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Selective stimulation of orexin receptor type 2 promotes wakefulness in freely behaving rats
Brain Research 1048 (2005) 138 – 145 www.elsevier.com/locate/brainres
Research report
Selective stimulation of orexin receptor type 2 promotes wakefulness in freely behaving rats Moses A. Akanmu1, Kazuki Honda* Department of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Accepted 22 April 2005 Available online 24 May 2005
Abstract Orexins A and B are a pair of neuropeptides implicated in the regulation of feeding and arousal behavior mediated through two orexin receptors type 1 and type 2. We have determined the arousal effects of newly developed selective orexin receptor type 2 agonist, [Ala11]orexin-B, on the sleep – wake cycle in rats. The effects of third ventricle intracerebroventricular (ICV) infusion of the novel orexin receptor type 2 selective agonist, [Ala11]orexin-B, on the sleep – wake cycle were investigated. ICV infusion of [Ala11]orexin-B (1, 10 and 40 nmol) during the light period (11:00 – 16:00) dose-dependently resulted in a significant increase in wake duration by 46.9% (n = 5, P < 0.05), 159.2% (n = 4, P < 0.01) and 163.6% (n = 7, P < 0.01)), respectively, and a significant decrease in rapid eye movement (REM) (P < 0.01) and non-REM sleep (P < 0.01) for all doses. In contrast, ICV infusion of orexin B at the same doses (1, 10 and 40 nmol) caused a 16.6% (n = 6, non-significant), 99.8% (n = 6, P < 0.05) and 72.0% (n = 4, P < 0.05) increase in wakefulness, respectively. Moreover, orexin-A, which exerts its effects through orexin receptor type 1 and orexin receptor type 2 with similar potency, resulted in a significant increase in wakefulness duration by 17.1% (n = 6, P < 0.05), 184.0% (n = 6, P < 0.01) and 228.6% (n = 6, P < 0.01) at doses of 0.1, 1 and 10 nmol, respectively. Further, the enhancement of wakefulness was accompanied by a marked reduction in REM and nonREM sleep. These findings suggest that orexin receptor type 2 plays an important role in the modulation of sleep – wake state and behavioral responses. D 2005 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Orexin; Orexin-B agonist; Behavior; Non-REM sleep; REM sleep; Wakefulness
1. Introduction
Abbreviations: ICV, intracerebroventricular; REM, rapid eye movement; non-REM, non-rapid eye movement; LHA, lateral hypothalamic area; OX1R, orexin receptor type 1; OX2R, orexin receptor type 2; LC, locus coeruleus; VTA, ventral tegmental area; DRN, dorsal raphe´ nucleus; LDT, laterodorsal tegmental nuclei; PPT, pedunculopontine tegmental nuclei; TMN, tuberomammilary nucleus; GABA, gamma amino butyric acid; 5HT, 5-hydroxyltryptamine; VLPO, ventrolateral preoptic nucleus; EEG, electroencephalogram; EMG, electromyogram; CSF, cerebrospinal fluid * Corresponding author. Fax: +81 3 5280 8098. E-mail address: [email protected] (K. Honda). 1 Present address: Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria. 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.04.064
Orexins, also called hypocretins, are a pair of neuropeptides expressed by a specific population of neurons in the lateral hypothalamic area (LHA), a region of the brain implicated in feeding and arousal [7,9,32,44]. Orexin-A (hypocretin-1) and orexin-B (hypocretin-2) are derived from a common precursor peptide [8,32]. The actions of orexins are mediated by two G protein-coupled receptors termed orexin receptor type 1 (OX1R) and orexin receptor type 2 (OX2R) [32]. OX1R is expressed in the locus coeruleus (LC), ventral tegmental area (VTA), dorsal raphe nucleus (DRN), laterodorsal tegmental nuclei (LDT),
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pedunculopontine tegmental nuclei (PPT) and hypothalamus, while OX2R is expressed in the tuberomammilary nucleus (TMN), VTA, DRN, LDT, PPT and hypothalamus [19,42]. Orexin-containing neurons project from the LHA to numerous brain regions, with the limbic system, hypothalamus, monoaminergic LC, dopaminergic VTA, serotonergic DRN, cholinergic nuclei of brainstem and histaminergic TMN receiving particularly strong innervation [8,24,29]. Amygdala is highly interconnected with a number of brain regions involved in sleep regulation, such as the basal forebrain, hypothalamus and brainstem [2] and also connected with the parabrachial region, DRN, PPT and LDT. Bilateral lesions of the amygdala produced substantial effects on sleep in monkeys [4]. Moreover, lesions of the posterior lateral hypothalamus where the tuberomammillary neurons reside and rostral midbrain in the brains of monkeys, rats and cats caused sleepiness while the lesions of lateral preoptic and basal forebrain in rats and cats caused sleep suppression [20,25,31,39,43]. Interestingly, these terminal areas such as the posterior lateral hypothalamic and preoptic and basal forebrain are involved in the homeostatic and circadian control of sleep [5,33]. Furthermore, the sleep – wake mechanism involves a positive neuronal feedback mechanism in which orexin excites other arousal systems, including serotonin (5-hydroxyltryptamine; 5HT) and noradrenaline. This ascending arousal system sends projection from the brainstem and posterior hypothalamus throughout the forebrain. Orexin neurons in the lateral hypothalamic area innervate all of the components of ascending arousal system and may stabilize behavioral state by increasing the activity of aminergic neurons, thus maintaining consistent inhibition of sleep-promoting neurons in the ventrolateral preoptic nucleus (VLPO) and rapid eye movement (REM)-promoting neurons in the PPT-LDT [6,10,35,38]. Indeed, central orexin administration in rodents results in increases in sympathetic tone, plasma corticosterone levels [13], metabolic rate [18], food intake [32], locomotor activity [23] and wakefulness [13]. Further, mice lacking either the orexin gene (prepro-orexin knockout mice) or orexin neurons (orexin/ataxin-3 transgenic mice), as well as mice and dogs with null mutations in the orexin receptor type 2 gene, all have phenotypes remarkably similar to those of the human sleep disorder, narcolepsy [7,14,17,44,45], and recent reports suggest that human narcolepsy is accompanied by a specific destruction of orexin neurons in the hypothalamus [30,41]. While these studies suggest that orexin stabilizes prolonged periods of wakefulness by opposing homeostatic sleep propensity [33], the differential roles of the two orexin receptors remain unclear. Thus, the goal of the present study was to determine the effects of intracerebroventricular administration of the OX2R-selective agonist, [Ala11]orexin-B, on the sleep – wake cycle in rats.
