The latency of rapid eye movement (REM) sleep is notably reduced in narcolepsy patients, and the existence of ‘sleep onset REM periods’ (that is, REM sleep directly preceded by an awake period) is one of the diagnostic criteria for narcolepsy. Nocturnal sleep is often disturbed by sleep fragmentation combined with the occurrence of hypnagogic hallucinations, vivid dreaming and sleep paralysis, which usually occur when patients fall asleep. Narcolepsy patients often suffer from a condition called ‘cataplexy’, which is characterized by a sudden weakening of muscle tone, ranging from jaw dropping and speech slurring to complete bilateral collapse of the postural muscles. These attacks are triggered by emotional stimuli. Consciousness is preserved during cataplexy. Narcolepsy with cataplexy is sometimes referred as ‘narcolepsy–cataplexy’. Symptoms of narcolepsy–cataplexy can be divided into two pathological phenomena. One is an inability to maintain a long awake period, characterized by abrupt transition to non-REM (NREM) sleep (dysregulation of NREM sleep onset). This phenomenon manifests clinically as excessive daytime sleepiness or a sleep attack. Recent studies suggested that it 42 largely results from lack of OX2R activation . Psychostimulant drugs, such as modafinil, methyl phenidate, amphetamine and caffeine are used to treat these symptoms. The other key phenomenon is the pathological intrusion of REM sleep into wakefulness (dysregulation of REM sleep onset); it is during these periods that the patient might experience cataplexy, hypnagogic hallucinations and sleep paralysis. Available therapy for this symptom consists of tricyclic antidepressants such as imipramine and selective serotonin reuptake inhibitors101. Lack of signalling from both receptors is critically associated with this symptom.

In addition, orexin neurons project directly to the However, one should not disregard the importance

laterodorsal tegmental/pedunculopontine tegmental of OX1R in the regulation of sleep and wakefulness. The nucleus (LDT/PPT) cholinergic neurons, some popu- behavioural and electroencephalographic phenotype lations of which are implicated in the in the mainte- of prepro-orexin-knockout mice and double-receptor 36 41 nance of wakefulness . Other populations of LDT/PPT knockout (OX1R- and OX2R-null) mice , which seem neurons are implicated in the regulation of REM sleep to have similar phenotypes41, is more severe than that of 36 42 and muscle atonia during REM sleep . Direct injection OX2R-knockout mice , supporting an important but less (FIG. 2; of orexin A into the LDT of cats results in an increased significant contribution of OX1R Supplementary 37 awake time and a decreased REM sleep time . In addi- information S1 (table)). OX2R-knockout and pre- tion, several reports have shown that orexin induces pro-orexin-knockout mice are similarly affected by long-lasting excitation of cholinergic neurons in the behaviourally abnormal attacks of NREM sleep (sleep 38 42 LDT . However, more recent work has shown that attacks) , but OX2R-knockout mice show a lower degree orexin A inhibits cholinergic neurons in the PPT through of disrupted wakefulness compared with double-receptor γ 42 (FIG. 2) activation of GABA ( -aminobutyric acid)-containing knockouts . In particular, OX2R-knockout mice local interneurons and GABA-containing neurons in the are only mildly affected by cataplexy and direct transi- substantia nigra pars reticulata39. These results indicate tions to REM sleep from awake states42, whereas prepro- that hypothalamic orexin neurons affect the activity of orexin-knockout mice and double-receptor knockout LDT/PPT cholinergic neurons both directly and indi- mice are severely affected7,41,42 (FIG. 2). These observations

rectly to regulate arousal and REM sleep. However, fur- indicate that OX1R has additional effects on sleep–wake ther studies are needed to understand the precise effects regulation, especially the regulation of REM sleep. –/– of orexins on LDT/PPT cholinergic neurons. So, despite the lack of an overt OX1R phenotype, loss of signalling through both receptor pathways seems to Orexin receptors in sleep and wakefulness. Some reports be necessary for the emergence of a complete narcoleptic have indicated that the effect of orexin on wakefulness is phenotype, indicating that both receptors are involved in largely mediated by activation of the histaminergic sys- the regulation of sleep and wakefulness.

tem through the OX2R. In rats, ICV injection of orexin during the light period potently increases the duration Orexinergic activity in the sleep–wake cycle. In trans- of wakefulness, and this effect is markedly attenuated genic mice with constitutive activation of orexinergic 32 by the histamine H1 receptor antagonist, pyrilamine . tone (CAG/orexin mice), orexin is expressed in a dif- This pharmacological effect of orexin A on awake time fuse, ectopic pattern in the brain in an unregulated 40 43 is almost completely absent in H1-deficient mice . fashion . The mice exhibited abnormal sleep and wake-

Furthermore, whereas OX1R-knockout mice show only fulness patterns, including fragmented NREM sleep 41 a mild fragmentation of sleep and awake states , OX2R- in the light period and incomplete REM sleep atonia knockout mice exhibit a narcoleptic phenotype42 (FIG. 2; with abnormal myoclonic activity during REM sleep

Supplementary information S1 (table)). OX2R is abun- (T.S., unpublished observations). These results indicate dantly expressed in the histaminergic TMN, whereas that orexin neurons need to be switched off to maintain

OX1R is highly expressed in the noradrenergic LC, indi- consolidated NREM sleep and the muscle atonia that cating that the TMN might be an important effector site accompanies REM sleep, but have to be activated during of orexin for the regulation of sleep and wakefulness. awake periods.

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Orthodromic and Consistent with this idea, Fos expression (a marker of waking, decreased discharge during quiet waking, and antidromic activation neuronal activity) in orexin neurons in rats is increased virtually ceased firing during both REM and NREM Neural stimulation in the same during the dark, active period in which the awake state sleep. Orexin neurons increased firing before the end of and the opposite direction of is dominant44. Moreover, orexin levels in cerebrospinal REM sleep and thereby heralded by several seconds the the physiological nerve fluid peak during the dark period and decrease during return of the awake state. Although the numbers of cells conductance, respectively. the light period in which the sleep state is dominant45. examined are too small to provide a complete picture Recent in vivo recording studies revealed further changes of orexin neurons’ activity across the sleep–wake cycle, of orexin neuronal activity across the sleep–wake cycle. these seminal studies provide the strongest evidence Mileykovskiy et al. recorded orthodromic and antidromic that these cells are activated during wakefulness, and activity of VTA and LC to identify orexin neurons in inhibited during sleep. unanaesthetized, unrestrained rats46. They found that orexin neurons were relatively inactive in quiet waking Other functions of orexins but were transiently activated during sensory stimula- Feeding behaviour and energy homeostasis. Narcolepsy tion. Furthermore, the neurons were silent during NREM patients have a decreased caloric intake but an increased sleep and tonic periods of REM sleep, with occasional body mass index, indicating that the abnormality that burst discharges during phasic REM sleep. gives rise to narcolepsy has links to a reduced energy Lee et al. also recorded from orexin neurons, identi- expenditure or a low metabolic rate48,49. Orexin neurons fied using a combination of neurobiotin labelling and have been shown to have a role in the regulation of energy immunohistochemistry, in the LHA of head-fixed rats47. homeostasis. For example, orexin neuron-ablated mice They found that the orexin neurons fired during active exhibit hypophagia and late-onset obesity, although the extent to which this is the case critically depends on the genetic backgrounds of the mice9,50. Supporting the physiological relevance of orexin in the control of feeding, ICV administration of an anti-

orexin antibody or an OX1R-selective antagonist reduced food intake3,51, and prepro-orexin-knockout mice and transgenic mice lacking orexin neurons ate less than 9,41 control wild-type mice . Moreover, an OX1R-selective antagonist reduced food intake and ameliorated obesity in leptin-deficient ob/ob mice2. Consistent with the dense projection of orexin neurons to the arcuate nucleus24,26,52, several studies have suggested that the increased food intake following orexin A administration is at least partly mediated by the activation of neuropeptide Y neurons in the arcuate nucleus52,53. Other events involved in orexin-induced feeding behaviour include the inhibition of pro- LHA/PH opiomelanocortin neurons in the arcuate nucleus, which

