Review articles

Hypocretin/, and

Marcel Hungs and Emmanuel Mignot*

Summary Introduction The discovery that hypocretins are involved in narco- The function of sleep and why this behavior was selected lepsy, a disorder associated with excessive daytime by natural evolution is one of the remaining mysteries in sleepiness, and unusually rapid transitions to rapid-eye-movement sleep, opens a new field of investi- physiology today. Sleep is a vital behavior that consumes one gation in the area of sleep control physiology. Hypo- third of any given life; animals die if totally deprived of cretin-1 and -2 (also called orexin-A and -B) are newly sleep.(1,2) Electrophysiological studies have long shown that discovered processed from a common sleep is a heterogeneous state, most classically separated into precursor, preprohypocretin. Hypocretin-containing cells are located exclusively in the lateral , rapid eye movement (REM) and non-REM sleep. Non-REM with widespread projections to the entire neuroaxis. Two sleep can also be subdivided into light non-REM sleep (stage known receptors, Hcrtr1 and Hcrtr2, have been reported. I and II) and slow wave sleep (SWS, stage III and IV). The functional significance of the hypocretin system is Independent of this organization by sleep stages, the rapidly emerging in both animals and . Hypo- propensity to sleep or to stay awake is regulated independently cretin abnormalities cause narcolepsy in dogs, human and mice. The role of the hypocretin system in normal by homeostatic (-dependent) and circadian (clock- sleep regulation is more uncertain. We believe hypocretin dependent) processes. Finally, sleep is associated with a host cells drive cholinergic and monoaminergic activity across of peripheral changes that have a physiological impact. These the . Input from the suprachiasmatic nucleus include established sleep-state-specific or circadian-con- to hypocretin-containing may explain the occur- trolled changes in endocrine release, convulsive thresholds, rence of clock-dependent alertness. Other functions are suggested by pharmacological and regulation of breathing, cardiovascular control, gastrointesti- experiments. These include regulation of food intake, nal physiology and muscle tone. neuroendocrine function, autonomic nervous system There has been spectacular progress in the last decade activity and energy balance. BioEssays 23:397±408, in our understanding of how the generates circadian 2001. ß 2001 John Wiley & Sons, Inc. rhythms.(3) In mammals, circadian rhythmicity is generated by the suprachiasmatic nucleus (SCN), a discrete hypothalamic region. At the genetic level, key circadian factors and Stanford Center for Narcolepsy, Department of Psychiatry & (3) Behavioral Sciences, Stanford University Medical Center. generating circadian rhythmicity have now been isolated. In Funding agencies: NIH (Grant Nos: NS23724, NS33797, and contrast, there has been little progress in understanding the HL59601 to E.M.), and Deutsche Forschungsgemeinschaft regulation of sleep. The most accepted neuroanatomical (Grant No: HU 827/2-1 to M.H.). model involves reciprocal inhibitory interactions between *Correspondence to: Emmanuel Mignot, Stanford Center for cholinergic [e.g. laterodorsal tegmental (LDT) area and Narcolepsy, Stanford University Medical Center, Department of Psychiatry & Behavioral Sciences, 1201 Welch Road, P-114, Palo pedunculopontine (PPT) nuclei] and monoaminergic [e.g. Alto, CA 94305-5485. E-mail: [email protected] (LC), raphe and tuberomamillary (TMN) nuclei] cells groups of the brainstem.(4,5) In this model, ascending cholinergic and monoaminergic projections mediate EEG changes by modulating thalamocortical Abbreviations: CSF, cerebrospinal fluid; DB, diagonal band of Brocca; loops.(4,5) Monoaminergic tone is high during EEG, electroencephalogram; Hcrtr1, hypocretin--1; Hcrtr2, (generating EEG desynchronization) and decreases across hypocretin-receptor-2; HLA, ; icv, intra- cerebroventricular; LC, locus coeruleus; LDT, laterodorsal tegmental the sleep cycle (promoting EEG synchronization). Decreased nucleus; MCH, Major Histocompatibility Complex; NPY, Y; monoaminergic tone desinhibits cholinergic neurons during OX1R, orexin-receptor-1; OX2R, orexin-receptor-2; PAG, periaque- the later part of the sleep cycle, resulting in REM sleep and ductal gray; PPT, pedunculopontine tegmental nucleus; PVN, paraven- associated EEG desynchronization. Little progress has tricular nucleus; REM, rapid eye movement; SCN, suprachiasmatic been achieved in elucidating the genetics of sleep control. nucleus; TMN, tuberomammillary nucleus; VLPO, ventrolateral pre- optic area; VMN, ventromedial nucleus; VTA/SN, ventral tegmental The two major limitations in the area have been the lack of area and substantia nigra pars compacta single mutant models with sleep abnormalities and the difficulties of defining ``sleep'' in non-mammalian species