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2. Materials and methods 2.1. Animal usage Sixteen to eighteen male Sprague – Dawley rats (330 – 360 g) were obtained from Crea Japan, Inc. (Tokyo, Japan) and were housed in cages for one week. The room was maintained at a temperature of 25 T 1 -C, a relative humidity of 54 T 6%, and a light–dark cycle of 12:12 h (lights on 8:00 h). All experimental protocols were performed in accordance with the Guidelines for Animal Experimentation of Tokyo Medical and Dental University. 2.2. Preparation of [Ala11]orexin-B and orexins A- and -B The OX2R selective agonist, [Ala11]orexin-B, was obtained from Banyu pharmaceutical Co. Ltd., Tsukuba, and orexin-A and orexin-B were purchased from Peptide institute, Inc, Osaka. The peptides were dissolved in normal saline, and either peptides or vehicle were continuously infused intracerebroventricularly (icv) at a rate of 10 Al/h for the sleep –wake analysis. 2.3. Electroencephalogram (EEG) and electromyogram (EMG) Simultaneous EEG and EMG recordings were performed as described previously [15]. Briefly, 60- to 70-day-old male Sprague – Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and fixed on a stereotaxic apparatus. A stainless steel cannula (0.35 mm o.d., inclined at 20- from the vertical line, 2 mm posterior, 3.4 mm lateral to the bregma, 8.5 mm depth from the surface of cortex) was implanted in the third cerebral ventricle for continuous icv infusion. Three cortical gold-plated screw electrodes and paired stainless-steel electrodes were fixed to the skull with dental acrylic resin for recording of the EEG and EMG, respectively, and the cable from the electrodes was attached to a socket. After implantation of the cannula and attachment of the electrodes, animals were administered penicillin G potassium (20,000 U, subcutaneously) and were allowed to recover for 10 days. Following the recovery period, animals were transferred to an individual experimental cage that allowed continuous icv infusion and monitoring of EEG and EMG. The experimental cages were placed in a soundproof, electromagnetically shielded room with the same environmental conditions described above. Polyethylene tubing (PE10, 0.28 mm, i.d.) was used to connect the cannula to an infusion pump (Eicom, ESP-32, Kyoto), which was set for continuous infusion of normal saline with or without the experimental peptides at a rate of 10 Al/h. During the 7 days preceding the experiment, animals underwent daily continuous infusion of normal saline in the experimental chamber to acclimatize the animals to the infusion and recording conditions. For measurements, lead wires of the
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electrodes were connected to an EEG/EMG amplifier (Nihon-Kohden, MEG-6116, Tokyo) through a 5-strand cable with a slip-ring that allowed free movement of the rats. The amplifier was connected to a personal computer with an AD converter and software (Kissei Comtec, SleepSign, Nagano) for acquiring and processing data. Data were sampled at 128 Hz and subjected to on-line spectral analysis by Fast Fourier Transformation over 8-s epochs. Data were stored on a magnetic optical (MO) disk that was subsequently visually analyzed offline after autoanalysis of the sleep – wake stages. EEG and EMG were measured, starting at lights on, for two consecutive days: a baseline day and experimental day. Animals received either [Ala11]orexin-B or orexin-B at doses of 1, 10, 40 nmol or orexin A at doses of 0.1, 1 and 10 nmol for a 5-h period (11:00 – 16:00) in random order. During the resting phase, all animals received icv infusion of saline without experimental peptides. Sleep –waking state was classified as wakefulness (W), rapid eye movement (REM) sleep and non-REM sleep from the EEG and EMG recordings, and the results were verified visually according to the standard criteria [1,15]. Discrimination of wakefulness (high EMG amplitude, low EEG amplitude), non-REM sleep (low EMG amplitude, high EEG amplitude with high power density in the delta band (0.5 –4.0 Hz)) and rapid-eye-movement (REM) sleep (silent low EMG amplitude, low EEG amplitude with high values in the theta band (4.0 – 8.0 Hz)) was performed. The analyzed sleep variables included the amount of non-REM and REM sleep, and number and duration of each sleep parameter episode. The duration of wakefulness, non-REM sleep and REM sleep was determined for each hour in the 24-h recording. 2.4. Statistical analysis Statistical analyses were performed using repeated measures analyses of variance (ANOVA) followed by post hoc analysis of significance with the Student – Newman – Keuls test. Probability (P) values less than 0.05 were considered to indicate statistical significance.
3. Results Vigilance states and EEG of rats were characterized at baseline and during icv administration of [Ala11]orexin-B, orexin-B and orexin-A. Daily icv infusion (11:00 – 16:00 h) of [Ala11]orexin-B at doses of 1, 10 and 40 nmol resulted in a significant and dose-dependent increase in the duration of wakefulness (Table 1), primarily due to a marked increase in episode duration for all three doses, with significant increases at doses of 10 nmol (P < 0.05) and 40 nmol (P < 0.01). Icv administration of [Ala11]orexin-B also resulted in a significant and dose-dependent decrease in REM sleep (P < 0.01), primarily via a decrease in the episode frequency at doses of 10 and 40 nmol. Further, non-REM sleep was significantly (P < 0.01) reduced at doses of 10 and 40 nmol of [Ala11]orexin-B. Compared with baseline, 40 nmol of [Ala11]orexin-B significantly (P < 0.01, n = 7) reduced REM and non-REM sleep episode frequency and duration and increased wakefulness episode duration (Table 1). When analyzing the hourly subsets of the infusion period, 10 nmol of [Ala11]orexin-B produced a four-fold increase in wakefulness during the first hour of infusion (Fig. 1), and this arousal effect was maintained during the first three hours of the infusion period. Further, the arousal effect of [Ala11]orexinB resolved immediately after completion of the infusion, indicating that the effect of [Ala11]orexin-B on arousal is transient. All three doses of [Ala11]orexin-B significantly increased wakefulness and suppressed REM sleep (Fig. 2). In contrast, 10 nmol of orexin-A produced a three-fold increase in wakefulness during the first hour of infusion and a five-fold increase in wakefulness during the second hour of infusion. The increase was maintained at the three-fold level throughout the remainder of the infusion period (Fig. 3). ICV administration of orexin-A also significantly suppressed REM and non-REM sleep, with a more profound effect on REM sleep. REM sleep was suppressed throughout the infusion period and was completely suppressed by the second hour of infusion. In contrast, non-REM sleep was significantly suppressed only during the first two hours of drug infusion. Orexin-A administration also resulted in a significant increased in wake duration by 17.1%, 184.0%, 228.6%,
Table 1 Effects of diurnal (11:00 – 16:00) icv infusion of [Ala11]orexin-B on each sleep parameters in freely behaving rats (mean T SEM) 1 nmol (n = 5) Baseline Wakefulness
REM
Non-REM
Frequency Duration (s) Total time (min) Frequency Duration (s) Total time (min) Frequency Duration (s) Total time (min)
60.2 62.2 59.6 26.8 103.6 42.5 61.8 196.4 193.2
T T T T T T T T T
10 nmol (n = 4) Experiment
7.4 6.9 4.2 5.0 11.6 2.2 6.5 22.2 6.0
71.8 76.4 88.2 12.6 103.6 21.8 73.2 162.4 185.5
T T T T T T T T T
9.1 9.2 9.5# 1.6 9.6 3.2# 9.0 22.8 8.7
Baseline 68.8 53.8 61.7 29.0 96.0 45.2 69.8 166.0 189.9
T T T T T T T T T
4.9 5.0 7.3 4.0 6.4 4.0 4.2 15.5 7.0
40 nmol (n = 7) Experiment
Baseline
64.0 T 3.7 141.5 T 22.2* 147.2 T 15.2* 5.8 T 2.5# 85.0 T 22.6 8.2 T 4.1# 64.5 T 3.4 118.3 T 5.6* 126.8 T 8.0#
70.0 73.3 84.2 20.1 104.4 35.1 72.0 153.7 178.4
T 5.3 T 6.1 T 6.9 T 1.2 T 6.9 T 3.4 T 5.0 T 13.1 T 5.1
Experiment 46.1 298.9 207.4 3.1 75.4 5.6 46.6 100.4 74.5
T T T T T T T T T
5.0# 47.4# 11.3# 1.3# 24.4 2.6# 5.1# 11.4* 7.5#
* P < 0.05 vs. Baseline (Vehicle-treated); # P < 0.01 vs. Baseline (Vehicle-treated); ICV, intracerebroventricular; REM, rapid eye movement; Non-REM, nonrapid eye movement.