LC OX1R are thought to have an important role in leptin-mediated inhibition of food intake53. Recent reports also showed TMN OX2R that infusions of orexin A into the shell of the nucleus Raphe accumbens (NAc) increase feeding behaviour54. In addi- LDT tion, infusions of the GABA receptor agonist muscimol OX1R and OX2R A VTA into the NAc shell strongly induced food intake and PPT simultaneously increased Fos expression specifically in 55 Figure 1 | Schematic drawing showing main projections of orexin neurons. orexin neurons . These findings indicate that reciprocal This figure summarizes predicted orexinergic projections in the human brain. Please interactions between the orexin and limbic systems have a role in the regulation of feeding. note that distributions of orexin fibres and receptors (OX1R, OX2R) are predicted from the results of studies on rodent brains, as it is on rodents that most histological studies Orexin-mediated maintenance of consolidated wake- on the orexin system have been carried out. Circles show regions with strong receptor fulness might also be important in feeding behaviour, expression and dense orexinergic projections. Orexin neurons originating in the lateral because maintenance of arousal during food search- hypothalamic area (LHA) and posterior hypothalamus (PH) regulate sleep and ing and intake is essential for an animal’s survival. For wakefulness and the maintenance of arousal by sending excitatory projections to the example, when faced with reduced food availability, entire CNS, excluding the cerebellum, with particularly dense projections to animals adapt with a longer awake period, which dis- monoaminergic and cholinergic nuclei in the brain stem and hypothalamic regions24–32,38, rupts the normal circadian pattern of activity11,56,57. This including the locus coeruleus (LC, containing noradrenaline), tuberomammillary nucleus (TMN, containing histamine), raphe nuclei (Raphe, containing serotonin) and response is absent in transgenic mice with ablated orexin 11 laterodorsal/pedunclopontine tegmental nuclei (LDT/PPT), containing acetylcholine). neurons , indicating that these neurons are crucial for Orexin neurons also have links with the reward system through the ventral tegmental evoking adaptive maintenance of arousal during fast- area (VTA, containing dopamine) and with the hypothalamic nuclei that stimulate ing. In other words, if energy stores are low, the activ- feeding behaviour. Anatomical image adapted, with permission, from REF. 108 © (1996) ity of orexin neurons could be modulated to maintain Appleton & Lange. wakefulness, allowing more time to search for food.

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R WT DMH and orexin neurons has a key role, as a central S W food-entrainable oscillator, in the feeding-mediated regulation of circadian behaviours. R S OX1R KO Orexin and the autonomic nervous system. Several stud- W ies have clearly shown that orexins have a role in regulat- R ing autonomic function. It has been demonstrated that OX2R KO S ICV orexin injections increase blood pressure and heart

Sleep–wake state Sleep–wake W rate, and that these effects are abolished by the admin- OX1R KO/OX2R DKO 60 R istration of drugs that block α- or β-adrenoceptors . S Moreover, blood pressure in orexin-deficient mice is W 10–15 mmHg lower than in wild-type littermates61,62. 20:00 22:00 24:00 02:00 04:00 06:00 These results indicate that orexins physiologically Time stimulate sympathetic outflow and provide a pos- Figure 2 | Sleep state abnormalities in orexin receptor-knockout mice. Typical sible explanation for the increased body mass index representative 12 hour dark period (20:00–08:00) hypnograms for wild-type (WT), OX R- 1 observed in conditions of low orexin: orexin deficiency knockout (OX R KO), OX R-knockout (OX R KO) and double-receptor knockout mice 1 2 2 might decrease sympathetic tone, which could result (OX R/OX R DKO), all on a C57B/6J background, are shown. The different levels above 1 2 in decreased energy expenditure. As might be expected in the baseline indicate states of sleep and wakefulness (R, rapid eye movement (REM) sleep; S, non-REM (NREM) sleep; W, awake) of the mouse at the time. Episodes of direct a system geared for weight gain, orexins do not slow the transition from wakefulness to REM sleep are shown by red arrowheads. Note the greater metabolic rate. Instead, they increase both food intake 63 awake/NREM sleep episode fragmentation and reduced duration of wakefulness in the and metabolic rate . Because animals must be vigilant and active when they seek and eat food, an orexin-induced hypnograms of OX2R-knockout and double-receptor knockout mice compared with wild-type and OX1R-knockout mice. Episodes of direct transition from wakefulness to increase in sympathetic nerve activity might be important

REM sleep were not observed in OX1R-knockout mice, and barely in OX2R-knockout for feeding behaviour. mice, whereas they were frequently observed in double-receptor knockout mice. As discussed later, the orexin-mediated increase in Hypnograms were obtained by simultaneous electroencephalography (EEG) and sympathetic tone could also be involved in the mechanisms electromyography (EMG) recording for 4 weeks (N = 18–40). by which the limbic system modulates the sympathetic outflow responding to emotional stimuli61,62. The activity of orexin neurons also contributes to the promotion and maintenance of food anticipatory Orexin and the reward system. Anatomically, orexin activity (FAA)12,13. Daily restricted feeding produces an neurons are well-positioned to alter reward function- anticipatory locomotor activity rhythm and entrains a ing. Orexin neurons project to reward-associated brain molecular oscillator that is independent of the central regions, including the NAc and VTA, and orexin directly 29 clock, which is located in the suprachiasmatic nucleus activates VTA dopaminergic neurons through OX1R (SCN). Restricted feeding shifts the peak of Fos (FIG. 3). This indicates a possible role for orexins in expression in orexin neurons from night to the period reward function and motivation, consistent with previ- during which feeding was restricted, indicating that ous studies implicating orexins in feeding. In fact, the orexin neurons are activated when animals need to be activation of orexin neurons was shown to be strongly awake and seek food12,13. The establishment of FAA linked to preferences for cues associated with drug and was severely impaired in orexin/ataxin 3 transgenic food rewards16. Dopaminergic neurons that originate mice, in which orexin neurons are ablated12,13. The in the VTA and project into the forebrain, particularly transgenic mice also showed reduced expression of the NAc, have classically been identified as the ‘reward mRNA for murine period 1 (mPer1), brain and muscle pathway’. Drugs of abuse stimulate this pathway. ICV arnt-like protein 1 (Bmal1) and neuronal PAS domain or local VTA infusions of orexin have been shown to protein 2 (Npas2), a transcription factor thought to be reinstate drug-seeking or food-seeking behaviour in Food anticipatory activity 10,16 (FAA). Behavioural activation involved in regulating the food-entrainable oscillator. rodents . Conversely, the subcutaneous morphine induced by restricted access These observations indicate that orexin neurons con- (μ-opioid receptor agonist)-induced place preference to food; a manifestation of the vey an efferent signal from a putative food-entrainable and hyperlocomotion observed in wild-type mice were food-entrained oscillator. oscillator or oscillators to increase wakefulness and abolished in mice that lacked the prepro-orexin gene17, mPer1 locomotor activity. Recently, a part of the dorsomedial and injections of an OX1R antagonist into the VTA block The PER1 gene is a core clock hypothalamic nucleus (DMH) was shown to have a the development of morphine-conditioned place pref- factor that has an essential role robust oscillation of mPer gene expression only under erence17. These observations indicate the strong func- in generating circadian restricted feeding58. The oscillation persisted for tional interaction between orexinergic pathways and the rhythms. mPer1 is the mouse at least 2 days, even when mice were given no food dopaminergic system. counterpart of the human PER1 gene. during the expected feeding period after the establish- Recent work has provided interesting insights into ment of the FAA. It has also been demonstrated that the cellular and molecular mechanisms underlying Bmal1 lesions in the DMH in rats blocked food entrainment these effects by showing that orexin A input to the Bmal1 (brain and muscle arnt- of wakefulness, locomotor activity and core body VTA potentiates NMDAR (N-methyl-d-aspartate like protein 1) is a putative 59 clock gene which encodes a temperature . Taken in conjunction with recent find- receptor)-mediated neurotransmission through a 14,15 basic helix-loop-helix-PAS ings that DMH neurons project to orexin neurons , protein kinase C-dependent insertion of NMDARs in transcription factor. these results indicate that the connection between the VTA dopamine neuron synapses in slice preparations64.