BioEssays 23:397±408, ß 2001 John Wiley & Sons, Inc. BioEssays 23.5 397 Review articles

(e.g. Drosophila) that may be more amendable to genetic Hypocretins/ and their discovery studies. Hypocretins/orexins were discovered by two independent This situation is now changing rapidly. Investigators are groups only two years ago.(12,13) In 1998, de Lecea and now trying to equate sleep and locomotion activity control in colleagues selectively enriched a cDNA library for hypotha- lower organisms.(6) Our own work has focussed on the only lamic-specific transcripts and identified a clone selectively known single gene mammalian mutant in the field, canine expressed in the called H35.(12) Their narcolepsy. Narcolepsy is a unique model as it is one of the few goal was to find novel genes expressed primarily in the disorders with a well-defined and dramatic sleep phenotype. A hypothalamus, a structure well known to be a major regulatory positional cloning project identified two exon skipping Hcrtr2 center of autonomic and endocrine . The H35 causing canine narcolepsy in Dobermans and clone was sequenced and shown to contain the precursor of Labradors.(7) This discovery was followed by the observation two related neuropeptides, hypocretin-1 and -2 (Fig. 1). of narcolepsy-like symptoms in preprohypocretin knockout Neuroanatomical characterizations as well as electron micro- mice(8) and by the more recent demonstration of generalized scopy and electrophysiological studies indicated that the hypocretin deficiency in human narcolepsy.(9±11) In this hypocretins were genuine . Of note, high review, we will briefly summarize the emerging knowledge concentrations of hypocretin-2 were found to be generally regarding this newly described system and excitatory in hypothalamic cultures. Based on the neurotrans- argue that hypocretins may complement monoaminergic and mitters' hypothalamic origin and their similarity to the gut cholinergic systems as major contributors for the generation of (contested by others) de Lecea et al. named the sleep cycle. these hypocretin-1 and hypocretin-2.

Figure 1. Preprohypocretin sequences in selected mammalian species(12,13,38,101) and Xenopus.(102) Variable residues are marked in light blue letters. Hypocretin-1 and hypocretin-2 domains are indicated in gray. Note that hypocretin-1 is identical across all mammalian species studied while hypocretin-2 differs by two residues. The prohormone contains a sequence where a dominant narcolepsy (16L ! R) has been described.(10) Note dibasic (RR) prohormone convertase site between the two hypocretin peptide domains and at the C-terminal end of hypocretin-2. The residue located in the C-terminal end of both peptides is converted to an amide indispensable to biological activity.(13,103) The C-terminal regions of hypocretin-1 and hypocretin-2 have high homology areas that are the most critical for biological activity based on recent studies.(103) Hypocretin-1 is also transaminated in the N-terminal region to create a cyclic pyroglutamyl residue and has two disulfide bridges.

