Melanin concentrating hormone in central hypersomnia

Sleep Medicine 12 (2011) 768–772

Contents lists available at ScienceDirect

Sleep Medicine journal homepage: www.elsevier.com/locate/sleep

Original Article

Melanin concentrating hormone in central hypersomnia Christelle Peyron a, Françoise Valentin c, Sophie Bayard b, Lucie Hanriot a, Christophe Bedetti a, Bernard Rousset c, Pierre-Hervé Luppi a, Yves Dauvilliers b,⇑ a

Centre National de la Recherche Scientifique UMR5167, Université Claude Bernard Lyon 1, Université de Lyon, Institut Fédératif des Neurosciences de Lyon, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France b Department of Neurology, Gui de Chauliac Hospital, CHU Montpellier, INSERM U1061, National Reference Network for Orphan Diseases (Narcolepsy, Hypersomnia, Kleine–Levin Syndrome), Neurologie B, 80 Ave., Augustin Fliche, 34295 Montpellier, France c INSERM U664, Université Claude Bernard Lyon 1, Université de Lyon, Institut Fédératif 62, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France

a r t i c l e

i n f o

Article history: Received 25 August 2010 Received in revised form 15 March 2011 Accepted 23 April 2011 Available online 21 June 2011 Keywords: Narcolepsy Cataplexy MCH Orexin Hypersomnia Hypothalamus

a b s t r a c t Background: Narcolepsy with cataplexy (NC) is a disabling disorder characterized by excessive daytime sleepiness and abnormal rapid eye movement (REM) sleep manifestations, due to a deficient hypocretin/orexin neurotransmission. Melanin concentrating hormone (MCH) neurons involved in the homeostatic regulation of REM sleep are intact. We hypothesized that an increased release of MCH in NC would be partly responsible for the abnormal REM sleep manifestations. Methods: Twenty-two untreated patients affected with central hypersomnia were included: 14 NC, six idiopathic hypersomnia with long sleep time, and two post-traumatic hypersomnia. Fourteen neurological patients without any sleep disorders were included as controls. Using radioimmunoassays, we measured hypocretin-1 and MCH levels in cerebrospinal fluid (CSF). Results: The MCH level was slightly but significantly lower in patients with hypersomnia (98 ± 32 pg/ml) compared to controls (118 ± 20 pg/ml). After exclusion of patients affected with post-traumatic hypersomnia the difference became non-significant. We also failed to find any association between MCH level and hypocretin level, the severity of daytime sleepiness, the number of SOREMPs, the frequency of cataplexy, and the presence of hypnagogic hallucinations or sleep paralysis. Conclusion: This study reports the first measurement of MCH in CSF using radioimmunoassay technology. It appears to be a non-informative tool to differentiate etiologies of central hypersomnia with or without REM sleep dysregulation. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Narcolepsy-cataplexy (NC) is a disabling disorder characterized by excessive daytime sleepiness (EDS) and abnormal rapid eye movement (REM) sleep manifestations [1]. These include cataplexy (sudden loss of muscle tone triggered by strong emotions), sleep paralysis, hypnagogic hallucinations, and sleep-onset REM periods (SOREMP). Recent pathophysiological insights have demonstrated that NC is caused by the early loss of hypothalamic neurons producing hypocretin/orexin [2–5]. A striking decrease in cerebrospinal fluid (CSF) hypocretin-1 concentrations has been noted in most of those patients with a high (94%) positive predictive value for the diagnosis of NC [6,7]. In addition, NC is one of the diseases most tightly associated with a specific HLA allele, DQB10602, found in 85–95% of sporadic patients [1]. In contrast, little is known regarding the pathophysiology of other central hypersomnia, especially narcolepsy without cata⇑ Corresponding author. Tel.: +33 0 467 33 63 61; fax: +33 0 467 33 72 85. E-mail address: [email protected] (Y. Dauvilliers). 1389-9457/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.sleep.2011.04.002

