Selective stimulation of orexin receptor type 2 promotes wakefulness in freely behaving rats

doi:10.1016/j.brainres.2005.04.064

Brain Research

Volume 1048, Issues 1–2, 28 June 2005, Pages 138-145

https://doi.org/10.1016/j.brainres.2005.04.064 Get rights and content

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 non-REM sleep. These findings suggest that orexin receptor type 2 plays an important role in the modulation of sleep–wake state and behavioral responses.

Introduction

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), 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.

Section snippets

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 ± 1 °C, a relative humidity of 54 ± 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.

Preparation of [Ala¹¹]orexin-B and orexins A- and -B

The OX₂R selective agonist, [Ala¹¹]orexin-B, was obtained from Banyu

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

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 Fig. 1, Fig. 3, Fig. 5, respectively. Compared to baseline, during the first 1 h of ICV administration, [Ala¹¹]orexin B, orexin-A 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

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

Acknowledgment

This study was supported by a Grant-in-Aid for Scientific Research (C) (No. 14570912, 16614003) to K.H.

References (46)

S. Asahi et al.
Development of an Orexin-2 receptor selective agonist, [Ala¹¹, D-Leu¹⁵] Orexin-B
Bioorg. Med. Chem. Lett.
(2003)
R.M. Benca et al.
Effects of amygdala lesions on sleep in rhesus monkeys
Brain Res.
(2000)
R.E. Brown et al.
Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat
Neuropharmacology
(2001)
R.M. Chemelli et al.
Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation
Cell
(1999)
E. Eggermann et al.
Orexins/orexins excite basal forebrain cholinergic neurons
Neuroscience
(2001)
R.A. Espana et al.
Wake-promoting and sleep-suppressing actions of hypocretin (orexin): basal forebrain sites of action
Neuroscience
(2001)
J. Hara et al.
Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity
Neuron
(2001)
K. Honda et al.
Oxidized glutathione regulates physiological sleep in unrestrained rats
Brain Res.
(1994)
T.S. Kilduff et al.
The hypocretin/orexin ligand–receptor system: implications for sleep and sleep disorders
Trends Neurosci.
(2000)
L. Lin et al.
The sleep disorder canine narcolepsy is caused by a mutation in the Hypocretin (orexin) receptor 2 gene
Cell
(1999)

M. Lubkin et al.

Independent feeding and metabolic actions of orexins in mice

Biochem. Biophys. Res. Commun.

(1998)

T. Nakamura et al.

Orexin-induced hyperlocomotion and stereotypy are mediated by the dopaminergic system

Brain Res.

(2000)

T. Nambu et al.

Distribution of orexin neurons in the adult rat brain

Brain Res.

(1999)

S. Nishino et al.

Pharmacological aspects of human and canine narcolepsy

Prog. Neurobiol.

(1997)

S. Nishino et al.

Hypocretin (orexin) deficiency in human narcolepsy

Lancet

(2000)

C.B. Saper et al.

The sleep switch: hypothalamic control of sleep and wakefulness

Trends Neurosci.

(2001)

J.M. Siegel

Narcolepsy: a key role for hypocretins (orexins)

Cell

(1999)

F. Spadaro et al.

Corticotropin-releasing factor acts via a third ventricle site to reduce exploratory behavior in rats

Pharmacol. Biochem. Behav.

(1990)

T. Thannickal et al.

Reduced number of hypocretin neurons in human narcolepsy

Neuron

(2000)

P. Trivedi et al.

Distribution of orexin receptor mRNA in the rat brain

FEBS Lett.

(1998)

J.T. Willie et al.

Distinct narcolepsy syndromes in orexin receptor-2 and orexin null mice: molecular genetic dissection of non-REM and REM sleep regulatory processes

Neuron

(2003)

A. Yamanaka et al.

Orexins activate histaminergic neurons via the orexin 2 receptor

Biochem. Biophys. Res. Commun.

(2002)

M.A. Akanmu et al.

Hypnotic effects of total aqueous extracts of Vervain hastata (Verbenaceae) in rats

Psych. and Clin. Neurosci.

(2002)

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¹: Present address: Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.

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Research reportSelective stimulation of orexin receptor type 2 promotes wakefulness in freely behaving rats

Abstract

Introduction

Section snippets

Animal usage

Preparation of [Ala11]orexin-B and orexins A- and -B

Results

Discussion

Conclusions

Acknowledgment

Bioorg. Med. Chem. Lett.

Brain Res.

Neuropharmacology

Cell

Neuroscience

Neuroscience

Neuron

Brain Res.

Trends Neurosci.

Cell

Biochem. Biophys. Res. Commun.

Brain Res.

Brain Res.

Prog. Neurobiol.

Lancet

Trends Neurosci.

Cell

Pharmacol. Biochem. Behav.

Neuron

FEBS Lett.

Neuron

Biochem. Biophys. Res. Commun.

Hypnotic effects of total aqueous extracts of Vervain hastata (Verbenaceae) in rats

Psych. and Clin. Neurosci.

Research report
Selective stimulation of orexin receptor type 2 promotes wakefulness in freely behaving rats

Preparation of [Ala¹¹]orexin-B and orexins A- and -B