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The same neuropeptide system was discovered concomi- The discovery that hypocretin-containing cell bodies were tantly by Sakurai et al. using a different approach.(13) The focus exclusively located in the lateral hypothalamus, an area where of their investigation was on discovering novel ligands for lesions are known to dramatically reduce food intake,(14) orphan G--coupled receptors. Two peptides (Orexin-A, initially suggested a role for these peptides in the regulation of Orexin-B) and two receptors (OX1R and OX2R) were food intake.(12,13) Further neuroanatomical studies using biochemically characterized in this study. Importantly, the immunocytochemical and neurochemical techniques indi- exact location of proteolytic cleavage sites for the precursor cated that, while cells bodies were restricted to the hypo- and relevant post-translational modifications (dilsufide bonds, , projections were widespread, suggesting more N-pyroglutamyl cyclization and C-terminal amidation) were complex functions (Fig. 2).(16±23) Intrahypothalamic projec- also reported (see Fig. 1 for description). Intrahypothalamic tions were dense and included (ARC), localization of the peptides was demonstrated. The observa- paraventricular nucleus (PVN) and ventromedial nucleus tion that preprohypocretin transcripts were upregulated by (VMN), structures known to integrate feeding. Limbic system fasting while central administration of these peptides stimu- and associated structures (, , lated feeding led these authors to suggest a primary role in septum, basal area, nucleus of the stria feeding regulation.(13) Hcrtr1 has a significantly higher affinity terminalis), specific area of the thalamus (paraventricular for hypocretin-1 while Hcrtr2 binds hypocretin-1 and -2 with and reticular thalamus) and the brainstem also received dense similar affinity. Hypocretin-1/orexin-A, hypocretin-2/orexin-B, fibers (Fig. 2). These contrasted with a lack of projections to Hcrtr1/OX1R and Hcrtr2/OX2R refer to the same biological motor nuclei such as caudate nucleus, putamen, globus entities. pallidus and sensory thalamic nuclei. Dense projections to all monoaminergic cell groups (locus coeruleus, raphe nucleus, Neuroanatomical studies of the substantia nigra, and tuberomamillary hypocretin/orexin system nucleus) were also noted. Studies measuring hypocretin The hypothalamus is one of the most complex brain regions at content in various nuclei indicated a similar profile, with higher both the functional and neuroanatomical level.(14) This brain hypocretin-2 versus hypocretin-1 levels noted in all brain structure has long been recognized as a vegetative center regions examined.(10,22±26) regulating body homeostasis. This regulation involves the Hypocretin receptor localization has been examined using integration of functions as diverse as energy balance and body in situ hybridization by three groups.(27±29) Both receptor fluid homeostasis, cardiovascular and autonomic regulation, subtypes were generally expressed differentially in the central circadian rhythms and sleep, and reproduction.(14,15) The nervous system. Intrahypothalamically, Hcrtr1 was preferen- intrahypothalamic circuitry underlying these functions is tially expressed in the VMN, while Hcrtr2 was mostly rapidly emerging, thanks to functional studies and improved expressed in the PVN and arcuate nucleus. Both hypocretin neurochemical characterization of hypothalamic subgroups. receptors were thus expressed in hypothalamic regions known

Figure 2. Hypocretin-containing neurons are exclusively located in the posterior hypothalamus, in a discrete region including and surrounding the perifornical nucleus. In contrast, hypocretin-positive fibers and terminals are widely distributed in the brain. Selected projections with corresponding hypocretin receptor subtypes are indicated. Intrahypothalamic projections are not displayed in details. Projections with predominantly Hcrtr2 and Hcrtr1 receptors are denoted in red and blue, respectively. Regions containing both receptors and/or without any information are displayed in black.