plexy, and idiopathic hypersomnia (IH) with long or without long sleep time. It remained difficult in many cases to clinically differentiate patients affected with IH without long sleep time and those with narcolepsy without cataplexy [8]. Despite short latencies, the two latter diagnoses differ mainly on the number of SOREMPs observed on the multiple sleep latency tests (MSLT). Another chronic central hypersomnia etiology may be diagnosed in a context of a traumatic brain injury. Indeed, a recent prospective study has reported frequent hypersomnia following traumatic brain injury with the rare occurrence of REM sleep manifestations [9]. In contrast to NC, no biological or genetic tools have helped to understand and differentiate those central hypersomnias. Melanin concentrating hormone (MCH) neurons localized within the same hypothalamic areas as hypocretin neurons are intact in patients with NC, as shown in post-mortem samples [4,10]. It is interesting to note that hypocretin and MCH neuropeptides are involved in similar physiological functions but often in an antagonistic way. Hypocretins promote wakefulness [11] while MCH induces REM sleep [12] (for review [13]). In vivo electrophysiological recordings showed that hypocretin neurons are active during

C. Peyron et al. / Sleep Medicine 12 (2011) 768–772

waking [14,15] while MCH neurons are active during REM sleep [16]. Furthermore, neurotransmitters involved in wakefulness such as noradrenalin, serotonin and acetylcholine exert a direct inhibitory action on MCH neurons [17–19]. As decreased hypocretinergic and monoaminergic tones have been described in NC [20], a lack of inhibition of MCH neurons may occur with an increased release of MCH neuropeptide as a consequence. CSF hypocretin-1 levels represent a major biological tool to diagnose NC. We hypothesize that CSF MCH levels may be an additional interesting biological marker involved in central hypersomnia with high REM sleep pressure. The aim of the present study was to compare hypocretin and MCH CSF levels between patients with NC, patients affected with central hypersomnia without REM sleep dysregulation, and neurological controls.

769

ported an increased sleep need of more than 2 h per 24 h compared to the pre-traumatic period. Polysomnographies and MSLT were performed 3 and 5 years after the occurrence of EDS and mean sleep latencies on MSLT were, respectively, 10 and 10.2 min with one patient presenting one SOREMP. None were HLA DQB10602 positive. Fourteen patients (7 males and 7 females, mean age at 63.8 ± 18.3, range from 27 to 84 years old) affected with well-defined neurological disorders classically associated with a preserved hypocretin system (including normal pressure hydrocephalus, polyneuropathy, spinal cord lesions, multiple sclerosis, and Alzheimer’s disease) were included as controls. None of them had significant complaint of EDS, insomnia or parasomnia. All patients with chronic hypersomnia or neurological disorder gave informed consent to take part in the study, which was approved by the Local Ethics Committee.

2. Methods 2.2. Sample collection 2.1. Patients Twenty-two unrelated patients (mean age at 39.4 ± 14.9, range from 12 to 58 years old) were included with a central hypersomnia diagnosis according to revised criteria [21]. All patients underwent one night of polysomnographic recording followed by the MSLT in the sleep laboratory as required in ICSD-2 [21]. Patients also filed a sleep diary with a particular focus on assessing good sleep hygiene the week before the polysomnography recording in order to avoid sleep deprivation and large variation in sleep onset and offset. A HLA class II typing was performed in all patients. None of the patients had any psychiatric disorder based on the DSM-IV criteria. Subjects with an index of respiratory events (apneas, hypopneas) >10, were excluded from the study. Patients were not taking psychostimulants, anticataplectic medications or any other medication known to influence sleep for at least one month prior to sleep recording. Sleep was recorded and scored based on standard method [22]. The following polysomnographic variables were measured: total sleep time, sleep latency, sleep efficiency, duration of wake after sleep onset, number of awakenings, percentage of stages N1, N2, N3 and REM sleep, REM latency, and REM efficiency. The mean sleep latency and the number of SOREMPs on the MSLT were also reported. Fourteen (8 males and 6 females) patients with narcolepsy, clear-cut cataplexy and positive HLA DQB10602 typing were included. The frequency of cataplexy attacks was assessed on a scale from 1 to 5, where 1 represents one or fewer attacks per year, 2 represents more than one per year but fewer than one per month, 3 stands for more than one cataplexy attack per month but fewer than one per week, 4 represents more than one per week but fewer than one per day and 5 represents severe cases with at least one cataplexy attack per day [23]. All patients with NC had two or more SOREMPs and a mean sleep latency of <8 min on the MSLT, except one patient aged 52 with clear-cut cataplexies but a mean sleep latency at 10 min and only one SOREMP as frequently reported in that age-group [24]. Six patients (2 males and 4 females) were affected with IH with long sleep time. They all reported a complaint of constant EDS (Epworth sleepiness scale >12) and non-refreshing naps irrespective of their duration, an uninterrupted and prolonged nighttime sleep and sleep drunkenness. Sleep efficiencies on the polysomnography were always above 90% and none carried the HLA DQB10602 allele. Two patients had mean sleep latency below 8 min on the MSLT and four above 8 min, including one patient with one SOREMP. Two patients (1 male and 1 female) were diagnosed as posttraumatic hypersomnia in a context of EDS occurring within 4 months after the traumatic brain injury. Those two patients re-