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Fig. 1. Effects of 10 nmol [Ala11]orexin-B infusion into the third ventricle on total time spent in rapid eye movement (REM) and non-REM sleep. Vertical lines on each hourly value stand for SEM. Baseline: closed circle and [Ala11]orexin-B: open circle. *P < 0.05; #P < 0.01 compared to baseline (vehicle-treated) rats (n = 4).
respectively; reduced REM sleep by 27.5%, 75.7%, 96.2%, respectively; and reduced NREM sleep by 5.0%, 49.2% and 88.0%, respectively (Fig. 4) at doses of 0.1 nmol (P < 0.05), 1 nmol (P < 0.01) and 10 nmol (P < 0.01).
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Fig. 3. Effects of 10 nmol orexin-A infusion into the third ventricle on total time spent in REM and non-REM sleep. Vertical lines on each hourly value stand for SEM. Baseline: closed circle, and Orexin-A: open circle. *P < 0.05; #P < 0.01 compared to baseline (vehicle-treated) rats (n = 6).
ICV infusion of orexin-B also significant increases in wakefulness during the infusion period (Fig. 5). Orexin-B increased the total amount of wakefulness by 16.6%, 99.8%, 72.0%, respectively; reduced REM sleep by 14.0%, 64.2% and 74.5%, respectively; and reduced non-REM sleep by
Fig. 2. Total time spent in wake, REM and non-REM sleep during the 5-h infusion of [Ala11]orexin-B into the third ventricle of rats. Line and deep-shaded columns represent baseline and experimental day profiles, respectively. Values are mean T SEM (n = 5, 4, and 7 for 1, 10 and 40 nmol, respectively). *P < 0.05, # P < 0.01 compared to baseline (vehicle-treated) rats.
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Fig. 4. Total time spent in wake, REM and non-REM-sleep during the 5-h infusion of different doses of orexin-A (1, 10 and 40 nmol) into the third ventricle of rats. Line and deep-shaded columns represent baseline and experimental day profiles, respectively. Values are mean T SEM (n = 6). *P < 0.05, #P < 0.01 compared to baseline (vehicle-treated) rats.
0%, 34.1% and 36.9%, respectively, at doses of 1, 10 and 40 nmol (Fig. 6). The magnitude of orexin-B-induced increases in wakefulness and suppression of REM and nonREM was substantially less than that observed following icv infusions of [Ala11]orexin-B at the same dose levels. The results also showed that there was REM sleep rebound effect after the cessation of infusion of orexins during the dark period. This effect may be due to compensatory behavioral response to earlier REM sleep suppression effects observed with infusion of the orexins administered during the day or due to unknown mechanism.
showing a 10-fold higher affinity at OX2R than at OX1R [32], while [Ala11]orexin-B showed a 120-fold selectivity for OX2R over OX1R [3]. Orexin increases arousal via its action on basal forebrain areas, which are comprised of a number of distinct regions. A relatively high density of
4. Discussion In this study, following [Ala11]orexin-B, orexin-A and orexin-B icv administration, there were significant changes in the amount of wake, NREM and REM sleep as shown in Figs. 1, 3 and 5, respectively. Compared to baseline, during the first 1 h of ICV administration, [Ala11]orexin B, orexinA significantly (P < 0.01) increased wakefulness immediately to 413.6% and 153.4%, respectively, while orexin-B only had an increase of 32.6% above the baseline value when the percentage change was analyzed indicating the importance of OX2R in sleep – wake regulation. However, orexin-B increased wakefulness to 258.1% by the second hour of infusion. This further suggests a possible early OX2R stimulation in the orexin– ligand –receptor system in sleep – wake regulation. Indeed, 10 nmol [Ala11]orexin-B produced an immediate increase in wakefulness and caused total REM sleep suppression by the second hour of infusion (Fig. 1). [Ala11] orexin-B appears more potent than orexin-B and equally as potent as orexin-A at inducing waking and decreasing REM sleep. This is consistent with reports that [Ala11]orexin-B has a 120-fold selectivity for OX2R over OX1R [3]. Alternatively, [Ala11]orexin-B may be more metabolically stable than orexin-B. Orexin-A has a high affinity for both receptors (OX1R and OX2R) with equal potency, whereas orexin-B acts more selectively on OX2R
Fig. 5. Effects of 10 nmol orexin-B infusion into the third ventricle on total time spent in rapid eye movement (REM) and non-REM sleep. Vertical lines on each hourly value stand for SEM. Baseline: closed circle and orexin-B: open circle. *P < 0.05; #P < 0.01 compared to baseline (vehicletreated) rats (n = 6).
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Fig. 6. Total time spent in wake, REM and non-REM sleep during the 5-h infusion of orexin-B into the third ventricle of rats. Line and deep shaded columns represent baseline and experimental day profiles, respectively. Values are mean T SEM (n = 6, 6, and 4 for 1, 10 and 40 nmol, respectively). *P < 0.05, #P < 0.01 compared to baseline (vehicle-treated) rats.