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to be important for feeding regulation. Another important The low penetrance of HLA-DQB1Ã0602 in producing finding was the preferential localization of Hcrtr1 in the clinically significant symptoms suggests that factors other adrenergic locus coeruleus while Hcrtr2 was densely located than HLA genes are involved in triggering narcolepsy. Family on tuberomamillary histaminergic cells and within the diagonal and twin studies have shown that both environmental factors band and septum, possibly on cholinergic cells.(29) and non-HLA genes are important.(34) The familial risk of a first degree relative of a narcoleptic patient developing Human and canine narcolepsy the disorder is approximately 20±40 times higher than in the Narcolepsy has fascinated clinicians and researcher alike for general population, a result that cannot be explained by the more than 100 years.(30) A canine model was first described in sharing of HLA alone.(34,36) Finally, familial cases are more the 1970s and has been the subject of intense pharmacolo- frequently HLA-DQB1Ã0602 negative than sporadic cases gical and neurochemical investigations.(31) Narcolepsy is the (e.g. 30% DQB1Ã0602-negative in probands of multiplex only frequent neurological disorder characterized by a primary families), suggesting that these cases might involve highly disorganization of sleep and wakefulness.(32) The disorder penetrant non-HLA genes. These data suggest that human affects approximately 0.05% of the general population in narcolepsy is both genetically complex and etiologically Western Europe and North American countries. In normal heterogeneous.(10,34) subjects, REM sleep occurs in the middle of the night and is associated with dreaming and muscle paralysis. Patients with Genetic alteration of the hypocretin system narcolepsy are chronically sleepy and have an abnormal causes narcolepsy in dogs and mice tendency to enter (REM) sleep prematurely. Nocturnal sleep is The first clue suggesting hypocretin involvement in narcolepsy also frequently disturbed by , and came from studies in a canine model of the disorder.(7) As in vivid dreaming. The pathognomonic symptom of narcolepsy is humans, most cases of canine narcolepsy are sporadic but cataplexy, a sudden onset of muscle atonia trigged by emo- autosomal recessive narcolepsy was also observed in some tions such as laughing and anger.(33) Cataplexy is believed breeds (canarc-1).(30,37) A ten year long positional cloning to represent abnormal REM sleep paralysis in reaction to project led to the isolation of the canine narcolepsy gene in .(33) 1999.(7) Three Hcrtr2 mutations causing narcolepsy in Human narcolepsy is a disabling disorder that necessitates Labradors, Dobermans and Dachshunds have been identified life-long therapy.(4) It usually starts during and is (Fig. 3).(7,38) Two of these mutations are exon skipping treated symptomatically using -like alterations while the third is a single change in the and antidepressants. These drugs have been shown to act N-terminal part of the Hcrtr2 gene.(7,38) Functional analysis of by stimulating and adrenergic transmission, the three mutations indicate complete loss of function.(38) Exon respectively.(31) Disturbed nocturnal sleep may be treated skipping resulted in truncated Hcrtr2 receptors that are not with antidepressants, -like normally translocated on the plasma membrane.(38) The point medications or gamma-hydroxybutyrate. Studies in a well- mutation changes a to a lysine, and produces a established animal model, canine narcolepsy, have shown loss of binding.(38) Hcrtr2-mutated narcoleptic canines that the disorder is associated with cholinergic and mono- have normal hypocretin levels and neurons, suggesting a aminergic abnormalities.(31) These results are generally primary role of Hcrtr2-mediated transmission in generating the consistent with decreased monoaminergic tone, cholinergic narcolepsy phenotype.(39,40) hyperactivity and cholinergic hypersensitivity, a result that These results have now been extended to other animal may explain daytime sleepiness and REM abnormalities in models. The positional cloning of canarc-1 was independently narcolepsy.(31) followed by the observation that preprohypocretin knockout mice have a narcolepsy-like phenotype.(8) These animals Genetic aspects of human narcolepsy exhibit episodes of behavioral arrest similar to cataplexy and Human narcolepsy is primarily a sporadically occurring have disrupted sleep/wake during their activity period.(8) The disorder but familial clustering has been observed since its phenotype is more difficult to study and cataplexy is hard to initial description.(34) The disorder is tightly associated with quantify in mice.(41) Further developments of mouse models HLA-DQB1Ã0602 and HLA-DQA1Ã0102, suggesting the in- revealed that Hcrtr2 knockout mouse also have cataplexy but volvement of the in the pathophysiology of the the phenotype may be less severe than that of prepro- disorder.(34) HLA-DQB1Ã0602 is present in a large portion of hypocretin knockout mice.(42,43) In contrast, Hcrtr1 knockout the general population (e.g. 25% in North European Cauca- mice have almost no obvious behavioral changes, with the sians) who do not have narcolepsy-cataplexy.(34) Interest- exception of a possible milder sleep fragmentation.(42,44) ingly, HLA-DQB1Ã0602-positive healthy controls have shorter This suggests that Hcrtr2 may be critical to the narcolepsy REM sleep latency implicating an increased susceptibility to phenotype, while Hcrtr1 acts as a modifying gene enhancing narcolepsy in subjects carrying this antigen.(35) the severity of the phenotype. Additional studies in Hcrtr1/

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Figure 3. Genomic organization of the canine hypocretin receptor 2 gene (Hcrtr2) and identified narcolepsy-causing mutations. The gene encodes a 7 transmembrane G-protein coupled receptor. Sequence analysis revealed exon skipping mutations in narcoleptic Dobermans (exon 4) and Labradors (exon 6). Deleted exons result in frameshifts and premature stop codons. In Dachshunds, a point mutation in exon 1 results in an aminoacid change from glutamic acid to lysine at the transition point from the N-terminal domain to the first transmembrane region. Functional analysis indicates that all these mutations result in a complete loss of Hcrtr2 function.(38)