CSF samples were collected by lumbar puncture between 14:00 and 18:00 and stored immediately at 80 °C until use. The samples were thawed only two times for CSF hypocretin-1 and MCH measurements. 2.3. Radioimmunoassay (RIA) measures CSF hypocretin-1 (orexin-A) level was determined in duplicate from crude CSF samples using 125I RIA kits (Phoenix Europe GmbH, Kalsruhe, Germany) in accordance with the manufacturer’s instructions. The detection limit was 40 pg/ml and intra-assay variability was less than 10%. CSF hypocretin-1 levels below 110 pg/ml were considered as low and normal over 200 pg/ml [6]. All values were back-referenced to stanford reference samples (HHMI Stanford University Center for Narcolepsy, Palo Alto CA). MCH was measured with commercially available 125I RIA kits (Phoenix Europe GmbH) following the manufacturer’s instructions with little modification. MCH concentrations were determined against a known standard curve (10–1280 pg). The detection limit of the assay was 50 pg/ml with less than 5% of intra-assay variability. The activity level of the 125I-MCH radioisotope provided by Phoenix Europe GmbH (Germany) was adjusted to introduce 8500–9500 cpm/tube. Briefly, two measures from 550 ll of crude CSF were done for each patient. Samples were concentrated with a Speedvac, reconstituted in 100 ll of RIA buffer, and then incubated with 100 ll of provided rabbit anti-MCH antibody at 4 °C for 24 h. Then, 100 ll of 125I-MCH was introduced and samples were incubated at 4 °C for 24 h. On the third day, 100 ll of provided secondary antibody (goat anti-rabbit IgG) and 100 ll of provided normal rabbit serum were added to the mix and incubated at room temperature for 90 min. Finally, samples were centrifuged at 1700g for 20 min at 4 °C and supernatants were immediately removed. To increase sensitivity, an additional wash was performed with 500 ll of washing buffer. Again, samples were centrifuged (1700g, 20 min, 4 °C), supernatant was removed and the level of radioactivity left in the pellet was measured with a RIASTAR counter (Packard). Similar fractions of the CSF were used to measure both hypocretin-1 and MCH levels for all patients with central hypersomnia. 2.4. Validation procedure As we report here the first RIA measurement of MCH in human CSF, we performed some validation steps. Increasing amounts of standard MCH (0, 51, and 154 pg) were added to 600 ll of pooled CSF samples. MCH measures reflected MCH added amounts with 48, 95, and 190 pg/tube, respectively. Further, MCH was detected