orexin fibers have been observed in close proximity to the lateral and third ventricles [11], including the organum vasculosum laminae terminalis, a site that is particularly sensitive to the behavioral actions of various peptides [37]. Thus, ventricular orexin infusion may act on these particular areas to induce waking behavioral effects. Orexin also acts on multiple brainstem systems to modulate behavioral state [36]. The influence of orexins on arousal state has been suggested by experiments that demonstrated that canine narcolepsy results from a mutation in the OX2R gene [17] or a deficiency of orexin [7]. Similarly, brains from human narcoleptics exhibit a nearly complete loss of neurons expressing orexin mRNA or protein [30,41], and orexin is below the detectable limits in the cerebrospinal fluid of humans with narcolepsy [27,30]. Orexin neurons heavily innervate aminergic nuclei that promote wakefulness and inhibit REM sleep. Moreover, previous work suggested that activation of orexin neurons may contribute to the promotion or maintenance of wakefulness [12]. Indeed, canine narcolepsy is associated with hyperactivity of cholinergic neurotransmission and decreased monoaminergic tone, which would produce daytime sleepiness and REM abnormalities [21,26]. Further, wakefulness produced by modafinil, a drug used to treat the excessive daytime sleepiness of narcolepsy, is associated with an increase in orexin neuron activity in rodents, as indicated by increased expression of the transcription factor, Fos [7,34]. The importance of OX2R in the sleep – wakefulness regulation was demonstrated by the finding that narcoleptic dogs carry mutations in the OX2R gene [17]. Further, icv administration of orexin-A significantly increased wakefulness in control dogs but not in narcoleptic dogs [28]. Indeed, recent reports have demonstrated that increased orexinergic tone induces wakefulness, while reduced orexinergic tone resulting from knockouts of the prepro-orexin gene or the OX2R gene decreases wakefulness and increases premature entry into REM sleep. Homozygous knockout mice showed frequent narcoleptic/cataplectic attacks, significant disruptions of sleep –wakefulness cycling during the dark phase, frequent direct transitions into REM sleep from wakeful-
ness, marked fragmentation of waking, decreased waking time, increased non-REM and REM sleep time, decreased REM sleep latency, and a decreased duration of waking episodes during the dark phase [7,8]. OX2R knockout mice have also been reported to show characteristics of narcolepsy, albeit with an EEG and behavioral phenotype that is less severe than that found in the orexin knockout mouse, while OX1R knockouts only showed increased fragmentation of sleep –wakefulness cycles [7,16,22,30,40,44]. More importantly, OX2R activation is essential for the normal regulation of wake/non-REM sleep transitions, and the profound dysregulation of REM sleep control unique to the narcolepsy – cataplexy syndrome results from loss of signaling through both OX2R-dependent and OX2R-independent pathways [45]. Therefore, phenotypic differences between OX1R knockout mice and OX2R knockout mice suggest that lack of an orexin signal via OX2R contribute to the pathogenesis of narcolepsy. This is consistent with observations from the present study that icv administration of [Ala11]orexin-B resulted in a potent, rapid and dosedependent wake-promoting effect. Further, a recent study reported the existence of a direct synaptic connection between orexin neurons and histaminergic neurons in the TMN and demonstrated that orexins increased firing rates of isolated histaminergic neurons via OX2R [46]. Thus, the orexin –histamine pathway may mediate the effects elicited by [Ala11]orexin-B.
5. Conclusions Daily icv infusion of the novel orexin-2 receptor selective agonist, [Ala11]orexin-B, into the third ventricle induced a significant arousal effect in freely behaving rats, and the efficacy of [Ala11]orexin-B was comparable to that of native peptides at the same dose. These results indicate that [Ala11]orexin-B dose-dependently increased wakefulness and reduced REM and non-REM sleep and suggest that [Ala11]orexin-B plays an important role in the modulation of sleep – wake state. Exogenous administration of the OX2R
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agonist, [Ala11]orexin-B, may be a useful tool for the future characterization of the physiological role of OX2R in the regulation of sleep –waking. Acknowledgment This study was supported by a Grant-in-Aid for Scientific Research (C) (No. 14570912, 16614003) to K.H. References [1] M.A. Akanmu, K. Honda, S. Inoue, Hypnotic effects of total aqueous extracts of Vervain hastata (Verbenaceae) in rats, Psych. and Clin. Neurosci. 56 (2002) 309 – 310. [2] D.G. Amaral, J.L. Price, A. Pitkanem, S.T. Carmichael, Anatomical organization of the primate amygdaloid complex, in: J. Aggleton (Ed.), The Amygdala, Wiley-Liss, New York, 1992, pp. 1 – 67. [3] S. Asahi, S. Egashira, M. Matsuda, et al., Development of an Orexin-2 receptor selective agonist, [Ala11, D-Leu15] Orexin-B, Bioorg. Med. Chem. Lett. 13 (2003) 111 – 113. [4] R.M. Benca, W.H. Obermeyer, S.E. Shelton, J. Droster, N.H. Kalin, Effects of amygdala lesions on sleep in rhesus monkeys, Brain Res. 879 (2000) 130 – 138. [5] A.A. Borbely, P. Achermann, Sleep homeostasis and models of sleep regulation, J. Biol. Rhythms 14 (1999) 557 – 568. [6] R.E. Brown, O. Sergeeva, K.S. Ericksson, H.L. Haas, Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat, Neuropharmacology 40 (2001) 457 – 459. [7] R.M. Chemelli, J.T. Willie, C.M. Sinton, et al., Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation, Cell 98 (1999) 437 – 451. [8] Y. Date, Y. Ueta, H. Yamashita, et al., Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 748 – 753. [9] L. de Lecea, T.S. Kilduff, C. Peyron, et al., The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 322 – 327. [10] E. Eggermann, M. Serafin, L. Bayer, et al., Orexins/orexins excite basal forebrain cholinergic neurons, Neuroscience 108 (2001) 177 – 181. [11] R.A. Espana, B.A. Baldo, A.E. Kelley, C.W. Berridge, Wakepromoting and sleep-suppressing actions of hypocretin (orexin): basal forebrain sites of action, Neuroscience 106 (2001) 699 – 715. [12] I.V. Estabrooke, M.T. McCarthy, E. Ko, et al., Fos expression in orexin neurons varies with behavioral state, J. Neurosci. 21 (2001) 1656 – 1662. [13] J.J. Hagan, R.A. Leslie, S. Patel, et al., Orexin A activates locus coeruleus cell firing and increases arousal in the rat, Proc. Natl. Acad. Sci. 96 (1999) 10911 – 10916. [14] J. Hara, C.T. Beuckmann, J. Nambu, et al., Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity, Neuron 30 (2001) 345 – 354. [15] K. Honda, Y. Komoda, S. Inoue´, Oxidized glutathione regulates physiological sleep in unrestrained rats, Brain Res. 636 (1994) 253 – 258. [16] T.S. Kilduff, C. Peyron, The hypocretin/orexin ligand – receptor system: implications for sleep and sleep disorders, Trends Neurosci. 23 (2000) 359 – 365. [17] L. Lin, J. Faraco, R. Li, et al., The sleep disorder canine narcolepsy is caused by a mutation in the Hypocretin (orexin) receptor 2 gene, Cell 98 (1999) 365 – 376. [18] M. Lubkin, A. Stricher-Krongrad, Independent feeding and metabolic actions of orexins in mice, Biochem. Biophys. Res. Commun. 253 (1998) 241 – 245.