Hcrtr2 double knockout mice are currently in progress and arginine change in the hydrophobic core of the signal peptide seem to confirm this hypothesis.(42) of the hypocretin peptide (Fig. 1). Functional analysis indicates Interestingly, hypocretin gene mutations were not ob- impaired hypocretin trafficking and processing for the mutant served in sporadic cases of canine narcolepsy.(38) This result polypeptide, with accumulation in the smooth endoplasmic does not necessarily mean that abnormalities in the hypocretin reticulum and most probably cell death in vivo.(10) system are not involved in these cases. In fact, hypocretin As in canines, however, the absence of hypocretin levels were found to be dramatically decreased or absent in the mutations in most human cases does not indicate a lack of cerebrospinal fluid (CSF) and the brain of sporadic canine involvement of this system in human narcolepsy. A study in narcolepsy cases.(39) The fact that hypocretin-ligand-defi- nine narcoleptic patients showed low CSF hypocretin levels in cient, sporadic cases of canine narcolepsy are generally more seven narcoleptic patients (Fig. 4).(9) More recently, post- severely affected with narcolepsy than Hcrtr2 mutated familial mortem studies in ten narcoleptic subjects indicated unde- cases(37) also agree with the suggestion that Hcrtr1 has an tectable hypocretin-1 and -2 peptides in projection sites such enhancing role. as cortex and pons and a 80±100% reduction of hypocretin- containing cells in the hypothalamus as measured by in situ Hypocretin abnormalities in human narcolepsy hybridization and immunocytochemistry (Fig. 4).(10,11) Six Mutation screening of the hypocretin, Hcrtr1 and Hcrtr2 genes subjects in one study were all HLAÃ0602 positive, had was first carried out in a large number of human patients.(10) definitive cataplexy and included a case with familial occur- These cases included unusual patients with familial occur- rence.(10) Taken together, the CSF and postmortem studies rence and/or HLA-DQB1Ã0602 negativity.(10) Surprisingly, indicate that hypocretin deficiency causes narcolepsy in most only one mutation was identified in an HLA-negative patient human cases with cataplexy.(9±11) with atypical onset at 6 months of age (Fig. 1). This result indicates etiologic and genetic heterogeneity in familial Hypocretin and sleep regulation narcolepsy-cataplexy.(10) The mutation with early onset Pharmacological studies indicate potent wake-promoting and narcolepsy is a T to G transversion causing a to REM sleep reduction effects after central administration of

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Figure 4. Most cases of human narcolepsy are associated with undetectable CSF hypocretin-1 levels (left). Measuring CSF hypocretin levels may become an established diagnostic procedure.(9) In situ hybridization studies revealed a greatly reduced level or absence of hypocretin neurons in the hypothalamus (right).(10) These results indicate that human narcolepsy is caused by an hypocretin deficiency. The loss of hypocretin neurons is generally believed to be secondary to an autoimmune process but this hypothesis has not been formally proven.

hypocretins.(45) Intracerebroventricular (icv) administration of more consistent with a global stimulatory effect of hypocretins hypocretin-1 but not hypocretin-2 induces wakefulness and on monoaminergic tone than preferential adrenergic effects. reduces REM sleep.(45) At high doses, increased locomotion, Interestingly, hypocretin-1 is often more effective than grooming and stereotypes are even observed.(45,46) The lack hypocretin-2 in stimulating monoaminergic activity in vitro, of effects of hypocretin-2 in vivo is generally believed to be due suggesting Hcrtr1 mediation.(45,46) This last result is surprising to biological instability for this peptide. considering the primary importance of Hcrtr2 in expressing the The wake-promoting effects of hypocretins are hypothe- narcolepsy phenotype in genetic studies. Hcrtr2 is mostly sized to be mediated by a stimulation of monoaminergic located in dopaminergic, histaminergic and, possibly, choli- transmission via excitatory hypocretin receptors. In favor of nergic neurons.(27±29) Hcrtr1 is coupled with Gq and known to this hypothesis, administration of hypocretins directly onto the be excitatory. In contrast, Hcrtr2 has been suggested to be locus coeruleus increases wakefulness and reduces REM both excitatory and inhibitory and may coupled to either Gq or sleep in .(47) This, together with the observation that Gi/o.(42) Hypocretins have excitatory effects on laterodorsal hypocretin application strongly stimulates firing rate in the tegmental cholinergic(50) and tuberomamillary histaminergic locus coeruleus (LC, Fig. 2) suggests a primary role for this neurons (Haas, personal communication) in slices, consistent structure in the wake-promoting effects of hypocretins.(18,45) with Hcrtr2-mediated excitation rather than inhibition. Other authors have, however, shown similar in vitro excitatory While a role for hypocretins in narcolepsy is well estab- effects of hypocretins on dopaminergic VTA,(46) serotoniner- lished, very little data to date suggest the involvement of gic raphe magnus(48) and histaminergic TMN cells (Haas, hypocretins in normal sleep or circadian regulation. Based on personal communication). Hypocretin-1 effects on locomotion the results indicated above, we hypothesize that hypocretin are also blocked by and sulpiride, suggesting cells are driving monoaminergic and cholinergic tone during secondary dopaminergic effects.(46) These results are thus the sleep cycle, with maximal and minimal activity during