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Furthermore, in vivo administration of an OX1R the periventricular hypothalamic nucleus (PVN) and antagonist blocks locomotor sensitization to cocaine the central nucleus of the amygdala (CeA)68. The link and occludes cocaine-induced potentiation of excita- between the CRF system and orexin neurons is recipro- tory currents in VTA dopamine neurons64. These cal69, and might maintain wakefulness during stressful results suggest an important role for orexin signal- events. ling in the VTA in the neural plasticity associated with reward, and indicate that orexins also contribute Mechanisms regulating orexin neuron activity to cocaine-induced psychomotor sensitization and To establish the physiological relevance of orexins, reward-seeking. These findings highlight the key understanding the system that regulates the activity of role of orexin in the mechanisms of reward and drug orexin neurons is key. This section discusses several addiction. Consistently, prepro-orexin-knockout mice factors and systems involved in this regulation. are less susceptible than wild-type animals to develop- ing morphine dependence, as measured by physical Neurotransmitters and neuromodulators. Electro- withdrawal responses65. Interestingly, some narcolepsy physiological studies have identified several neuro- patients with daytime sleepiness who were treated with transmitters and neuromodulators that activate or amphetamine-like stimulants and/or sodium oxybate inhibit the activity of orexin neurons (TABLE 1). By (γ-hydroxybutyrate, also known as GHB) for a long recording from hypothalamic slices of transgenic mice time rarely developed drug abuse66. that express green fluorescent protein (GFP) selectively in orexin neurons, it was shown that agonists Orexin and the stress response. Orexin influences of ionotropic glutamate receptors (AMPA (α-amino- neuroendocrine function, and thereby affects arousal 3-hydroxy-5-methyl-4-isoxazole propionic acid) and and the stress response. For example, ICV injection of NMDA) excite orexin neurons, whereas glutamate orexin stimulates the hypothalamic–pituitary–adrenal antagonists (AP5 (D(–)-2-amino-5-phosphonovaleric (HPA) axis67 and decreases prolactin secretion23. ICV acid), CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) administration of orexin A strongly activates cortico- or NBQX (6-nitro-7-sulphamoylbenzo(f)quinoxaline- tropin releasing factor (CRF)-expressing neurons in 2,3-dione)) reduce their activity70,71. These results indicate that orexin neurons are tonically activated by glutamatergic neurons. SCN LDT/PPT In addition, several other neurotransmitters Acetylcholine have been shown to influence the activity of orexin Amygdala VTA neurons. Importantly, both noradrenaline and serotonin and BST DMH Dopamine (5-hydroxytryptamine, 5-HT) hyperpolarize and LC inhibit GFP-expressing orexin neurons through the Noradrenaline activation of G-protein-regulated inwardly rectifying K+ (GIRK or Kir3) channels by α -adrenoceptors and LHA DR 2 VLPO 70–72 Orexin Serotonin 5-HT receptors, respectively . The cholinergic GABA 1A PH agonist carbachol activates 27% and inhibits 6% of TMN orexin neurons14,71, whereas histamine seems to have Histamine no effect on orexin neurons. These observations SNr OX1R indicate that serotonin and noradrenaline neurons Glucose Ghrelin Leptin GABA OX2R might send inhibitory feedback projections to orexin Excitatory Pheripheral neurons. Furthermore, although orexin neurons metabolic cues Inhibitory do not express functional dopamine receptors, dopamine can inhibit orexin neurons by acting on Interactions of orexin neurons with other brain regions implicated in Figure 3 | α -adrenoceptors71,72. sleep and wakefulness. Orexin neurons in the lateral hypothalamic area (LHA) and 2 posterior hypothalamus (PH) are anatomically well placed to provide a link between the A recent study showed that a short 2 hour period limbic system, systems involved in energy homeostasis and monoaminergic and of total sleep deprivation changed the action of cholinergic neurons in the brain stem. Solid arrows show excitatory projections, and noradrenaline on orexin neurons from excitation to broken lines inhibitory ones. Wake-active regions, sleep-active regions and REM-active inhibition. This mechanism might contribute to the regions are shown by red, blue and green boxes, respectively. Orexin neurons promote growing sleepiness that accompanies sleep depriva- wakefulness through the monoaminergic nuclei that are wake-active. Stimulation of tion73, although this phenomenon was not observed dopaminergic centres by orexins can modulate reward systems (purple). Peripheral in mice72. metabolic signals such as leptin, ghrelin and glucose influence orexin neuronal activity to Using transgenic mice in which orexin neurons spe- coordinate arousal and energy homeostasis. The nucleus suprachiasmaticus (SCN), the cifically express a genetically encoded intracellular cal- central body clock, sends signals to orexin neurons via the dorsomedial hypothalamus cium indicator (Yellow Cameleon, Yc2.1), we screened (DMH). The DMH acts as a food-entrainable ossilator, and influences orexin neuronal activity. Input from the limbic system (amygdala and bed nucleus of the stria terminalis for factors that affect the activity of orexin neurons and (BST)) might regulate the activity of orexin neurons upon emotional stimuli to evoke found that a sulphated octapeptide form of cholecysto- emotional arousal or fear-related responses61,62. VLPO, ventrolateral preoptic area; DR, kinin (CCK-8S), as well as neurotensin, oxytocin and dorsal raphe; GABA, γ-aminobutyric acid; LC, locus coeruleus; LDT, laterodorsal tegmental vasopressin activate orexin neurons74, whereas GABA, nucleus; PPT, pedunculopontine tegmental nucleus; SNr, substantia nigra pars reticulata; glucose, serotonin, noradrenaline and leptin inhibit TMN, tuberomammillary nucleus. them (TABLE 1). Finally, a recent paper described how