[19] J.N. Marcus, C.J. Aschkenasi, C.E. Lee, et al., Differential expression of orexin receptors 1 and 2 in the rat brain, J. Comp. Neurol. 435 (2001) 6 – 25. [20] D.J. McGintty, M.B. Sterman, Sleep suppression after basal forebrain lesions in the cat, Science 160 (1968) 12253 – 12255. [21] E. Mignot, S. Nishino, L.H. Sharp, et al., Heterozygosity at the canarc-1 locus can confer susceptibility for narcolepsy: induction of catalexy in heterozygous asymptomatic dogs after administration of a combination of drugs acting on monoaminergic and cholinergic systems, J. Neurosci. 13 (1993) 1057 – 1064. [22] R.Y. Moore, E.A. Abrahamson, P.A. Van Den, The hypocretin neuron system: an arousal system in the human brain, Arch. Ital. Biol. 139 (2001) 195 – 205. [23] T. Nakamura, K. Uramura, T. Nambu, et al., Orexin-induced hyperlocomotion and stereotypy are mediated by the dopaminergic system, Brain Res. 873 (2000) 181 – 187. [24] T. Nambu, T. Sakurai, K. Mizukami, et al., Distribution of orexin neurons in the adult rat brain, Brain Res. 827 (1999) 243 – 260. [25] W.J.H. Nauta, Hypothalamic regulation of sleep in rats: an experimental study, J. Neurophysiol. 9 (1946) 285 – 316. [26] S. Nishino, E. Mignot, Pharmacological aspects of human and canine narcolepsy, Prog. Neurobiol. 52 (1997) 27 – 78. [27] S. Nishino, B. Ripley, S. Overeem, et al., Hypocretin (orexin) deficiency in human narcolepsy, Lancet 355 (2000) 39 – 40. [28] S. Nishino, N. Fujiki, Y. Yoshida, E. Mignot, The effects of hypocretin-1 (orexin A) in hypocretin receptor 2 gene mutated and hypocretin ligand-deficient narcoleptic dogs, Sleep 24 (2001) 287A. [29] C. Peyron, D.K. Tighe, P.A. Van den, et al., Neurons containing hypocretin (orexin) project to multiple neuronal systems, J. Neurosci. 18 (1998) 9996 – 10015. [30] C. Peyron, J. Faraco, W. Rogers, et al., A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains, Nat. Med. 6 (2000) 991 – 997. [31] S.W. Ranson, Somnolence caused by hypothalamic lesions in the monkey, Arch. Neurol. Psychiatry 41 (1939) 1 – 23. [32] T. Sakurai, A. Amemiya, M. Ishii, et al., Orexins and orexin receptors: a family of hypothalamic neuropeptides and G proteincoupled receptors that regulates feeding behavior, Cell 92 (1998) 573 – 578. [33] C.B. Saper, T.C. Chou, T.E. Scammell, The sleep switch: hypothalamic control of sleep and wakefulness, Trends Neurosci. 24 (2001) 726 – 731. [34] T. Scammell, I. Estabrooke, M. McCarthy, et al., Hypothalamic arousal regions are activated during modafinil-induced wakefulness, J. Neurosci. 20 (2000) 8620 – 8628. [35] J.E. Sherin, P.J. Shromani, R.W. McCarley, C.B. Saper, Activation of ventrolateral preoptic neurons during sleep, Science 271 (1996) 216 – 219. [36] J.M. Siegel, Narcolepsy: a key role for hypocretins (orexins), Cell 98 (4) (1999) 409 – 412. [37] F. Spadaro, C.W. Berridge, H.A. Baldwin, et al., Corticotropinreleasing factor acts via a third ventricle site to reduce exploratory behavior in rats, Pharmacol. Biochem. Behav. 36 (1990) 305 – 309. [38] T.L. Steininger, H. Gong, D. McGintty, et al., Subregional organization of preoptic area and anterior hypothalamic projections to arousal-related monoaminergic cell groups, J. Comp. Neurol. 429 (2001) 638 – 653. [39] C. Swett, J. Hobson, The effects of posterior hypothalamic lesions on behavioural and electrographic manifestations of sleep and waking in cats, Arch. Ital. Biol. 106 (1968) 270 – 282. [40] S. Taheri, S. Bloom, Orexin/hypocretins: waking up the scientific world, Clin. Endocrinol. (Oxf.) 54 (2001) 421 – 429. [41] T. Thannickal, R.Y. Moore, R. Nienhuis, et al., Reduced number of hypocretin neurons in human narcolepsy, Neuron 27 (2000) 469 – 474. [42] P. Trivedi, H. Yu, D.J. MacNeil, et al., Distribution of orexin receptor mRNA in the rat brain, FEBS Lett. 438 (1998) 71 – 75.
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syndromes in orexin receptor-2 and orexin null mice: molecular genetic dissection of non-REM and REM sleep regulatory processes, Neuron 38 (2003) 715 – 730. [46] A. Yamanaka, N. Tsujino, H. Funahashi, et al., Orexins activate histaminergic neurons via the orexin 2 receptor, Biochem. Biophys. Res. Commun. 290 (2002) 1237 – 1245.
Research report
Selective stimulation of orexin receptor type 2 promotes wakefulness in freely behaving rats Moses A. Akanmu1, Kazuki Honda* Department of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Accepted 22 April 2005 Available online 24 May 2005
Abstract Orexins A and B are a pair of neuropeptides implicated in the regulation of feeding and arousal behavior mediated through two orexin receptors type 1 and type 2. We have determined the arousal effects of newly developed selective orexin receptor type 2 agonist, [Ala11]orexin-B, on the sleep – wake cycle in rats. The effects of third ventricle intracerebroventricular (ICV) infusion of the novel orexin receptor type 2 selective agonist, [Ala11]orexin-B, on the sleep – wake cycle were investigated. ICV infusion of [Ala11]orexin-B (1, 10 and 40 nmol) during the light period (11:00 – 16:00) dose-dependently resulted in a significant increase in wake duration by 46.9% (n = 5, P < 0.05), 159.2% (n = 4, P < 0.01) and 163.6% (n = 7, P < 0.01)), respectively, and a significant decrease in rapid eye movement (REM) (P < 0.01) and non-REM sleep (P < 0.01) for all doses. In contrast, ICV infusion of orexin B at the same doses (1, 10 and 40 nmol) caused a 16.6% (n = 6, non-significant), 99.8% (n = 6, P < 0.05) and 72.0% (n = 4, P < 0.05) increase in wakefulness, respectively. Moreover, orexin-A, which exerts its effects through orexin receptor type 1 and orexin receptor type 2 with similar potency, resulted in a significant increase in wakefulness duration by 17.1% (n = 6, P < 0.05), 184.0% (n = 6, P < 0.01) and 228.6% (n = 6, P < 0.01) at doses of 0.1, 1 and 10 nmol, respectively. Further, the enhancement of wakefulness was accompanied by a marked reduction in REM and nonREM sleep. These findings suggest that orexin receptor type 2 plays an important role in the modulation of sleep – wake state and behavioral responses. D 2005 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Orexin; Orexin-B agonist; Behavior; Non-REM sleep; REM sleep; Wakefulness
1. Introduction
Abbreviations: ICV, intracerebroventricular; REM, rapid eye movement; non-REM, non-rapid eye movement; LHA, lateral hypothalamic area; OX1R, orexin receptor type 1; OX2R, orexin receptor type 2; LC, locus coeruleus; VTA, ventral tegmental area; DRN, dorsal raphe´ nucleus; LDT, laterodorsal tegmental nuclei; PPT, pedunculopontine tegmental nuclei; TMN, tuberomammilary nucleus; GABA, gamma amino butyric acid; 5HT, 5-hydroxyltryptamine; VLPO, ventrolateral preoptic nucleus; EEG, electroencephalogram; EMG, electromyogram; CSF, cerebrospinal fluid * Corresponding author. Fax: +81 3 5280 8098. E-mail address: [email protected] (K. Honda). 1 Present address: Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria. 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.04.064
Orexins, also called hypocretins, are a pair of neuropeptides expressed by a specific population of neurons in the lateral hypothalamic area (LHA), a region of the brain implicated in feeding and arousal [7,9,32,44]. Orexin-A (hypocretin-1) and orexin-B (hypocretin-2) are derived from a common precursor peptide [8,32]. The actions of orexins are mediated by two G protein-coupled receptors termed orexin receptor type 1 (OX1R) and orexin receptor type 2 (OX2R) [32]. OX1R is expressed in the locus coeruleus (LC), ventral tegmental area (VTA), dorsal raphe nucleus (DRN), laterodorsal tegmental nuclei (LDT),