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wakefulness and REM sleep, respectively (Fig. 5). In this GABAergic inhibition from the VLPO and the PAG, a model, the reversal of cholinergic tone observed during REM mechanism that has been suggested to also regulate might be secondary to changes in monoaminergic activity, as monoaminergic activity across the sleep cycle.(58) A small predicted by the reciprocal interaction model(4,5) and various additional decrease in monoaminergic tone on a depressed pharmacological experiments.(49,51) In favor of this hypoth- baseline could lead to cholinergic desinhibition and acetylcho- esis, electrophysiological studies have indicated the existence line release on supersensitive receptors, thus promoting REM- of wake-on, REM-off cells in the perifornical area(52) but this sleep-like transitions at . Most strikingly, canine finding is not a very specific without associated neurochemi- narcolepsy is associated with decreased (59) and cal characterization. Similarly, double-staining studies have (60) levels in the brain, two systems with known shown increased c-fos production in hypocretin-containing Hcrtr2 localization. neurons with wakefulness but other neurons in the region These models are still speculative. Hypocretin projections display similar, but weaker, fluctuations.(53,54) to non-monoaminergic, non-cholinergic cell groups are also In our model, high hypocretin tone during wakefulness likely to play a role. For example, hypocretin-1 injection in the activates both cholinergic and monoaminergic tone. In non- lateral preoptic hypothalamus increases wakefulness.(61) REM sleep, decreased hypocretin activity reduces monoami- Unknown projections, for example to the limbic system/basal nergic and cholinergic tone. In REM sleep, the decreased forebrain area may be involved in the triggering of cataplexy by monoaminergic tone reaches such a low level that a des- emotions.(31) Other experiments indicate surprisingly small inhibition of cholinergic systems occur. Cholinergic activa- state- and circadian-associated changes in hypocretin stores tion in REM would thus occur in spite of a depressed excitatory and mRNA.(62) does not affect hypocretin hypocretin tone on these cells (Fig. 5). The recent report of mRNA levels in rats.(63) Hypocretin mRNA level fluctuates with direct projections from the suprachiasmatic nucleus, onto circadian time, but the highest levels are observed during hypocretin cells(55) also suggest that hypocretin activity may the inactive period, in opposition with the model proposed be modulated by the biological clock. This projection could above.(62) Similarly, hypocretin-1 peptide content does not provide a biological substrate for the concept of SCN- exhibit circadian variations in most brain regions examined, dependent alertness, a system that has been suggested to with the possible exception of preoptic/anterior hypothalamic be abnormal in hypocretin-deficient narcolepsy.(56) nuclei (maximal levels at 9 am, when rats are mostly sleeping) An alternative hypothesis proposed by Kilduff and Peyron and pons (maximal levels at 1 am, when rats are most postulates increased hypocretin tone during both REM and active).(62) Finally, in our clinical CSF studies, we also did not wakefulness.(57) In this model, inhibitory GABAergic projec- observe any significant change in CSF hypocretin-1 levels tions from the preoptic hypothalamus and periaqueductal gray between early morning and late afternoon (unpublished (PAG) to monoamine- and hypocretin-containing cells plays a results). These results suggest minimal changes in global complementary role in the driving of the sleep cycle.(57) We hypocretin transmission but do not exclude more localized but believe that the REM sleep desinhibition observed in relevant changes in . Of note, hypocretin-2, hypocretin-deficient narcolepsy is more compatible with the the most abundant of the two hypocretin peptides, was hypothesis that hypocretin tone is depressed during REM generally not studied. In vivo dialysis studies of the local sleep. Other investigators have also speculated that hypocre- release of these neurotransmitters in functionally significant tin may inhibit cholinergic tone during REM sleep as the result sites of projections is now critically needed. Additional of direct inhibitory Hcrtr2 effects.(42) Recent studies indicating electrophysiological studies of fully characterized hypocretin excitatory effects of hypocretins on laterodorsal tegmental neurons across the sleep cycle will also in time answer if cholinergic neurons in slices,(50) are also inconsistent with this electrical or metabolic changes in hypocretin neurons are last hypothesis. functionally relevant to sleep regulation. The concept that hypocretin neurotransmission is excita- tory to both cholinergic and monoaminergic systems parallels Hypocretins as orexinogenic agents neurochemical data reported in narcolepsy. In the sleep Hypocretins were initially described as neuromodulator of food disorder, symptoms are best explained by baseline mono- intake. Central administration of hypocretin-1 stimulates food aminergic hyperactivity, hyperactivity of cholinergic systems intake, while data are more inconsistent after hypocretin-2 and cholinergic receptor hypersensitivity.(31,49) Monoaminer- administration.(13,64±75) Recentpharmacologicalstudiesusing gic hypoactivity and hypersensitivity to cholinergic stimulation a Hcrtr1 antagonist, SB-334867-A, suggest a Hcrtr1 mediation are consistent with a reduced cholinergic and monoaminergic for this effect.(76) A reduced food intake was also observed tone across the 24 hour day. The depressed monoaminergic when an anti-orexin polyclonal antibody was applied intracis- tone would also have the tendency to deactivate the ternally in fasted rats.(77) Hypocretin-1 microinjections in the cholinergic system more easily, especially at sleep onset. At perifornical hypothalamus, the lateral hypothalamus, the this time, monoaminergic cell activity may be decreased by dorsomedial nucleus and, possibly, the paraventricular