770

C. Peyron et al. / Sleep Medicine 12 (2011) 768–772

in different volumes (600, 1000, and 1100 ll) of the same pool of CSF. A linear correlation (r = 0.75; p < 0.01) between detected immunoreactive MCH (MCH-ir) and CSF initial volume was found (50, 80, and 95 pg/tube, respectively). However, concentration of a larger amount of CSF (above 1200 ll) did not give linear correlation anymore. 2.5. Statistical analysis All results are reported as mean ± SD. Statistical analyses were performed to compare patients to controls and to compare patients with at least 2 SOREMPs (n = 13) and patients with 1 or 0 SOREMPs (n = 9) during the MSLT indices using Fisher exact test. The comparison of patient group characteristics on the CSF hypocretin and MCH-ir levels was performed using the Kruskall–Wallis test. Correlation coefficients were calculated according to Pearson’s method when done on the 36 patients and controls. Spearman’s method was used, however, when correlation was looked for in smaller samples such as for patients with narcolepsy–cataplexy or controls groups. Statistical significance was set at p < 0.05. 3. Results Table 1 presents demographic, clinical, biological and sleep characteristics of patients with hypersomnia and neurological controls. Between-group comparison revealed a significantly higher severity of objective sleepiness in NC compared to patients with IH and post-traumatic hypersomnia (p < 0.01). CSF hypocretin-1 levels were significantly lower in patients with NC compared to patients with IH and post-traumatic hypersomnia (p < 0.0001). We found 98 ± 32 pg/ml of MCH-ir in CSF of patients affected with central hypersomnia (n = 22). This value is significantly lower than in the control population (n = 14, 118 ± 20 pg/ml) that includes patients affected with various neurological conditions (p < 0.026). The level of MCH-ir was similar for patients with NC (n = 14, 98 ± 36 pg/ml) and IH with long sleep time (n = 6, 104 ± 26 pg/ml), and slightly higher compared to post-traumatic hypersomnia (n = 2, 76 ± 6 pg/ml), but without statistical significance (p = 0.079) (Fig. 1 and Table 1). CSF MCH-ir level was not different between patients with central hypersomnia (100 ± 33 pg/ml) and neurological controls when values from patients with post-traumatic hypersomnia are not considered. Table 1 shows the mean sleep latency on the MSLT but also the number of SOREMPs for each hypersomnia etiology. Two or more SOREMPs were observed in all patients with NC except one, but not in the other etiologies. We failed to find any difference in CSF MCH-ir levels between patients with SOREMP-associated hypersomnia (95 ± 37 pg/ml, n = 13) and other hypersomnia (102 ± 28 pg/ml, n = 9). In contrast, we found significant CSF hypocretin1 difference between SOREMP-associated hypersomnia and other hypersomnias (50 ± 27 vs. 436 ± 61 pg/ml, p < 0.0001). When considering patients with central hypersomnia (n = 22), we found no correlation between the CSF levels of MCH-ir and hypocretin-1. We reported significant differences in CSF MCH-ir level between genders in hypersomnia groups, with higher levels in males (114 ± 32 pg/ml, n = 11) than females (82 ± 24 pg/ml, n = 11) (p = 0.013), although no sex ratio difference was found between patient groups (Table 1). However, we failed to find such gender difference in the neurological controls (males: 122 ± 4 pg/ml, n = 7 vs. females: 114 ± 10 pg/ml, n = 7). No correlation was found between CSF MCH-ir levels and age, body mass index, percent of NREM or REM sleep stages, EDS (as assessed with the Epworth sleepiness scale or mean sleep latencies on the MSLT), the frequency of cataplexy and the number of SOREMPs. In addition, we failed to report any difference in CSF

Table 1 Clinical features of the studied populations.

Sex ratio (M/F) Age (years) Body Mass Index Age at onset of EDS (years) Epworth sleepiness scale Frequency of cataplexy Patients with hypnagogic hallucinations, n Patients with sleep paralysis, n MSLT latency (min) SOREMPs, n Patients 62 SOREMPs, n Patients with 1 SOREMP, n CSF hypocretin-1 level (pg/ml), mean CSF hypocretin-1 <110 pg/ml,% CSF MCH level (pg/ml)

Narcolepsy Idiopathic with hypersomnia cataplexy n = 6 n = 14

Neurological Postcontrols traumatic hypersomnia n = 14 n=2

8/6 40.2 16.6a 25.5 5.4 25.4 16.1

2/4 33. 7 11d 22.4 4.1 21.8 7.3

1/1 50.5 4.9 22.9 0.3 38 7.1

7/7 63.8 18.3 n/a n/a

18.6 3.7

17.7 3.3

23 1.4

n/a

0

0

0

3

1

0

1

1

0

4.5 2.5 3.4 1.2b 13

8.4 4.6 0.17 0.4 0

10.1 0.1 0.5 0.7 0

n/a n/a n/a

1

1

1

n/a

26 27

475 142

505 80

n/a

100

0

0

n/a

98 36

104 26

76 6

118 20

4.1 1.2

a,b,c

12

9 c

b,c

Values are reported as mean ± standard deviation. n/a stands for non available. EDS, excessive daytime sleepiness; MSLT, multiple sleep latency tests. Significance is indicated for p < 0.05. a narcolepsy with cataplexy versus controls; b narcolepsy with cataplexy versus idiopathic hypersomnia; c narcolepsy with cataplexy versus post-traumatic hypersomnia; d idiopathic hypersomnia versus controls.

