Orexin is a neuropeptide that plays a highly important role in mechanisms that regulate sleep/wake states. Lack of the orexin gene or orexin-producing neurons (orexin neurons) results in narcolepsy in several mammalian species, suggesting that orexin is an important factor for the maintenance of wakefulness. Constitutive, ectopic expression of orexin in transgenic mice resulted in severe fragmentation of non–rapid eye movement sleep, along with abnormal muscle tone regulation during REM sleep, suggesting that activity of orexin neurons should be appropriately decreased during sleep to maintain consolidated sleep states. This review will discuss the mechanisms by which the orexin system is regulated during sleep.
Sleep–wake regulation is achieved through reciprocal interplay of wakefulness-promoting and sleep-promoting regions in the brain. Included in the wake-promoting system are cholinergic, histaminergic (HIST), setoronergic (5-HT) and noradrenergic (NA) neurons that are located in the posterior hypothalamus/brain stem regions and diffusely innervate the forebrain to regulate cortical function. Conversely, the ventrolateral preoptic neurons (VLPO) in the anterior hypothalamus is shown to play a critical role in sleep initiation and maintenance. Neurons in the VLPO that carry GABA and galanin as inhibitory neurotransmitters play important roles in this function. These neurons send inhibitory projections to neurons that release the aforementioned wake-promoting neurotransmitters. Some neurons in the VLPO fire at a rapid rate during sleep with attenuation of firing during wakefulness. Conversely, brain stem wake-promoting neurons fire rapidly during wakefulness and are relatively quiescent during sleep, with the exception of the cholinergic neurons, which are firing rapidly during both wakefulness and rapid eye movement (REM) sleep.
Recently, evidence from multiple sources has clearly shown that members of a novel neuropeptide family, orexin-A and orexin-B, are critically involved in the neuronal circuitry regulating sleep/wake states. The finding that orexin deficiency causes narcolepsy in humans and animals suggests that these hypothalamic neuropeptides play a critical role in maintenance of wakefulness.1–5 Recent studies have suggested further roles for orexin in the coordination of emotion, energy homeostasis, reward, drug addiction and arousal.6–13 Orexin neurons have been shown to have interaction with various brain regions regulating such functions (Fig. 1). Orexin neurons receive abundant input from the limbic system,10,11 which might be important for increasing arousal during emotional stimuli. In addition, they are regulated by peripheral metabolic cues, including ghrelin, leptin and glucose, suggesting that orexin neurons might provide a link between energy homeostasis and vigilance states.7 Together, these observations suggest that orexin neurons are involved in sensing the body's external and internal environments, and regulate states of sleep and wakefulness accordingly, which is beneficial for survival.14 Among these various inputs, this review focuses on the interaction between sleep-promoting centers and orexin neurons, and discusses the mechanisms that maintain sleep states. Please see our recent reviews for further references regarding other aspects of orexin functions.14,15
OREXINS AS WAKE-PROMOTING SUBSTANCES
Narcolepsy is a sleep disorder characterized by intolerable daytime sleepiness and outbreak of sudden REM sleep. An increasing body of evidence suggests that this disorder is associated with a postnatal loss of orexin neurons.14 In most people with narcolepsy, orexin levels in the cerebrospinal fluid (CSF) are very low or undetectable.16
Orexin neurons are almost exclusively localized in the lateral hypothalamic area (LHA) and posterior hypothalamus (PH).17–19 From these regions, orexin neurons send axonal projections widely to the entire neuroaxis except the cerebellum.17–19 Particularly dense staining of orexin-immunoreactive nerve endings in the brain is found in the paraventricular nucleus of the thalamus, the arcuate nucleus (Arc), and, most notably, the locus coeruleus (LC, containing NA neurons), the dorsal raphe (DR, containing 5-HT neurons) and the tuberomammillary nucleus (TMN, containing HIST neurons).5,17,18 The distribution of the orexin receptors mRNA is consistent with these projection sites; within the brain, OX1R is most abundantly expressed in the LC, while OX2R is highly expressed in the TMN.20 The DR, laterodorsal tegmental nucleus (LDT), pedunculopontine tegmental nucleus (PPT) and ventral tegmental area (VTA) contain both OX1R and OX2R.20 These observations suggest that these monoaminergic/cholinergic regions are major effector sites of orexins. In fact, electrophysiological experiments using brain-slice preparations or isolated cells have shown that cells of these nuclei are potently activated by orexins in vitro.
SLEEP AND OREXIN NEURONAL ACTIVITY
We previously analysed transgenic mice with constitutive activation of orexinergic tone (CAG/orexin mice), in which orexin is expressed in a diffuse, ectopic pattern in the brain in unregulated fashion.21 The mice exhibited abnormal sleep and wakefulness patterns, including fragmented non–rapid eye movement (NREM) sleep in the light period and incomplete REM sleep atonia with abnormal myoclonic activity during REM sleep (Willie et al., in press). These results suggest that orexin neurons need to be switched off to maintain consolidated NREM sleep and the muscle atonia that accompanies REM sleep.