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nucleus but not in the ventral tegmental area, the arcuatus block partially the hypocretin-induced nucleus, central nucleus of the amygdala, the preoptic area or feeding behavior, suggesting that the orexinogenic effect of the nucleus tractus solitarius induce feeding.(64,71) Hypocretin- hypocretins may be mediated secondarily via NPY activa- 1 administration in the lateral hypothalamus also stimulates tion.(69,74) Several recent experiments, however, suggest c-fos expression in projection areas involved in and more minor pharmacological effects of hypocretins on food body weight regulation.(78) Recent data showed that NPY intake. First, the orexigenic effects of hypocretin-1 are far

Figure 5.

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weaker than those of NPY, and more similar to those of MCH releasing hormone secretion also suggests a more minor and .(66,70) Chronic administration of hypocretins also contribution to the regulation of reproductive functions.(86,87) never leads to .(67,73) Second, these effects are more These effects generally result in increasing global energy pronounced when hypocretins are administered during the consumption. In mice, an icv injection of low doses of rest period.(67,73,75) a result that may partially reflect effects of hypocretin-1 induces an increase in the respiratory quotient wakefulness that would secondarily affect food intake. Third, without increasing activity or feeding, indicating an increased preprohypocretin knockout mice are not lean as would be metabolic rate.(70) expected if a purely orexinogenic signal was removed.(42) In Central administration of hypocretin increases drinking and fact, recent data suggest that preprohypocretin knockout mice preprohypocretin mRNA is upregulated when rats are de- can have normal body weight with reduced food intake.(42) prived of water.(88) The presence of hypocretin-immunoreac- This last result, together with the observation of increased tive fibers in the medulla of the brainstem also indicates obesity inhuman narcolepsy,(79) suggestthe existence ofother cardiovascular regulatory effects.(89,90) Hypocretin injections downstream metabolic effects quantitatively more important to intracisternally or into the ventrolateral medulla increase mean general body metabolism than the control of food intake. arterial in rats suggesting strong influences on sympathetic outflow.(91±93) Interestingly, the hypocretin sys- tem also modulates vagal tone, stimulating gastric acid Food intake and metabolic status regulates secretion after icv administration.(94) Finally, icv injection of hypocretin systems hypocretin stimulates corticosterone and adrenergic secretion In contrast with these results, many experiments suggest that while decreasing plasma and food and energy balance may regulate the hypocretin system levels.(45,95,96) Some of these effects occur after direct under physiological conditions. Preprohypocretin mRNA application onto peripheral organs, for example with hypocre- transcript levels are upregulated 2.4-fold in the lateral tin-1 stimulation of corticosterone release.(97) Subcutaneously hypothalamus during fasting or .(13,80,81) Inhibi- injected hypocretin-1 also increase blood and blood tory receptors have been reported on hypocretin- in rats, an effect partially reconstituted by direct containing cells.(82) Hypocretin neurons are also activated application onto the pancreas.(98) during hypoglycemia.(83,84) Not only hypocretin but also hypocretin receptors expression is modulated by food Perspectives deprivation.(28) Quantitative in situ hybridization after fasting The finding that hypocretins are absent and/or greatly revealed a time-dependent and region-specific Hcrtr1 and diminished in the brain and CSF of narcoleptic patients opens Hcrtr2 mRNA expression.(28) These results are consistent with a new area of investigation for this disabling condition. the idea that hypocretin systems are activated by starvation Measuring hypocretin levels in the CSF may become a while their activity may be reduced by satiety. standard diagnostic procedure. Of note, however, our current investigation indicates that narcolepsy can occur occasionally Hypocretin and other functions with normal or elevated hypocretin levels, independently of Other experiments also suggest a broader role for hypocretins HLA status and family history, suggesting disease hetero- in the homeostatic regulation of energy metabolism, auto- geneity.(9,10) Finding the cause of these other rarer cases may nomic function, hormonal balance and the regulation of body in time further our understanding of the molecular pathway fluids.(42,85) A modulation effect on - implicated in narcolepsy.