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Table 1 | Factors that influence the activity of orexin neurons tion and an increase in action potential frequency11. By contrast, bath-application of leptin was found to Factor Receptor involved References robustly inhibit most of the orexin neurons examined, Excitatory causing hyper polarization and a decrease in firing Glutamate AMPAR, NMDAR, mGluRs 11,70 rate11. Notably, insulin exerted no direct effect on orexin 11 Ghrelin GHSR 71 neurons . The above findings show that peripheral humoral Cholecystokinin CCK-A 74 factors that are related to energy metabolism influ- Neurotensin ND 74 ence the activity of orexin neurons. In addition, orexin Vasopressin V1a 74 expression in wild-type and ob/ob mice is negatively Oxytocin V1a 74 correlated with changes in blood glucose, leptin and food intake11. This is consistent with the idea that orexin Glucagon-like peptide 1 ND 105 neurons act as sensors of the nutritional status of the CRF CRFR1 69 body1,11,41. mACh (effect in 27% of orexin neurons) M3 14 Orexin neurons have been shown to be stimulated by hypoglycemia at least partly via the nucleus of the soli- ATP P2X 106 tary tract (NTS)78,79, indicating that peripheral metabolic Inhibitory cues might also influence the activity of orexin neurons Glucose Unknown 11 indirectly through vagal afferents and the NTS. GABA GABA , GABA 11,70,89 What is the physiological relevance of the regulation A B of orexin neurons by factors that act as indicators of an Serotonin 5-HT 71,91 1A animal’s nutritional state? When faced with a negative Noradrenaline α 71,72 2 energy balance due to reduced food availability, mam- Dopamine α 71 mals respond behaviourally with phases of increased 2 wakefulness and alertness that presumably enhance the Neuropeptide Y Y 107 1 ability to find food56,57. Orexin neuron-ablated mice fail Leptin OB-R 11 to exhibit this fasting-induced arousal11, indicating that mACh (effect in 6% of orexin neurons) ND 14,71 orexin neurons are necessary for evoking adaptive behavioural arousal during fasting. So, nutritional depletion- Adenosine A1 75 α α α induced metabolic cues activate orexin neurons, and 2, 2 adrenergic receptor; 5-HT1A, 5-hydroxytryptamine receptor 1A; AMPAR, -amino-3- hydroxy-5-methyl-4-isoxazole propionic acid receptor; CCK-A, cholecystokinin receptor A; orexin increases arousal, thereby reinforcing food- CRFR1, corticotropin-releasing factor receptor 1; GABA, γ-aminobutyric acid; GHSR, growth- seeking/feeding pathways. These mechanisms might hormone secretagogue receptor; mACh, muscarinic acetylcholine; mGluRs, metabotropic glutamate receptors; ND, not determined; NMDAR, N-methyl-d-aspartate receptor; also be important in the maintenance of prolonged OB-R, leptin receptor; P2X, purinocepter. wakefulness during the active period; in the regulation of energy homeostasis that helps to ensure survival; and, adenosine inhibits orexin neurons via the adenosine interestingly, might hinder attempts to treat obesity by 75 A1 receptor . This mechanism might relate to the food restriction. This might also explain why orexin sleep-promoting effect of adenosine75. receptor antagonists decrease food intake3.

Humoral factors. Metabolic signals also contribute to Neuronal input. Until recently, very little was known the regulation of orexin neuron activity: decreasing the about the synaptic input into hypothalamic orexin extracellular glucose concentration produced depolari- neurons, and this seems to be largely because of the zation and increased the frequency of action potentials challenges associated with the cells being dispersed in orexin neurons, whereas increasing it induced marked mediolaterally within the LHA. In mice with a geneti- hyperpolarization and cessation of action potentials in cally encoded retrograde tracer, the neuronal popula- the same neurons11,76. Importantly, this mechanism is tions that send afferent innervations to orexin neurons sufficiently sensitive to encode variations in glucose were mapped14. Labelled cells were identified in multiple levels reflecting those occurring physiologically between brain regions, including the basal forebrain cholinergic normal meals11,76. neurons, GABA-containing neurons in the ventrolateral A recent study demonstrated that the inhibition of preoptic nucleus (VLPO), neurons in the posterior/ orexin neurons by glucose is mediated by tandem-pore dorsomedial hypothalamus and serotonergic neurons in + 77 K (K2P) channels . Glucose seemed to act at an extra- the raphe nuclei. Labelled neurons were also found cellular site on orexin neurons, as it inhibited orexin in regions associated with emotion including the amyg- neurons only when applied extracellularly77. An undeter- dala, infralimbic cortex, NAc shell, lateral septum and mined intracellular messenger that was not ATP, Ca2+ or the bed nucleus of the stria terminalis (BST). glucose itself transmitted this information to the chan- By combining antero- and retrograde tracers, a study nels77. These results reveal an unexpected energy-sensing mapped afferents of orexin neurons in rats and found pathway in neurons that regulates states of wakefulness that hypothalamic orexin neurons received abundant and energy balance77. projections from the lateral septum, preoptic area, BST Ghrelin applied in a superfused solution activated and posterior hypothalamus15. In addition, it was found 60% of dispersed orexin neurons, with depolariza- that hypothalamic regions preferentially innervated

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83 Box 3 | The extended amygdala and emotion knockout mice . Similarly, air-jet stress-induced eleva- tions of blood pressure and heart rate were attenuated in A key component of the neural circuitry of emotion in animals is the amygdala and its conscious orexin/ataxin 3 transgenic mice, in which orexin related regions (extended amygdala), which consist of a well-defined subcortical nuclear neurons are ablated62. group that in vertebrates is a centre for emotional responses, including fear102–104. The Limbic inputs to orexin neurons include the CRF amygdala receives many kinds of sensory information directly from the periphery, or via 69 the thalamus and cortex. For example, sensory stimuli that predict an aversive outcome neurons that originate in the amygdala . They activate 69 will change neural transmission in the amygdala to produce the somatic, autonomic and orexin neurons through the CRF-R1 receptor . The endocrine signs of fear, as well as increased attention and arousal to that stimulus. reciprocal link between the CRF system and orexin Consolidation of emotional memory involves lateral and basolateral parts of the neurons might maintain wakefulness during stressful amygdala, where the association between incoming sensory stimuli leads to events. Indeed, activation of orexin neurons by foot shock potentiation of synaptic transmission. These parts project to the central amygdala stress is severely impaired in CRF-R1-deficient mice, (CeA), which in turn sends efferents to the hypothalamus and brain stem that trigger indicating that such activation is mediated by CRF69. the expression of emotions including arousal, autonomic and endocrine responses. The The neural input from the limbic system to orexin regions closely associated with the amygdala are also important for fear learning, and neurons might be implicated in the pathophysiology of include the bed nucleus of the stria terminalis (BST). Orexin neurons have been shown cataplexy, because strong, generally positive emotional to receive innervations from these regions14,15, indicating that these cells have a role in the emergence of emotional responses, such as increased arousal and sympathetic stimuli are known to trigger cataplexy in narcolepsy– outflow during fearful events. cataplexy patients. A local injection of orexin into the PPT strongly inhibited REM-related atonia in cats39. Cholinergic neurons in the LDT/PPT are implicated in the medial and perifornical parts of the orexin neuron REM-related atonia84, and the same pathway is implicated field, but most projections from the brainstem targeted in cataplexy. Therefore, emotional stimuli might increase the lateral part of the field, indicating a functional orexin release in the PPT to prevent muscle atonia dichotomy of orexin neurons. in wild type animals. Projections to orexin neurons However, tracing studies might not show all input to from the limbic system might also be important for orexin neurons. Monoaminergic and peptidergic systems maintaining orexin neuron activity during the active sometimes use ‘volume transmission’, which includes period by conveying various emotional stimuli to orexin short- (but larger than the synaptic cleft, that is, roughly neurons (FIG. 3). 20 nm) and long-distance diffusion of signals through the The limbic input to orexin neurons might also be extracellular and cerebrospinal fluid80. Therefore, although involved in the regulation of feeding behaviour, because tracing studies showed that projections of noradrenergic some of the affective content of the perception of food and dopaminergic neurons to orexin neurons are sparse, it is thought to be processed in the amygdala and limbic is important not to disregard the effects that these factors system85, and this information might be passed on to might have on orexin neurons (TABLE 1). orexin neurons. Food perception often evokes cataplexy Some studies have revealed that orexin neurons in narcoleptic dogs86, indicating that orexin signalling show apposition from peptidergic fibres, including is physiologically activated on perception of food, and neuro peptide Y, pro-opiomelanocortin and galanin-like that this system is necessary to evoke proper feeding peptide fibres81,82. Again, these data must be interpreted behaviour. carefully; such chemically defined ‘apposition’ does not necessarily mean that the nerve terminals functionally Input from preoptic areas. The preoptic area, especially synapse onto orexin neurons. the VLPO, seems to have a crucial role in NREM sleep Finally, not only synaptic receptors contribute to initiation and maintenance. Neurons in the VLPO fire regulating neuronal activity. Extrasynaptic receptors at a rapid rate during sleep, with attenuation of firing can sense ambient ligands such as CCK, leptin and during wakefulness. GABA and galanin are the primary glucose, which can act as a neuromodulators on orexin inhibitory neurotransmitters of the VLPO87, which sends neurons11,74 (TABLE 1). out multiple inhibitory projections to the LC, TMN and DR87,88. Interactions with other neuronal systems Orexin neurons, which are innervated by GABA- Orexin neurons interact with multiple neuronal systems containing cells in the VLPO14,15, are strongly inhibited (FIG. 3) . These interactions provide a key to understanding by both the GABAA agonist muscimol and the GABAB the physiological roles of orexin neurons. receptor agonist bacrofen11,89, indicating that the VLPO might be a source of GABA-containing inhibitory projec- Input from the limbic system. Arousal resulting from tions to orexin neurons. This pathway might be important emotional stimuli or fear-related responses increases for turning off orexin neurons during sleep (FIGS 3,4). sympathetic outflow. Orexin neurons receive input from the limbic system14,15,69, indicating a role for this Input from the SCN. Although direct input to system in the regulation of orexin neuron activity (BOX 3). orexin neurons from the SCN seems to be sparse, orexin Indeed, the importance of this connection is readily appar- neurons receive abundant innervations from the BST, ent in the defence, or ‘fight or flight’, response: mice tested supraventricular zone and DMH14,15, all of which receive in a resident–intruder paradigm show cardiovascular and input from the SCN. This indicates that orexin neurons locomotor responses to the emotional stress evoked by this might receive circadian influences indirectly from the test, but these responses are diminished in prepro-orexin- SCN via these regions90.