Consistent with this idea, Fos expression (a marker of neuronal activity) in orexin neurons in rats is increased during the dark, active period in which the awake state is dominant, while it is decreased during the light, rest period in which the sleep state is dominant.22 Moreover, orexin levels in cerebrospinal fluid (CSF) peak during the dark period and decrease during the light period.23 Recent in vivo recording studies using rats and mice further revealed that the orexin neurons fired during active waking, decreased discharge during quiet waking, and virtually ceased firing during both REM and NREM sleep.24–26 Orexin neurons increased firing before the end of REM sleep and thereby heralded over several seconds the return of the awake state. These studies provide evidence that these cells are activated during wakefulness, and inhibited during sleep. Collectively, orexin neurons, activated during wakefulness through various exciting inputs, exert an excitatory influence on wake-active neurons, thus sustaining their activity, while these neurons are inhibited during sleep.
SLEEP SUBSTANCES, SLEEP -PROMOTING MECHANISMS AND OREXIN NEURONS
These observations suggest that orexin neurons are inhibited during sleep. What mechanism inhibits orexin neurons? Evidence suggests that sleep-active neurons in the preoptic area (POA) might play a role in this inhibition. The hypnogenic function of the rostral hypothalamic region, particularly the VLPO, is well established on the basis of lesioning, neuronal unit recording, and neurochemical and thermal stimulation studies.27 The sleep-active GABAergic neurons in the VLPO have been shown to play a crucial role in the induction and maintenance of sleep by inhibiting monoaminergic neurons. Reciprocal interactions between VLPO and wake-promoting neurons are apparently important in sleep–wake and wake–sleep transitions. The putative sleep factor, adenosine (ADE), was found to excite sleep-active neurons in the VLPO.28–30 The hypnogenic properties of ADE were first recognized as sleep-like behavior induced by intracerebroventricular injection of ADE. It is considered that the adenosine receptors, A1 and A2A, are involved in sleep induction by adenosine.31 Adenosine analogs or inhibitors of its metabolism increase sleep, especially NREM sleep in the rodents.32 Caffeine, a widely used psychoactive stimulant, is an ADE receptor antagonist.
In the VLPO, Fos was expressed in VLPO neurons after subarachnoid infusion of A2A R agonist,33 while GABA release was increased in the histaminergic TMN, indicating that A2A R activation can cause GABAergic inhibition to wakefulness-promoting regions.34
Neuronal recording studies also found that there are many sleep-active neurons in the lateral preoptic area (LPO) and medial preoptic area (MPO).35,36 These sleep-active neurons contain GABA as the inhibitory neurotransmitter. GABA potently and directly inhibits the activity of orexin neurons.7,37,38 This suggests a possibility that sleep-active neurons in the POA send inhibitory GABAergic projections to orexin neurons, and cease firing of these cells during sleep.
Consistently, a recent report showed that microdialysis-perfusion of the POA area with a GABA agonist, muscimol, which decreased the sleep period of rats to less than 3% of the baseline value over a 90-min period, induced expression of Fos in 36% of orexin neurons, suggesting that subpopulations of the preoptic neurons send a tonic inhibitory input to orexin neurons.39 Furthermore, recent histological studies showed that neurons in the POA send innervations to orexin neurons.10,11 These observations suggest that the activity of orexin neurons is negatively regulated by GABAergic neurons in the POA sleep-active neurons. Consistent with this, a more recent study showed that selective deletion of GABAB1 receptor gene in orexin neurons resulted in severe fragmentation of NREM sleep, further suggesting the importance of GABA-mediated inhibition of orexin neurons during sleep.40
The sleep-active GABAergic neurons in the VLPO are thought to play a crucial role in the initiation and maintenance of sleep by inhibiting wake-promoting monoaminergic neurons. Also, wake-promoting substances like acetylcholine, noradrenaline and serotonin inhibit the VLPO, although it is insensitive to histamine. This reciprocal inhibitory mechanism seems to play an important role in sleep–wake and wake–sleep transitions. Orexin neurons exert strong excitatory influence on the wake-promoting monoaminergic neurons, while monoaminergic neurons send inhibitory projections back to orexin neurons.14 On the other hand, orexin neurons are innervated by GABA-containing neurons in the VLPO. This pathway might be important for turning off orexin neurons during sleep, while the triangular formation plays a highly important role in sleep–wake/wake–sleep transitions and the stability of each state14 (Fig. 2).
Taking into account the aforementioned GABAergic mechanism of sleep induction, successful manipulation of this system could be of extreme importance for the treatment of insomnia. The GABAA receptor complex binds benzodiazepines, which act in synergy with GABA, providing the mechanical basis of the use of these drugs as hypnotics. However, in the near future, direct pharmacological intervention in the orexin system, for example by using orexin receptor antagonists, could further help patients suffering from sleep disorders by paving a new way forward for the treatment of insomnia.41
CONFLICT OF INTERESTS
The authors indicated no potential conflict of interests.