Fig. 1. Box plot illustrating the level of MCH (median ± quartiles) in 4 different groups of patients, i.e. narcolepsy with cataplexy (n = 14), idiopathic hypersomnia (n = 6), post-traumatic hypersomnia (n = 2) and controls with neurological disease but without sleep disorders (n = 14).

MCH-ir levels regarding the presence of hypnagogic hallucinations or sleep paralysis. In contrast, CSF hypocretin-1 levels were significantly lower in patients with hypnagogic hallucinations (115 ± 177 vs. 397 ± 244 pg/ml, p < 0.004), sleep paralysis (98 ± 156 vs. 295 ± 261 pg/ml, p < 0.004) and frequent cataplexy (30 ± 27 vs. 332 ± 240 pg/ml, p < 0.05). Finally, in contrast to CSF hypocretin-1 level being clearly stated as abnormal below 110 pg/ml and normal over 200 pg/ml [6], no clear pathological level for CSF MCH-ir could be defined (Fig. 1).

C. Peyron et al. / Sleep Medicine 12 (2011) 768–772

4. Discussion The present study reports the first measurement of human CSF level of MCH-ir in patients affected with central hypersomnia compared to neurological controls. We found a slight but significantly lower CSF MCH-ir level in patients with hypersomnia compared to controls, results becoming non-significant after exclusion of patients with post-traumatic hypersomnia from analysis. We included patients with separate clear-cut central hypersomnia diagnosis only, avoiding narcolepsy without cataplexy and IH without long sleep time since they may have clinical and pathogenesis overlaps [1,8]. Since we aimed at determining whether MCH level would be increased in patients with central hypersomnia, and in particular when hypersomnia is associated with REM sleep abnormalities, we regarded the use of healthy controls as unnecessary. We rather preferred to take advantage of CSF samples from patients with neurological disease, but no sleep complaints, for which a lumbar puncture had to be performed. Several limitations in our study need to be addressed. First, the sample size used was relatively small, increasing the chance of type II errors. Second, no CSF hypocretin-1 measurement was available in neurological controls (no more CSF available). However, we may assume that those patients without any sleep complaints had all normal CSF hypocretin levels. Third, we were unable to perform a HPLC detection test on CSF. Nevertheless, we performed several validation tests that attested the reliability of the RIA method to measure MCH. Except for post-traumatic hypersomnia, CSF MCH-ir levels were in a similar range between patients with central hypersomnia and neurological controls, and no association was found between CSF MCH-ir levels and sleep parameters. In contrast, CSF hypocretin1 levels were lower in patients with NC than in other etiologies of hypersomnia and lower in presence of hypnagogic hallucinations, sleep paralysis and frequent cataplexy. These findings are in agreement with previous reports [25] and can be partially explained by the close association between cataplexy, hypnagogic hallucinations, sleep paralysis and the diagnosis of NC. We found no correlation between MCH-ir CSF levels and age of patients. This is in line with the absence of correlation between hypocretin-1 CSF level and age as observed in this study and by others [26]. But we found an association between gender and MCH-ir levels in patients with hypersomnias with a higher level in men. Supporting these data, it has been shown that the level of expression of prepro-MCH is reduced in presence of oestrogens [27]. No gender difference was noted in the older neurological controls with mean ages at 63.8 ± 18.3 years old. The reduced level of CSF MCH-ir in women affected with hypersomnias may thus be due to birth control treatment. Interestingly, the two patients affected with a post-traumatic hypersomnia present the lower CSF MCH-ir levels. This observation seems of major interest since histological data recently reported a 30% loss of MCH neurons in patients with traumatic brain injury [28]. Altogether, these data highlight a possible involvement of hypothalamic damage (including MCH neurons) in generating chronic sleepiness in traumatic brain injury as already suggested for Parkinson’s disease [29–31]. Recent data demonstrated both in animals and humans a correlation between CSF hypocretin concentrations and the number of hypocretin cells present in the hypothalamus [30,32]. Such a correlation is totally uncertain for the MCH system. Since the amount of CSF collected in rodents is not sufficient to reliably measure CSF MCH-ir level using the available technologies, we were unable to demonstrate that CSF MCH-ir level reflects MCH neurotransmission. We found, however, that MCH-ir level