Figure 5. Hypothetical model illustrating the influence of hypocretins on the regulation of sleep. The top panel displays hypothesized changes in monoaminergic, cholinergic and hypocretinergic activity across the sleep cycle. Red and blue lines indicate inhibitory and excitatory projections, respectively. In this simplified model (a), hypocretin systems are strongly excitatory to both monoaminergic and cholinergic system but have stronger effects on monoaminergic tone. During wakefulness (b), high hypocretin tone drives high cholinergic and monoaminergic tone. During non-REM sleep (c), decreased hypocretin tone reduces monoaminergic and cholinergic activity. During REM sleep (d), the depressed monoaminergic tone is so complete that cholinergic systems are desinhibited. Note that, in our model, hypocretin activity is mostly depressed during REM sleep, differentially from the Kilduff and Peyron's model.(57) Electrophysiological studies are needed to differentiate between these hypotheses. The influence of dopamine in this model might be more complex and could involve other inputs as the role of this system in the regulation of the sleep cycle is more uncertain. Differential effects of hypocretin/monoamines on brainstem and basal forebrain cholinergic systems may also explain a differential activation of these forebrain versus brainstem cholinergic systems across the sleep cycle, as suggested by some authors. Note that the presence of Hcrtr2 on cholinergic cells has not been finally established but high Hcrtr2 concentrations are observed in regions containing cholinergic neurons (e.g. basal forebrain). Inhibitory GABAergic projections from the preoptic hypothalamus or the periaqueductal gray to monoaminergic cell groups are also likely to be critical.(58)

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Most human have no preprohypocretin transcripts and Acknowledgments reduced immunocytochemical staining in the perifornical We thank the entire staff of the Center for Narcolepsy for area.(10,11) These results suggest either a lack of transcrip- scientific contributions and Claudia Gumbiner for editing the tion or a selective loss of hypocretin-containing cells. Our manuscript. histochemical studies did not detect any sign of acute inflammation using HLA-DR immunostaining(10) while another References study indicated residual gliosis in four brains examined in the 1. Rechtschaffen A, Gilliland MA, Bergmann BM, Winter JB. Physiological perifornical area.(11) This, together with the well-established correlates of prolonged sleep deprivation in rats. Science 1983;221: HLA association in narcolepsy, suggests an autoimmune 182±184. 2. Kushida CA, Bergmann BM, Rechtschaffen A. 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What rest in flies can tell us about sleep in mammals. enthusiasm. 2000;26:295±298. 7. Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ, Whether or not autoimmunity is involved in cell loss in Nishino S, Mignot E. The canine narcolepsy is caused by narcolepsy, the finding opens direct therapeutic opportunities. a mutation in the hypocretin (orexin) receptor 2 gene. Cell 1999; All currently available treatments act downstream of hypocre- 98:365±376. (31) 8. Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, tins, on monoaminergic systems. Supplementing hypocre- Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, tins in narcolepsy using with central penetration Hammer RE, Saper CB, Yanagisawa M. Narcolepsy in orexin knock- should be a better and more specific treatment for this out mice: molecular genetics of sleep regulation. Cell 1999;98:437± 451. disabling condition. Hypocretin receptors are classical G- 9. 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