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pedunculopontine tegmental nuclei (PPT) and hypothalamus, while OX2R is expressed in the tuberomammilary nucleus (TMN), VTA, DRN, LDT, PPT and hypothalamus [19,42]. Orexin-containing neurons project from the LHA to numerous brain regions, with the limbic system, hypothalamus, monoaminergic LC, dopaminergic VTA, serotonergic DRN, cholinergic nuclei of brainstem and histaminergic TMN receiving particularly strong innervation [8,24,29]. Amygdala is highly interconnected with a number of brain regions involved in sleep regulation, such as the basal forebrain, hypothalamus and brainstem [2] and also connected with the parabrachial region, DRN, PPT and LDT. Bilateral lesions of the amygdala produced substantial effects on sleep in monkeys [4]. Moreover, lesions of the posterior lateral hypothalamus where the tuberomammillary neurons reside and rostral midbrain in the brains of monkeys, rats and cats caused sleepiness while the lesions of lateral preoptic and basal forebrain in rats and cats caused sleep suppression [20,25,31,39,43]. Interestingly, these terminal areas such as the posterior lateral hypothalamic and preoptic and basal forebrain are involved in the homeostatic and circadian control of sleep [5,33]. Furthermore, the sleep – wake mechanism involves a positive neuronal feedback mechanism in which orexin excites other arousal systems, including serotonin (5-hydroxyltryptamine; 5HT) and noradrenaline. This ascending arousal system sends projection from the brainstem and posterior hypothalamus throughout the forebrain. Orexin neurons in the lateral hypothalamic area innervate all of the components of ascending arousal system and may stabilize behavioral state by increasing the activity of aminergic neurons, thus maintaining consistent inhibition of sleep-promoting neurons in the ventrolateral preoptic nucleus (VLPO) and rapid eye movement (REM)-promoting neurons in the PPT-LDT [6,10,35,38]. Indeed, central orexin administration in rodents results in increases in sympathetic tone, plasma corticosterone levels [13], metabolic rate [18], food intake [32], locomotor activity [23] and wakefulness [13]. Further, mice lacking either the orexin gene (prepro-orexin knockout mice) or orexin neurons (orexin/ataxin-3 transgenic mice), as well as mice and dogs with null mutations in the orexin receptor type 2 gene, all have phenotypes remarkably similar to those of the human sleep disorder, narcolepsy [7,14,17,44,45], and recent reports suggest that human narcolepsy is accompanied by a specific destruction of orexin neurons in the hypothalamus [30,41]. While these studies suggest that orexin stabilizes prolonged periods of wakefulness by opposing homeostatic sleep propensity [33], the differential roles of the two orexin receptors remain unclear. Thus, the goal of the present study was to determine the effects of intracerebroventricular administration of the OX2R-selective agonist, [Ala11]orexin-B, on the sleep – wake cycle in rats.
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2. Materials and methods 2.1. Animal usage Sixteen to eighteen male Sprague – Dawley rats (330 – 360 g) were obtained from Crea Japan, Inc. (Tokyo, Japan) and were housed in cages for one week. The room was maintained at a temperature of 25 T 1 -C, a relative humidity of 54 T 6%, and a light–dark cycle of 12:12 h (lights on 8:00 h). All experimental protocols were performed in accordance with the Guidelines for Animal Experimentation of Tokyo Medical and Dental University. 2.2. Preparation of [Ala11]orexin-B and orexins A- and -B The OX2R selective agonist, [Ala11]orexin-B, was obtained from Banyu pharmaceutical Co. Ltd., Tsukuba, and orexin-A and orexin-B were purchased from Peptide institute, Inc, Osaka. The peptides were dissolved in normal saline, and either peptides or vehicle were continuously infused intracerebroventricularly (icv) at a rate of 10 Al/h for the sleep –wake analysis. 2.3. Electroencephalogram (EEG) and electromyogram (EMG) Simultaneous EEG and EMG recordings were performed as described previously [15]. Briefly, 60- to 70-day-old male Sprague – Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and fixed on a stereotaxic apparatus. A stainless steel cannula (0.35 mm o.d., inclined at 20- from the vertical line, 2 mm posterior, 3.4 mm lateral to the bregma, 8.5 mm depth from the surface of cortex) was implanted in the third cerebral ventricle for continuous icv infusion. Three cortical gold-plated screw electrodes and paired stainless-steel electrodes were fixed to the skull with dental acrylic resin for recording of the EEG and EMG, respectively, and the cable from the electrodes was attached to a socket. After implantation of the cannula and attachment of the electrodes, animals were administered penicillin G potassium (20,000 U, subcutaneously) and were allowed to recover for 10 days. Following the recovery period, animals were transferred to an individual experimental cage that allowed continuous icv infusion and monitoring of EEG and EMG. The experimental cages were placed in a soundproof, electromagnetically shielded room with the same environmental conditions described above. Polyethylene tubing (PE10, 0.28 mm, i.d.) was used to connect the cannula to an infusion pump (Eicom, ESP-32, Kyoto), which was set for continuous infusion of normal saline with or without the experimental peptides at a rate of 10 Al/h. During the 7 days preceding the experiment, animals underwent daily continuous infusion of normal saline in the experimental chamber to acclimatize the animals to the infusion and recording conditions. For measurements, lead wires of the
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electrodes were connected to an EEG/EMG amplifier (Nihon-Kohden, MEG-6116, Tokyo) through a 5-strand cable with a slip-ring that allowed free movement of the rats. The amplifier was connected to a personal computer with an AD converter and software (Kissei Comtec, SleepSign, Nagano) for acquiring and processing data. Data were sampled at 128 Hz and subjected to on-line spectral analysis by Fast Fourier Transformation over 8-s epochs. Data were stored on a magnetic optical (MO) disk that was subsequently visually analyzed offline after autoanalysis of the sleep – wake stages. EEG and EMG were measured, starting at lights on, for two consecutive days: a baseline day and experimental day. Animals received either [Ala11]orexin-B or orexin-B at doses of 1, 10, 40 nmol or orexin A at doses of 0.1, 1 and 10 nmol for a 5-h period (11:00 – 16:00) in random order. During the resting phase, all animals received icv infusion of saline without experimental peptides. Sleep –waking state was classified as wakefulness (W), rapid eye movement (REM) sleep and non-REM sleep from the EEG and EMG recordings, and the results were verified visually according to the standard criteria [1,15]. Discrimination of wakefulness (high EMG amplitude, low EEG amplitude), non-REM sleep (low EMG amplitude, high EEG amplitude with high power density in the delta band (0.5 –4.0 Hz)) and rapid-eye-movement (REM) sleep (silent low EMG amplitude, low EEG amplitude with high values in the theta band (4.0 – 8.0 Hz)) was performed. The analyzed sleep variables included the amount of non-REM and REM sleep, and number and duration of each sleep parameter episode. The duration of wakefulness, non-REM sleep and REM sleep was determined for each hour in the 24-h recording. 2.4. Statistical analysis Statistical analyses were performed using repeated measures analyses of variance (ANOVA) followed by post hoc analysis of significance with the Student – Newman – Keuls test. Probability (P) values less than 0.05 were considered to indicate statistical significance.