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a Awake state Figure 4 | Mechanisms by which the orexin system Limbic system stabilizes sleep and wakefulness. The figures represent Orexin functional interactions between orexin neurons, SCN neurons monoaminergic wake-active centres and the ventrolateral Energy balance preoptic area (VLPO) sleep-active centre during various states of sleep and wakefulness. Solid arrows show Monoaminergic excitatory input, and broken lines inhibitory input. The neurons VLPO thickness of arrows and lines represents the relative strength of excitatory and inhibitory input, respectively. Circle sizes represent relative activities of each region. a | Awake state. Orexin neurons send excitatory influences Thalamus Cortex to monoaminergic neurons, which send inhibitory b Sleep state Orexin feedback projections to orexin neurons. This system might neurons maintain the activity of monoaminergic neurons. A slight decrease in input to the monoaminergic neurons results in decreased inhibitory influence to orexin neurons. Orexin Monoaminergic neurons, therefore, are disinhibited and increase excitatory VLPO neurons influence to monoaminergic cells to maintain their activity. These monoaminergic cells send excitatory projections to the thalamus and cerebral cortex, and send inhibitory Thalamus projections to the VLPO sleep centre. These mechanisms Cortex maintain wakefulness states. b | Sleep state. VLPO sleep- c Narcolepsy Orexin active neurons are activated and send inhibitory neurons projections to monoaminergic neurons and orexin neurons to maintain sleep. c | Narcolepsy. If orexin neurons are Monoaminergic removed, monoaminergic neurons and VLPO neurons set VLPO neurons up a mutually inhibitory circuit, which can cause unwanted and abrupt transitions between the states. Activity in one of the competing sides shuts down inhibitory inputs from the other side, and therefore disinhibits its own action. So, Inhibitory Thalamus Excitatory Cortex when either side begins to overcome the other, the switch abruptly turns into the alternative state.

Mechanisms that stabilize sleep and wakefulness VLPO sleep-active neurons and vice versa97. In such a Orexin neurons are an important component of the circuit, when activity on either side begins to overcome neural circuits that regulate sleep and wakefulness. How, the other, the system will flip into one of two possible then, do these neurons stabilize sleep and wakefulness extremes, because when a small perturbation gives one through these circuits? side a sudden ‘advantage’, it will turn off the alternative side As previously discussed, a feedback loop between abruptly96. A circuit of this type is thought to underlie orexin neurons and monoaminergic neurons in the the pathology of narcolepsy (FIG. 4). brain stem including the LC and DR71,72,91 might maintain the activity of monoaminergic neurons. Decreases Sleep modulation using the orexin system in monoaminergic neuron activity will decrease the Because narcolepsy–cataplexy is a disorder of sleep–wake inhibitory influence on orexin neurons. This disinhi- cycle organization resulting from the absence of orexin, bition of orexin neurons then increases the excitatory it is perhaps logical to consider that replacement therapy influence on monoaminergic cells, thereby increasing using orexin receptor agonists could provide an effective their activity (FIG. 4). treatment for this disorder. Indications that this might be Sleep-active, GABA-containing neurons in the VLPO successful came from a study which showed that chronic send descending projections that terminate within overproduction of orexin peptides from an ectopically wake-promoting populations in the TMN, LC and DR88. expressed transgene prevented the development of a During sleep, VLPO sleep-active neurons are thought to narcolepsy syndrome in orexin neuron-ablated (orexin/ be activated by sleep substances such as adenosine92–94, ataxin 3 transgenic) mice43. Acute ICV administration of and send inhibitory influences to monoaminergic orexin A also maintained wakefulness, suppressed sleep neurons in the brain stem and hypothalamus. As discussed and inhibited cataplectic attacks in orexin/ataxin 3 mice43. above, the sleep-active neurons also send inhibitory In fact, ICV administration of orexin A had stronger projections to hypothalamic orexin neurons15,95. arousal effects in orexin/ataxin 3 transgenic mice than These circuits are important for the regulation in wild-type controls43. The greater effectiveness might of wakefulness. If orexin neurons are removed from not have resulted from increased expression of orexin this system, as is the case in narcolepsy–cataplexy, the receptors43. Rather, in the orexin/ataxin 3 mice, mono- sleep-active neurons in the VLPO and monoaminergic aminergic neurons in the brain stem became more sensi- neurons exhibit a ‘flip-flop’ property, owing to the mutual tive to various stimuli (T.S., unpublished observations). inhibition between these two neuronal elements96: This mechanism might explain why narcoleptics cannot monoaminergic neurons send inhibitory influences to maintain long, consolidated NREM sleep periods.

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The effectiveness of ICV-administered orexin in ani- systems, and send excitatory signals to monoaminergic mals with a narcoleptic phenotype indicates that orexin and cholinergic nuclei in the brain stem. Interactions receptor agonists would be of potential value for treating with these systems indicate physiological functions of narcolepsy. However, as mentioned above, chronic over- orexin neurons, as discussed in this review. These func- expression of orexin in an unregulated fashion results tions should be further explored in future studies using in disruption of NREM sleep, and therefore it will be conditional knockouts of receptors that are expressed beneficial for therapeutically relevant orexin agonists to in orexin neurons. A more precise understanding of the have a short half-life (< 12 hours). Conversely, orexin mechanisms that regulate orexin neurons might provide antagonists might be effective as a sleep-inducing drug. further insights into how the systems that regulate emotion, energy homeostasis and reward interact with the Conclusion and perspectives mechanism that regulates sleep and wakefulness. The symptoms and the cellular and systems-level bases of narcolepsy–cataplexy unequivocally show that orexins Note added in proof and orexin receptors are important regulators of sleep and A recent paper showed that a new, potent, orally avail- wakefulness and of arousal maintenance by regulating able dual orexin receptor antagonist, ACT-078573, which

monoaminergic and cholinergic nuclei in the brain. blocks both OX1R and OX2R, effectively promotes sleep Orexin neurons receive afferents from multiple neuronal in rats, dogs and humans109.