771

in the human CSF is twice as low as hypocretin level, although MCH neurons are twice as high numbered as hypocretin neurons [10]. Altogether these observations question whether CSF measurement of MCH-ir is a good index of MCH brain release. In conclusion, this study reports the first measurement of human CSF level of MCH-ir in patients with hypersomnias and controls. From a clinical perspective, however, we believe that the measurement of CSF MCH is not an informative tool to differentiate etiologies of central hypersomnia with or without REM sleep dysregulation. Finally, our results support the idea that a decreased MCH tone may be involved in the context of post-traumatic hypersomnia. Further studies replicating and extending our preliminary findings would be of clear interest. Conflict of interest The ICMJE Uniform Disclosure Form for Potential Conflicts of Interest associated with this article can be viewed by clicking on the following link: doi:10.1016/j.sleep.2011.04.002.

Acknowledgments The authors would like to thank Rachida Rabilloud for her advice and technical help. This study was financially supported by CNRS, Université Claude Bernard-Lyon1. LH was supported by the French Ministry of Research. References [1] Dauvilliers Y, Arnulf I, Mignot E. Narcolepsy with cataplexy. Lancet 2007;369:499–511. [2] Blouin AM, Thannickal TC, Worley PF, Baraban JM, Reti IM, Siegel JM. Narp immunostaining of human hypocretin (orexin) neurons: loss in narcolepsy. Neurology 2005;65:1189–92. [3] Crocker A, Espana RA, Papadopoulou M, Saper CB, Faraco J, Sakurai T, et al. Concomitant loss of dynorphin, NARP, and orexin in narcolepsy. Neurology 2005;65:1184–8. [4] Peyron C, Faraco J, Rogers W, Ripley B, Overeem S, Charnay Y, et al. A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains. Nat Med 2000;6:991–7. [5] Thannickal TC, Moore RY, Nienhuis R, Ramanathan L, Gulyani S, Aldrich M, et al. Reduced number of hypocretin neurons in human narcolepsy. Neuron 2000;27:469–74. [6] Ripley B, Overeem S, Fujiki N, Nevsimalova S, Uchino M, Yesavage J, et al. CSF hypocretin/orexin levels in narcolepsy and other neurological conditions. Neurology 2001;57:2253–8. [7] Mignot E, Lammers GJ, Ripley B, Okun M, Nevsimalova S, Overeem S, et al. The role of cerebrospinal fluid hypocretin measurement in the diagnosis of narcolepsy and other hypersomnias. Arch Neurol 2002;59:1553–62. [8] Dauvilliers Y, Baumann CR, Carlander B, Bischof M, Blatter T, Lecendreux M, et al. CSF hypocretin-1 levels in narcolepsy, Kleine–Levin syndrome, and other hypersomnias and neurological conditions. J Neurol Neurosurg Psychiatry 2003;74:1667–73. [9] Baumann CR, Werth E, Stocker R, Ludwig S, Bassetti CL. Sleep-wake disturbances 6 months after traumatic brain injury: a prospective study. Brain 2007;130:1873–83. [10] Thannickal TC, Nienhuis R, Siegel JM. Localized loss of hypocretin (orexin) cells in narcolepsy without cataplexy. Sleep 2009;32:993–8. [11] Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K, de Lecea L. Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 2007;450:420–4. [12] Verret L, Goutagny R, Fort P, Cagnon L, Salvert D, Leger L, et al. A role of melanin-concentrating hormone producing neurons in the central regulation of paradoxical sleep. BMC Neurosci 2003;4:19. [13] Peyron C, Sapin E, Leger L, Luppi PH, Fort P. Role of the melanin-concentrating hormone neuropeptide in sleep regulation. Peptides 2009;30:2052–9. [14] Lee MG, Hassani OK, Jones BE. Discharge of identified orexin/hypocretin neurons across the sleep–waking cycle. J Neurosci 2005;25:6716–20. [15] Mileykovskiy BY, Kiyashchenko LI, Siegel JM. Behavioral correlates of activity in identified hypocretin/orexin neurons. Neuron 2005;46:787–98. [16] Hassani OK, Lee MG, Jones BE. Melanin-concentrating hormone neurons discharge in a reciprocal manner to orexin neurons across the sleep–wake cycle. Proc Natl Acad Sci USA 2009;106:2418–22. [17] Bayer L, Eggermann E, Serafin M, Grivel J, Machard D, Muhlethaler M, et al. Opposite effects of noradrenaline and acetylcholine upon hypocretin/orexin