3. Results Vigilance states and EEG of rats were characterized at baseline and during icv administration of [Ala11]orexin-B, orexin-B and orexin-A. Daily icv infusion (11:00 – 16:00 h) of [Ala11]orexin-B at doses of 1, 10 and 40 nmol resulted in a significant and dose-dependent increase in the duration of wakefulness (Table 1), primarily due to a marked increase in episode duration for all three doses, with significant increases at doses of 10 nmol (P < 0.05) and 40 nmol (P < 0.01). Icv administration of [Ala11]orexin-B also resulted in a significant and dose-dependent decrease in REM sleep (P < 0.01), primarily via a decrease in the episode frequency at doses of 10 and 40 nmol. Further, non-REM sleep was significantly (P < 0.01) reduced at doses of 10 and 40 nmol of [Ala11]orexin-B. Compared with baseline, 40 nmol of [Ala11]orexin-B significantly (P < 0.01, n = 7) reduced REM and non-REM sleep episode frequency and duration and increased wakefulness episode duration (Table 1). When analyzing the hourly subsets of the infusion period, 10 nmol of [Ala11]orexin-B produced a four-fold increase in wakefulness during the first hour of infusion (Fig. 1), and this arousal effect was maintained during the first three hours of the infusion period. Further, the arousal effect of [Ala11]orexinB resolved immediately after completion of the infusion, indicating that the effect of [Ala11]orexin-B on arousal is transient. All three doses of [Ala11]orexin-B significantly increased wakefulness and suppressed REM sleep (Fig. 2). In contrast, 10 nmol of orexin-A produced a three-fold increase in wakefulness during the first hour of infusion and a five-fold increase in wakefulness during the second hour of infusion. The increase was maintained at the three-fold level throughout the remainder of the infusion period (Fig. 3). ICV administration of orexin-A also significantly suppressed REM and non-REM sleep, with a more profound effect on REM sleep. REM sleep was suppressed throughout the infusion period and was completely suppressed by the second hour of infusion. In contrast, non-REM sleep was significantly suppressed only during the first two hours of drug infusion. Orexin-A administration also resulted in a significant increased in wake duration by 17.1%, 184.0%, 228.6%,
Table 1 Effects of diurnal (11:00 – 16:00) icv infusion of [Ala11]orexin-B on each sleep parameters in freely behaving rats (mean T SEM) 1 nmol (n = 5) Baseline Wakefulness
REM
Non-REM
Frequency Duration (s) Total time (min) Frequency Duration (s) Total time (min) Frequency Duration (s) Total time (min)
60.2 62.2 59.6 26.8 103.6 42.5 61.8 196.4 193.2
T T T T T T T T T
10 nmol (n = 4) Experiment
7.4 6.9 4.2 5.0 11.6 2.2 6.5 22.2 6.0
71.8 76.4 88.2 12.6 103.6 21.8 73.2 162.4 185.5
T T T T T T T T T
9.1 9.2 9.5# 1.6 9.6 3.2# 9.0 22.8 8.7
Baseline 68.8 53.8 61.7 29.0 96.0 45.2 69.8 166.0 189.9
T T T T T T T T T
4.9 5.0 7.3 4.0 6.4 4.0 4.2 15.5 7.0
40 nmol (n = 7) Experiment
Baseline
64.0 T 3.7 141.5 T 22.2* 147.2 T 15.2* 5.8 T 2.5# 85.0 T 22.6 8.2 T 4.1# 64.5 T 3.4 118.3 T 5.6* 126.8 T 8.0#
70.0 73.3 84.2 20.1 104.4 35.1 72.0 153.7 178.4
T 5.3 T 6.1 T 6.9 T 1.2 T 6.9 T 3.4 T 5.0 T 13.1 T 5.1
Experiment 46.1 298.9 207.4 3.1 75.4 5.6 46.6 100.4 74.5
T T T T T T T T T
5.0# 47.4# 11.3# 1.3# 24.4 2.6# 5.1# 11.4* 7.5#
* P < 0.05 vs. Baseline (Vehicle-treated); # P < 0.01 vs. Baseline (Vehicle-treated); ICV, intracerebroventricular; REM, rapid eye movement; Non-REM, nonrapid eye movement.
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Fig. 1. Effects of 10 nmol [Ala11]orexin-B infusion into the third ventricle on total time spent in rapid eye movement (REM) and non-REM sleep. Vertical lines on each hourly value stand for SEM. Baseline: closed circle and [Ala11]orexin-B: open circle. *P < 0.05; #P < 0.01 compared to baseline (vehicle-treated) rats (n = 4).
respectively; reduced REM sleep by 27.5%, 75.7%, 96.2%, respectively; and reduced NREM sleep by 5.0%, 49.2% and 88.0%, respectively (Fig. 4) at doses of 0.1 nmol (P < 0.05), 1 nmol (P < 0.01) and 10 nmol (P < 0.01).
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Fig. 3. Effects of 10 nmol orexin-A infusion into the third ventricle on total time spent in REM and non-REM sleep. Vertical lines on each hourly value stand for SEM. Baseline: closed circle, and Orexin-A: open circle. *P < 0.05; #P < 0.01 compared to baseline (vehicle-treated) rats (n = 6).
ICV infusion of orexin-B also significant increases in wakefulness during the infusion period (Fig. 5). Orexin-B increased the total amount of wakefulness by 16.6%, 99.8%, 72.0%, respectively; reduced REM sleep by 14.0%, 64.2% and 74.5%, respectively; and reduced non-REM sleep by
Fig. 2. Total time spent in wake, REM and non-REM sleep during the 5-h infusion of [Ala11]orexin-B into the third ventricle of rats. Line and deep-shaded columns represent baseline and experimental day profiles, respectively. Values are mean T SEM (n = 5, 4, and 7 for 1, 10 and 40 nmol, respectively). *P < 0.05, # P < 0.01 compared to baseline (vehicle-treated) rats.