1. Sakurai, T. et al. Orexins and orexin receptors: a 14. Sakurai, T. et al. Input of orexin/hypocretin neurons 31. Brown, R. E., Sergeeva, O. A., Eriksson, K. S. & family of hypothalamic neuropeptides and G protein- revealed by a genetically encoded tracer in mice. Haas, H. L. Convergent excitation of dorsal raphe coupled receptors that regulate feeding behavior. Neuron 46, 297–308 (2005). serotonin neurons by multiple arousal systems (orexin/ Cell 92, 573–585 (1998). 15. Yoshida, K., McCormack, S., Espana, R. A., Crocker, A. hypocretin, histamine and noradrenaline). J. Neurosci. Describes the discovery of orexins and their two & Scammell, T. E. Afferents to the orexin neurons of the 22, 8850–8859 (2002). target receptors, the determination of their exact rat brain. J. Comp. Neurol. 494, 845–861 (2006). 32. Yamanaka, A. et al. Orexins activate histaminergic structures and the evidence that the peptides References 14–15 map the neuronal input to orexin neurons via the orexin 2 receptor. Biochem. Biophys. stimulate short-term food intake. neurons. Res. Commun. 290, 1237–1245 (2002). 2. Haynes, A. C. et al. Anorectic, thermogenic and anti- 16. Harris, G. C., Wimmer, M. & Aston-Jones, G. A role for 33. Vanni-Mercier, G., Sakai, K. & Jouvet, M. Neurons obesity activity of a selective orexin-1 receptor lateral hypothalamic orexin neurons in reward seeking. specifiques de l’eveil dans l’hypothalamus posterieur antagonist in ob/ob mice. Regul. Pept. 104, 153–159 Nature 437, 556–559 (2005). du chat. C. R. Acad. Sci., III 298, 195–200 (1984). (2002). 17. Narita, M. et al. Direct involvement of orexinergic 34. Eggermann, E. et al. Orexins/hypocretins excite basal 3. Haynes, A. C. et al. A selective orexin-1 receptor systems in the activation of the mesolimbic dopamine forebrain cholinergic neurones. Neuroscience 108, antagonist reduces food consumption in male and pathway and related behaviors induced by morphine. 177–181 (2001). female rats. Regul. Pept. 96, 45–51 (2000). J. Neurosci. 26, 398–405 (2006). 35. Alam, M. N., Szymusiak, R., Gong, H., King, J. & 4. Edwards, C. M. et al. The effect of the orexins on food References 16–17 demonstrate roles for orexin McGinty, D. Adenosinergic modulation of rat basal intake: comparison with neuropeptide Y, melanin- neurons and ventral tegmental orexin receptors in forebrain neurons during sleep and waking: neuronal concentrating hormone and galanin. J. Endocrinol. reward-based learning and memory. recording with microdialysis. J. Physiol. 521, 160, R7–R12 (1999). 18. Nishino, S., Ripley, B., Overeem, S., Lammers, G. J. & 679–690 (1999). 5. Peyron, C. et al. A mutation in a case of early onset Mignot, E. Hypocretin (orexin) deficiency in human 36. Shouse, M. N. & Siegel, J. M. Pontine regulation of narcolepsy and a generalized absence of hypocretin narcolepsy. Lancet 355, 39–40 (2000). REM sleep components in cats: integrity of the peptides in human narcoleptic brains. Nature Med. 9, 19. Mignot, E. et al. The role of cerebrospinal fluid pedunculopontine tegmentum (PPT) is important for 991–997 (2000). hypocretin measurement in the diagnosis of narcolepsy phasic events but unnecessary for atonia during REM 6. Thannickal, T. C. et al. Reduced number of hypocretin and other hypersomnias. Arch. Neurol. 59, 1553–1562 sleep. Brain Res. 571, 50–63 (1992). neurons in human narcolepsy. Neuron 27, 469–474 (2002). 37. Xi, M., Morales, F. R. & Chase, M. H. Effects on sleep (2000). 20. American Academy of Sleep Medicine, Diagnostic and wakefulness of the injection of hypocretin-1 References 5–6 provide evidence that, in most Classification Steering Committee. The International (orexin-A) into the laterodorsal tegmental nucleus of cases, human narcolepsy–cataplexy is probably a Classification of Sleep Disorders: Diagnostic and the cat. Brain Res. 901, 259–264 (2001). neurodegenerative disease of orexin neurons. Coding Manual. (American Academy of Sleep 38. Takahashi, K., Koyama, Y., Kayama, Y. & Yamamoto, M. 7. Chemelli, R. M. et al. Narcolepsy in orexin knockout Medicine, 2005). Effects of orexin on the laterodorsal tegmental neurones. mice: molecular genetics of sleep regulation. Cell 98, 21. Crocker, A. et al. Concomitant loss of dynorphin, NARP, Psychiatry Clin. Neurosci. 56, 335–336 (2002). 437–451 (1999). and orexin in narcolepsy. Neurology 65, 1184–1188 39. Takakusaki, K. et al. Orexinergic projections to the 8. Lin, L. et al. The sleep disorder canine narcolepsy is (2005). midbrain mediate alternation of emotional behavioral caused by a mutation in the hypocretin (orexin) 22. Kadotani, H., Faraco, J. & Mignot, E. Genetic studies states from locomotion to cataplexy. J. Physiol. 568, receptor 2 gene. Cell 98, 365–376 (1999). in the sleep disorder narcolepsy. Genome Res. 8, 1003–1020 (2005). References 7–8 provide evidence that a deficiency 427–434 (1998). 40. Huang, Z. L. et al. Arousal effect of orexin A depends of orexin or the orexin receptor 2 results in a 23. Hagan, J. J. et al. Orexin A activates locus coeruleus on activation of the histaminergic system. Proc. Natl. narcoleptic phenotype in mice and dogs. cell firing and increases arousal in the rat. Proc. Natl Acad. Sci. USA 98, 9965–9970 (2001). 9. Hara, J. et al. Genetic ablation of orexin neurons in Acad. Sci. USA 96, 10911–10916 (1999). 41. Willie, J. T., Chemelli, R. M., Sinton, C. M. & mice results in narcolepsy, hypophagia, and obesity. 24. Date, Y. et al. Orexins, orexigenic hypothalamic Yanagisawa, M. To eat or to sleep? Orexin in the Neuron 30, 345–354 (2001). peptides, interact with autonomic, neuroendocrine and regulation of feeding and wakefulness. Annu. Rev. 10. Boutrel, B. et al. Role for hypocretin in mediating neuroregulatory systems. Proc. Natl Acad. Sci. USA 96, Neurosci. 24, 429–458 (2001). stress-induced reinstatement of cocaine-seeking 748–753 (1999). 42. Willie, J. T. et al. Distinct narcolepsy syndromes in behavior. Proc. Natl Acad. Sci. USA 102, 25. Nambu, T. et al. Distribution of orexin neurons in the Orexin receptor-2 and Orexin null mice: molecular 19168–19173 (2005). adult rat brain. Brain Res. 827, 243–260 (1999). genetic dissection of Non-REM and REM sleep 11. Yamanaka, A. et al. Hypothalamic orexin neurons 26. Peyron, C. et al. Neurons containing hypocretin (orexin) regulatory processes. Neuron 38, 715–730 (2003). regulate arousal according to energy balance in mice. project to multiple neuronal systems. J. Neurosci. 18, Demonstrates distinct roles of each orexin receptor Neuron 38, 701–713 (2003). 9996–10015 (1998). subtype in the regulation of sleep and wakefulness. Shows that orexin neurons are directly regulated by 27. Marcus, J. N. et al. Differential expression of orexin 43. Mieda, M. et al. Orexin peptides prevent cataplexy glucose, leptin and ghrelin, and are necessary for receptors 1 and 2 in the rat brain. J. Comp. Neurol. and improve wakefulness in an orexin neuron-ablated augmenting arousal during fasting. 435, 6–25 (2001). model of narcolepsy in mice. Proc. Natl Acad. Sci. USA 12. Akiyama, M. et al. Reduced food anticipatory Comprehensive report on the distribution of orexin 101, 4649–4654 (2004). activity in genetically orexin (hypocretin) neuron- receptor mRNAs in the rat brain. Demonstrates rescue of the narcolepsy–cataplexy ablated mice. Eur. J. Neurosci. 20, 3054–3062 28. Horvath, T. L. et al. Hypocretin (orexin) activation and phenotype of orexin neuron-ablated mice by (2004). synaptic innervation of the locus coeruleus genetic and pharmacological means, providing 13. Mieda, M. et al. Orexin neurons function in an noradrenergic system. J. Comp. Neurol. 415, evidence that receptor agonists might be of efferent pathway of a food-entrainable circadian 145–159 (1999). potential value for treating human narcolepsy. oscillator in eliciting food-anticipatory activity and 29. Nakamura, T. et al. Orexin-induced hyperlocomotion 44. Estabrooke, I. V. et al. Fos expression in orexin wakefulness. J. Neurosci. 24, 10493–10501 and stereotypy are mediated by the dopaminergic neurons varies with behavioral state. J. Neurosci. 21, (2004). system. Brain Res. 873, 181–187 (2000). 1656–1662 (2001). References 12–13 show that orexin neurons 30. Liu, R. J., van den Pol, A. N. & Aghajanian, G. K. 45. Yoshida, Y. et al. Fluctuation of extracellular convey an efferent signal from a putative food- Hypocretins (orexins) regulate serotonin neurons in the hypocretin-1 (orexin A) levels in the rat in relation to entrainable oscillator to increase wakefulness and dorsal raphe nucleus by excitatory direct and inhibitory the light–dark cycle and sleep-wake activities. locomotor activity. indirect actions. J. Neurosci. 22, 9453–9464 (2002). Eur. J. Neurosci. 14, 1075–1081 (2001).