772

[18]

[19]

[20] [21] [22]

[23]

[24]

C. Peyron et al. / Sleep Medicine 12 (2011) 768–772 versus melanin concentrating hormone neurons in rat hypothalamic slices. Neuroscience 2005;130:807–11. Gao XB, Ghosh PK, van den Pol AN. Neurons synthesizing melaninconcentrating hormone identified by selective reporter gene expression after transfection in vitro: transmitter responses. J Neurophysiol 2003;90:3978–85. van den Pol AN, Acuna-Goycolea C, Clark KR, Ghosh PK. Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection. Neuron 2004;42:635–52. Nishino S, Mignot E. Pharmacological aspects of human and canine narcolepsy. Prog Neurobiol 1997;52:27–78. Medicine AAoS. The international classification of Sleep Disorders, revised 2005. Iber CA-IS, Chesson A, Quan S. The AASM manual for the scoring of sleep and associated events: rules, terminology and technical specifications. The American Academy of Sleep Medicine; 2007. Dauvilliers Y, Montplaisir J, Molinari N, Carlander B, Ondze B, Besset A, et al. Age at onset of narcolepsy in two large populations of patients in France and Quebec. Neurology 2001;57:2029–33. Dauvilliers Y, Gosselin A, Paquet J, Touchon J, Billiard M, Montplaisir J. Effect of age on MSLT results in patients with narcolepsy–cataplexy. Neurology 2004;62:46–50.

[25] Baumann CR, Khatami R, Werth E, Bassetti CL. Hypocretin (orexin) deficiency predicts severe objective excessive daytime sleepiness in narcolepsy with cataplexy. J Neurol Neurosurg Psychiatry 2006;77:402–4. [26] Kanbayashi T, Yano T, Ishiguro H, Kawanishi K, Chiba S, Aizawa R, et al. Hypocretin-1 (orexin-A) levels in human lumbar CSF in different age groups: infants to elderly persons. Sleep 2002;25:337–9. [27] Murray JF, Baker BI, Levy A, Wilson CA. The influence of gonadal steroids on pre-pro melanin-concentrating hormone mRNA in female rats. J Neuroendocrinol 2000;12:53–9. [28] Baumann CR, Bassetti CL, Valko PO, Haybaeck J, Keller M, Clark E, et al. Loss of hypocretin (orexin) neurons with traumatic brain injury. Ann Neurol 2009;66:555–9. [29] Baumann CR, Scammell TE, Bassetti CL. Parkinson’s disease, sleepiness and hypocretin/orexin. Brain 2008;131:e91. [30] Fronczek R, Overeem S, Lee SY, Hegeman IM, van Pelt J, van Duinen SG, et al. Hypocretin (orexin) loss in Parkinson’s disease. Brain 2007;130:1577–85. [31] Thannickal TC, Lai YY, Siegel JM. Hypocretin (orexin) and melanin concentrating hormone loss and the symptoms of Parkinson’s disease. Brain 2008;131:e87. [32] Gerashchenko D, Murillo-Rodriguez E, Lin L, Xu M, Hallett L, Nishino S, et al. Relationship between CSF hypocretin levels and hypocretin neuronal loss. Exp Neurol 2003;184:1010–6.