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Fig. 4. Total time spent in wake, REM and non-REM-sleep during the 5-h infusion of different doses of orexin-A (1, 10 and 40 nmol) into the third ventricle of rats. Line and deep-shaded columns represent baseline and experimental day profiles, respectively. Values are mean T SEM (n = 6). *P < 0.05, #P < 0.01 compared to baseline (vehicle-treated) rats.
0%, 34.1% and 36.9%, respectively, at doses of 1, 10 and 40 nmol (Fig. 6). The magnitude of orexin-B-induced increases in wakefulness and suppression of REM and nonREM was substantially less than that observed following icv infusions of [Ala11]orexin-B at the same dose levels. The results also showed that there was REM sleep rebound effect after the cessation of infusion of orexins during the dark period. This effect may be due to compensatory behavioral response to earlier REM sleep suppression effects observed with infusion of the orexins administered during the day or due to unknown mechanism.
showing a 10-fold higher affinity at OX2R than at OX1R [32], while [Ala11]orexin-B showed a 120-fold selectivity for OX2R over OX1R [3]. Orexin increases arousal via its action on basal forebrain areas, which are comprised of a number of distinct regions. A relatively high density of
4. Discussion In this study, following [Ala11]orexin-B, orexin-A and orexin-B icv administration, there were significant changes in the amount of wake, NREM and REM sleep as shown in Figs. 1, 3 and 5, respectively. Compared to baseline, during the first 1 h of ICV administration, [Ala11]orexin B, orexinA significantly (P < 0.01) increased wakefulness immediately to 413.6% and 153.4%, respectively, while orexin-B only had an increase of 32.6% above the baseline value when the percentage change was analyzed indicating the importance of OX2R in sleep – wake regulation. However, orexin-B increased wakefulness to 258.1% by the second hour of infusion. This further suggests a possible early OX2R stimulation in the orexin– ligand –receptor system in sleep – wake regulation. Indeed, 10 nmol [Ala11]orexin-B produced an immediate increase in wakefulness and caused total REM sleep suppression by the second hour of infusion (Fig. 1). [Ala11] orexin-B appears more potent than orexin-B and equally as potent as orexin-A at inducing waking and decreasing REM sleep. This is consistent with reports that [Ala11]orexin-B has a 120-fold selectivity for OX2R over OX1R [3]. Alternatively, [Ala11]orexin-B may be more metabolically stable than orexin-B. Orexin-A has a high affinity for both receptors (OX1R and OX2R) with equal potency, whereas orexin-B acts more selectively on OX2R
Fig. 5. Effects of 10 nmol orexin-B infusion into the third ventricle on total time spent in rapid eye movement (REM) and non-REM sleep. Vertical lines on each hourly value stand for SEM. Baseline: closed circle and orexin-B: open circle. *P < 0.05; #P < 0.01 compared to baseline (vehicletreated) rats (n = 6).
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Fig. 6. Total time spent in wake, REM and non-REM sleep during the 5-h infusion of orexin-B into the third ventricle of rats. Line and deep shaded columns represent baseline and experimental day profiles, respectively. Values are mean T SEM (n = 6, 6, and 4 for 1, 10 and 40 nmol, respectively). *P < 0.05, #P < 0.01 compared to baseline (vehicle-treated) rats.
orexin fibers have been observed in close proximity to the lateral and third ventricles [11], including the organum vasculosum laminae terminalis, a site that is particularly sensitive to the behavioral actions of various peptides [37]. Thus, ventricular orexin infusion may act on these particular areas to induce waking behavioral effects. Orexin also acts on multiple brainstem systems to modulate behavioral state [36]. The influence of orexins on arousal state has been suggested by experiments that demonstrated that canine narcolepsy results from a mutation in the OX2R gene [17] or a deficiency of orexin [7]. Similarly, brains from human narcoleptics exhibit a nearly complete loss of neurons expressing orexin mRNA or protein [30,41], and orexin is below the detectable limits in the cerebrospinal fluid of humans with narcolepsy [27,30]. Orexin neurons heavily innervate aminergic nuclei that promote wakefulness and inhibit REM sleep. Moreover, previous work suggested that activation of orexin neurons may contribute to the promotion or maintenance of wakefulness [12]. Indeed, canine narcolepsy is associated with hyperactivity of cholinergic neurotransmission and decreased monoaminergic tone, which would produce daytime sleepiness and REM abnormalities [21,26]. Further, wakefulness produced by modafinil, a drug used to treat the excessive daytime sleepiness of narcolepsy, is associated with an increase in orexin neuron activity in rodents, as indicated by increased expression of the transcription factor, Fos [7,34]. The importance of OX2R in the sleep – wakefulness regulation was demonstrated by the finding that narcoleptic dogs carry mutations in the OX2R gene [17]. Further, icv administration of orexin-A significantly increased wakefulness in control dogs but not in narcoleptic dogs [28]. Indeed, recent reports have demonstrated that increased orexinergic tone induces wakefulness, while reduced orexinergic tone resulting from knockouts of the prepro-orexin gene or the OX2R gene decreases wakefulness and increases premature entry into REM sleep. Homozygous knockout mice showed frequent narcoleptic/cataplectic attacks, significant disruptions of sleep –wakefulness cycling during the dark phase, frequent direct transitions into REM sleep from wakeful-
ness, marked fragmentation of waking, decreased waking time, increased non-REM and REM sleep time, decreased REM sleep latency, and a decreased duration of waking episodes during the dark phase [7,8]. OX2R knockout mice have also been reported to show characteristics of narcolepsy, albeit with an EEG and behavioral phenotype that is less severe than that found in the orexin knockout mouse, while OX1R knockouts only showed increased fragmentation of sleep –wakefulness cycles [7,16,22,30,40,44]. More importantly, OX2R activation is essential for the normal regulation of wake/non-REM sleep transitions, and the profound dysregulation of REM sleep control unique to the narcolepsy – cataplexy syndrome results from loss of signaling through both OX2R-dependent and OX2R-independent pathways [45]. Therefore, phenotypic differences between OX1R knockout mice and OX2R knockout mice suggest that lack of an orexin signal via OX2R contribute to the pathogenesis of narcolepsy. This is consistent with observations from the present study that icv administration of [Ala11]orexin-B resulted in a potent, rapid and dosedependent wake-promoting effect. Further, a recent study reported the existence of a direct synaptic connection between orexin neurons and histaminergic neurons in the TMN and demonstrated that orexins increased firing rates of isolated histaminergic neurons via OX2R [46]. Thus, the orexin –histamine pathway may mediate the effects elicited by [Ala11]orexin-B.
5. Conclusions Daily icv infusion of the novel orexin-2 receptor selective agonist, [Ala11]orexin-B, into the third ventricle induced a significant arousal effect in freely behaving rats, and the efficacy of [Ala11]orexin-B was comparable to that of native peptides at the same dose. These results indicate that [Ala11]orexin-B dose-dependently increased wakefulness and reduced REM and non-REM sleep and suggest that [Ala11]orexin-B plays an important role in the modulation of sleep – wake state. Exogenous administration of the OX2R
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