REVIEWS

46. Mileykovskiy, B. Y., Kiyashchenko, L. I. & Siegel, J. M. 69. Winsky-Sommerer, R. et al. Interaction between the 93. Arrigoni, E., Chamberlin, N. L., Saper, C. B. & McCarley, Behavioral correlates of activity in identified hypocretin/ corticotropin-releasing factor system and hypocretins R. W. Adenosine inhibits basal forebrain cholinergic and orexin neurons. Neuron 46, 787–798 (2005). (orexins): a novel circuit mediating stress response. noncholinergic neurons in vitro. Neuroscience 140, 47. Lee, M. G., Hassani, O. K. & Jones, B. E. Discharge of J. Neurosci. 24, 11439–11448 (2004). 403–413 (2006).

identified orexin/hypocretin neurons across the sleep– 70. Li, Y., Gao, X. B., Sakurai, T. & van den Pol, A. N. 94. Huang, Z. L. et al. Adenosine A2A, but not A1, receptors waking cycle. J. Neurosci. 25, 6716–6720 (2005). Hypocretin/Orexin excites hypocretin neurons via a mediate the arousal effect of caffeine. Nature Neurosci. References 46–47 report in vivo activity of orexin local glutamate neuron-A potential mechanism for 8, 858–859 (2005). neurons during states of sleep and wakefulness. orchestrating the hypothalamic arousal system. 95. Sakurai, T. Roles of orexins and orexin receptors in 48. Schuld, A., Hebebrand, J., Geller, F. & Pollmacher, T. Neuron 36, 1169–1181 (2002). central regulation of feeding behavior and energy Increased body-mass index in patients with narcolepsy. 71. Yamanaka, A., Muraki, Y., Tsujino, N., Goto, K. & homeostasis. CNS Neurol. Disord. Drug Targets 5, Lancet 355, 1274–1275 (2000). Sakurai, T. Regulation of orexin neurons by the 313–325 (2006). 49. Lammers, G. J. et al. Spontaneous food choice in monoaminergic and cholinergic systems. Biochem. 96. Saper, C. B., Chou, T. C. & Scammell, T. E. The sleep narcolepsy. Sleep 19, 75–76 (1996). Biophys. Res. Commun. 303, 120–129 (2003). switch: hypothalamic control of sleep and wakefulness. 50. Hara, J., Yanagisawa, M. & Sakurai, T. Difference in 72. Yamanaka, A. et al. Orexin neurons are directly and Trends Neurosci. 24, 726–731 (2001). obesity phenotype between orexin-knockout mice and indirectly regulated by catecholamines in a complex 97. Gallopin, T. et al. Identification of sleep-promoting orexin neuron-deficient mice with same genetic manner. J. Neurophysiol. 96, 284–298 (2006). neurons in vitro. Nature 404, 992–995 (2000). background and environmental conditions. Neurosci. 73. Grivel, J. et al. The wake-promoting hypocretin/orexin 98. de Lecea, L. et al. The hypocretins: hypothalamus- Lett. 380, 239–242 (2005). neurons change their response to noradrenaline after specific peptides with neuroexcitatory activity. 51. Yamada, H., Okumura, T., Motomura, W., Kobayashi, Y. sleep deprivation. J. Neurosci. 25, 4127–4130 Proc. Natl Acad. Sci. USA 95, 322–327 (1998). & Kohgo, Y. Inhibition of food intake by central (2005). Describes the independent discovery of the injection of anti-orexin antibody in fasted rats. 74. Tsujino, N. et al. Cholecystokinin activates orexin/ transcript that encodes orexins, the prediction that Biochem. Biophys. Res. Commun. 267, 527–531 hypocretin neurons through the cholecystokinin A two peptides are encoded by the transcript, and the (2000). receptor. J. Neurosci. 25, 7459–7469 (2005). detection of the peptides in dense-core vesicles at 52. Yamanaka, A. et al. Orexin-induced food intake involves 75. Liu, Z. W. & Gao, X. B. Adenosine inhibits activity of synapses. neuropeptide Y pathway. Brain Res. 24, 404–409 hypocretin/orexin neurons by the A1 receptor in the 99. Zhu, Y. et al. Orexin receptor type-1 couples exclusively (2000). lateral hypothalamus: a possible sleep-promoting to pertussis toxin-insensitive G-proteins, while orexin 53. Muroya, S. et al. Orexins (hypocretins) directly interact effect. J. Neurophysiol. 97, 837–848 (2007). receptor type-2 couples to both pertussis toxin- with neuropeptide Y, POMC and glucose-responsive 76. Burdakov, D., Gerasimenko, O. & Verkhratsky, A. sensitive and-insensitive G-proteins. J. Pharmacol Sci. neurons to regulate Ca2+ signaling in a reciprocal Physiological changes in glucose differentially modulate 92, 259–266 (2003). manner to leptin: orexigenic neuronal pathways in the the excitability of hypothalamic melanin-concentrating 100. Mignot, E. Genetic and familial aspects of narcolepsy. mediobasal hypothalamus. Eur. J. Neurosci. 19, hormone and orexin neurons in situ.J. Neurosci. 25, Neurology 50, S16–S22 (1998). 1524–1534 (2004). 2429–2433 (2005). 101. Zeitzer, J. M., Nishino, S. & Mignot, E. The 54. Thorpe, A. J. & Kotz, C. M. Orexin A in the nucleus 77. Burdakov, D. et al. Tandem-pore K+ channels mediate neurobiology of hypocretins (orexins), narcolepsy and accumbens stimulates feeding and locomotor activity. inhibition of orexin neurons by glucose. Neuron 50, related therapeutic interventions. Trends Pharmacol. Brain Res. 1050, 156–162 (2005). 711–722 (2006). Sci. 27, 368–374 (2006). 55. Baldo, B. A. et al. Activation of a subpopulation of Shows a novel mechanism by which glucose 102. Davis, M. & Whalen, P. The amygdala: vigilance and orexin/hypocretin-containing hypothalamic neurons by regulates the activity of orexin neurons. emotion. Mol. Psychiat. 6, 13–34 (2001).

GABAA receptor-mediated inhibition of the nucleus 78. Cai, X. J. et al. Hypoglycemia activates orexin neurons 103. LeDoux, J. The emotional brain, fear, and the accumbens shell, but not by exposure to a novel and selectively increases hypothalamic orexin-B levels: amygdala. Cell. Mol. Neurobiol. 23, 727–738 (2003). environment. Eur. J. Neurosci. 19, 376–386 (2004). responses inhibited by feeding and possibly mediated 104. Phelps, E. A. & LeDoux, J. E. Contributions of the 56. Challet, E., Pevet, P. & Malan, A. Effect of prolonged by the nucleus of the solitary tract. Diabetes 50, amygdala to emotion processing: from animal models fasting and subsequent refeeding on free-running 105–112 (2001). to human behavior. Neuron 48, 175–187 (2005). rhythms of temperature and locomotor activity in rats. 79. Williams, G. et al. The hypothalamus and the control of 105. Acuna-Goycolea, C. & van den Pol, A. N. Glucagon-like Behav. Brain. Res. 84, 275–284 (1997). energy homeostasis: different circuits, different peptide 1 excites hypocretin/orexin neurons by direct 57. Itoh, T. et al. Effects of 24-hr fasting on purposes. Physiol. Behav. 74, 683–701 (2001). and indirect mechanisms: implications for viscera- methamphetamine- and apomorphine-induced 80. Agnati, L. F., Zoli, M., Stromberg, I. & Fuxe, K. mediated arousal. J. Neurosci. 24, 8141–8152 locomotor activities, and on monoamine metabolism in Intercellular communication in the brain: wiring versus (2004). mouse corpus striatum and nucleus accumbens. volume transmission. Neuroscience 69, 711–726 106. Wollmann, G., Acuna-Goycolea, C. & van den Pol, A. N. Pharmacol. Biochem. Behav. 35, 391–396 (1990). (1995). Direct excitation of hypocretin/orexin cells by 58. Mieda, M., Williams, S. C., Richardson, J. A., Tanaka, K. 81. Elias, C. F. et al. Chemically defined projections linking extracellular ATP at P2X receptors. J. Neurophysiol. & Yanagisawa, M. The dorsomedial hypothalamic the mediobasal hypothalamus and the lateral hypo- 94, 2195–2206 (2005). nucleus as a putative food-entrainable circadian thalamic area. J. Comp. Neurol. 402, 442–459 (1998). 107. Fu, L. Y., Acuna-Goycolea, C. & van den Pol, A. N. pacemaker. Proc. Natl Acad. Sci. USA 103, 82. Takenoya, F. et al. Neuronal interactions between Neuropeptide Y inhibits hypocretin/orexin neurons by 12150–12155 (2006). galanin-like-peptide- and orexin- or melanin- multiple presynaptic and postsynaptic mechanisms: 59. Gooley, J. J., Schomer, A. & Saper, C. B. The concentrating hormone-containing neurons. Regul. tonic depression of the hypothalamic arousal system. dorsomedial hypothalamic nucleus is critical for the Pept. 126, 79–83 (2005). J. Neurosci. 24, 8741–8751 (2004). expression of food-entrainable circadian rhythms. 83. Kayaba, Y. et al. Attenuated defense response and low 108. Martin, J. H. Neuroanatomy: Text and Atlas 2nd edn Nature Neurosci. 9, 398–407 (2006). basal blood pressure in orexin knockout mice. (Appleton & Lange, Stamford, Connecticut, 1996). 60. Shirasaka, T., Nakazato, M., Matsukura, S., Takasaki, M. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, 109. Brisbare-Roch, C. et al. Promotion of sleep by targeting & Kannan, H. Sympathetic and cardiovascular actions R581–R593 (2003). the orexin system in rats, dogs and humans. Nature of orexins in conscious rats. Am. J. Physiol. 277, 84. Shiromani, P. J., Armstrong, D. M., Berkowitz, A., Jeste, Med. 13, 150–155 (2007). R1780–R1785 (1999). D. V. & Gillin, J. C. Distribution of choline acetyl- 61. Kayaba, Y. et al. Attenuated defense response and low transferase immunoreactive somata in the feline Acknowledgements basal blood pressure in orexin knockout mice. brainstem: implications for REM sleep generation. This study was supported in part by a grant-in-aid for scientific Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, Sleep 11, 1–16 (1988). research from The 21st Century COE Program from the R581–R593 (2003). 85. Berthoud, H. R. Mind versus metabolism in the control Ministry of Education, Culture, Sports, Science and Technology 62. Zhang, W., Sakurai, T., Fukuda, Y. & Kuwaki, T. Orexin of food intake and energy balance. Physiol. Behav. 81, (MEXT) of Japan; the University of Tsukuba Project Research; neuron-mediated skeletal muscle vasodilation and shift 781–793 (2004). the ERATO Yanagisawa Orphan Receptor Project from the of baroreflex during defense response in mice. 86. Reid, M. S. et al. Neuropharmacological Japan Science and Technology Corporation; and anorexia ner- Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, characterization of basal forebrain cholinergic vosa research from the Japanese Ministry of Health, Labour R1654–R1663 (2006). stimulated cataplexy in narcoleptic canines. and Welfare. 63. Lubkin, M. & Stricker-Krongrad, A. Independent feeding Exp. Neurol. 151, 89–104 (1998). and metabolic actions of orexins in mice. Biochem. 87. Sherin, J. E., Elmquist, J. K., Torrealba, F. & Saper, C. B. Competing interests statement Biophys. Res. Commun. 253, 241–245 (1998). Innervation of histaminergic tuberomammillary neurons The author declares no competing financial interests. 64. Borgland, S. L., Taha, S. A., Sarti, F., Fields, H. L. & by GABAergic and galaninergic neurons in the Bonci, A. Orexin A in the VTA is critical for the induction ventrolateral preoptic nucleus of the rat. J. Neurosci. of synaptic plasticity and behavioral sensitization to 18, 4705–4721 (1998). cocaine. Neuron 49, 589–601 (2006). 88. Lu, J. et al. Selective activation of the extended DATABASES 65. Georgescu, D. et al. Involvement of the lateral ventrolateral preoptic nucleus during rapid eye The following terms in this article are linked online to: hypothalamic peptide orexin in morphine dependence movement sleep. J. Neurosci. 22, 4568–4576 Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. and withdrawal. J. Neurosci. 23, 3106–3111 (2003). (2002). fcgi?db=gene 66. Guilleminault, C., Carskadon, M. & Dement, W. C. 89. Xie, X. et al. GABA receptor-mediated modulation of B OX1R | OX2R On the treatment of rapid eye movement narcolepsy. hypocretin/orexin neurones in mouse hypothalamus. OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. Arch. Neurol. 30, 90–93 (1974). J. Physiol. 574, 399–414 (2006). fcgi?db=OMIM 67. Kuru, M. et al. Centrally administered orexin/ 90. Leak, R. K. & Moore, R. Y. Topographic organization of narcolepsy hypocretin activates HPA axis in rats. Neuroreport 11, suprachiasmatic nucleus projection neurons. J. Comp. 1977–1980 (2000). Neurol. 433, 312–334 (2001). FURTHER INFORMATION 68. Sakamoto, F., Yamada, S. & Ueta, Y. Centrally 91. Muraki, Y. et al. Serotonergic regulation of the orexin/ Sakurai’s laboratory: administered orexin-A activates corticotropin-releasing hypocretin neurons through the 5-HT1A receptor. http://www.md.tsukuba.ac.jp/basic-med/pharmacology/ factor-containing neurons in the hypothalamic J. Neurosci. 24, 7159–7166 (2004). paraventricular nucleus and central amygdaloid nucleus 92. Morairty, S., Rainnie, D., McCarley, R. & Greene, R. SUPPLEMENTARY INFORMATION of rats: possible involvement of central orexins on Disinhibition of ventrolateral preoptic area sleep-active See online article: S1 (table) stress-activated central CRF neurons. Regul. Pept. 118, neurons by adenosine:a new mechanism for sleep Access to this links box is available online. 183–191 (2004). promotion. Neuroscience 123, 451–457 (2004).