Interaction between sleep mechanisms and orexin neurons

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.

     

Narcolepsy,orexins and respiratory regulation

Narcolepsy is a debilitating sleep disorder characterized by excessive daytime sleepiness, cataplexy and intrusive rapid–eye movement sleep. Deficits in endogenous orexins are a major pathogenic component of the disease. This disorder is also associated with the gene marker HLADQB1*0602. Orexins as hypothalamic neuropeptides have multiple physiological functions, and their primary functions are regulation of the sleep–wake cycle and feeding. Evidence from animal studies using orexin knockout mice and focal microdialysis of an orexin receptor antagonist at the retrotrapezoid nucleus and medullary raphe in rats demonstrated that orexins also contribute to respiratory regulation in a vigilance state–dependent manner, as animals with orexin dysregulation have attenuated hypercapnic ventilatory responses predominantly in wakefulness. These findings are consistent with the notion that the activity of orexinergic neurons is higher during wake than sleep periods. Orexin neurons seem to be a pivotal link between conscious and unconscious brain functions in animals. The human model of hypocretin deficiency is patients with narcolepsy–cataplexy. In contrast to the findings suggested by animal studies, we found significant decreases in hypoxic responsiveness, but not in hypercapnic responsiveness, in narcoleptics, and further analysis indicated that decreased ventilatory responses to hypoxia in human narcolepsy–cataplexy is in relation to HLA-DQB1*0602 status, not hypocretin deficiency. This is confirmed by the fact that the hypoxic responsiveness was lower in HLA positive versus negative controls. Unlike in mice, hypocretin-1 is not a major factor contributing to depressed hypoxic responses in humans. Species differences may exist.

     

Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation.

Neurons containing the neuropeptide orexin (hypocretin) are located exclusively in the lateral hypothalamus and send axons to numerous regions throughout the central nervous system, including the major nuclei implicated in sleep regulation. Here, we report that, by behavioral and electroencephalographic criteria, orexin knockout mice exhibit a phenotype strikingly similar to human narcolepsy patients, as well as canarc-1 mutant dogs, the only known monogenic model of narcolepsy. Moreover, modafinil, an anti-narcoleptic drug with ill-defined mechanisms of action, activates orexin-containing neurons. We propose that orexin regulates sleep/wakefulness states, and that orexin knockout mice are a model of human narcolepsy, a disorder characterized primarily by rapid eye movement (REM) sleep dysregulation.     

Orexin receptors: Multi-functional therapeutic targets for sleeping disorders,eating disorders,drug addiction,cancers and other physiological disorders

The orexin peptides (orexin A, orexin B) and their receptors (orexin receptor type 1, orexin receptor type 2) are involved in multiple physiological processes such as the regulation of sleep/wakefulness state, energy homeostasis and reward seeking. A result of this has been the development of small-molecule orexin receptor antagonists as novel therapies for the treatment of insomnia and drug addiction. Increased levels of signaling via the orexin peptide/receptor system may protect against obesity, while somewhat unexpectedly, orexins acting at orexin receptors induce dramatic apoptosis resulting in the significant reduction of cell growth in various cancer cell lines. Meanwhile, the orexin peptide/receptor system is also involved in cardiovascular modulation, neuroendocrine and reproduction regulation. This review summarizes the latest developments in deciphering the biology of orexin signaling as well as efforts to manipulate orexin signaling pharmacologically.     

Local network regulation of orexin neurons in the lateral hypothalamus

Obesity and inadequate sleep are among the most common causes of health problems in modern society. Thus, the discovery that orexin (hypocretin) neurons play a pivotal role in sleep/wake regulation, energy balance, and consummatory behaviors has sparked immense interest in understanding the regulatory mechanisms of these neurons. The local network consisting of neurons and astrocytes within the lateral hypothalamus and perifornical area (LH/PFA), where orexin neurons reside, shapes the output of orexin neurons and the LH/PFA. Orexin neurons not only send projections to remote brain areas but also contribute to the local network where they release multiple neurotransmitters to modulate its activity. These neurotransmitters have opposing actions, whose balance is determined by the amount released and postsynaptic receptor desensitization. Modulation and negative feedback regulation of excitatory glutamatergic inputs as well as release of astrocyte-derived factors, such as lactate and ATP, can also affect the excitability of orexin neurons. Furthermore, distinct populations of LH/PFA neurons express neurotransmitters with known electrophysiological actions on orexin neurons, such as melanin-concentrating hormone, corticotropin-releasing factor, thyrotropin-releasing hormone, neurotensin, and GABA. These LH/PFA-specific mechanisms may be important for fine tuning the firing activity of orexin neurons to maintain optimal levels of prolonged output to sustain wakefulness and stimulate consummatory behaviors. Building on these exciting findings should shed further light onto the cellular mechanisms of energy balance and sleep-wake regulation.     

Dynamic interactions between orexin and dynorphin may delay onset of functional orexin effects: a modeling study

Orexin (also known as hypocretin) neurons play a key role in regulating sleep-wake behavior, but the links between orexin neuron electrophysiology and function have not been explored. Orexin neurons are wake-active, and spiking activity in orexin neurons may anticipate transitions to wakefulness by several seconds. However, it is suggested that while the orexin system is necessary to maintain sustained wake bouts, orexin has little effect on brief wake bouts. In vitro experiments investigating the actions of orexin and dynorphin, a colocalized neuropeptide, on orexin neurons indicate that the dynamics of desensitization to dynorphin may represent a mechanism for modulating local network activity and resolving the apparent discrepancy between the onset of firing in orexin neurons and the onset of functional orexin effects. To investigate the role of dynorphin on orexin neuron activity, a Hodgkin-Huxley-type model orexin neuron was developed in which baseline electrophysiology, orexin/dynorphin action, and dynorphin desensitization were closely tied to experimental data. In this model framework, model orexin neuron responses to orexin/dynorphin action were calibrated by simulating the physiologic effects of static orexin and dynorphin bath application on orexin neurons. Then behavior in a small network of model orexin neurons was simulated with pure orexin, pure dynorphin, or combined orexin and dynorphin coupling based on the mechanisms established in the static case. It was found that the dynamics of desensitization to dynorphin can mediate a clear shift from a network in which firing is suppressed by dynorphin-mediated inhibition to a network of neurons with high firing rates sustained by orexin-mediated excitation. The findings suggest that dynamic interactions between orexin and dynorphin at transitions from sleep to wake may delay onset of functional orexin effects.     

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

Narcolepsy-cataplexy, a neurological disorder associated with the absence of hypothalamic orexin (hypocretin) neuropeptides, consists of two underlying problems: inability to maintain wakefulness and intrusion of rapid eye movement (REM) sleep into wakefulness. Here we document, using behavioral, electrophysiological, and pharmacological criteria, two distinct classes of behavioral arrests exhibited by mice deficient in orexin-mediated signaling. Both OX2R(-/-) and orexin(-/-) mice are similarly affected with behaviorally abnormal attacks of non-REM sleep ("sleep attacks") and show similar degrees of disrupted wakefulness. In contrast, OX2R(-/-) mice are only mildly affected with cataplexy-like attacks of REM sleep, whereas orexin(-/-) mice are severely affected. Absence of OX2Rs eliminates orexin-evoked excitation of histaminergic neurons in the hypothalamus, which gate non-REM sleep onset. While normal regulation of wake/non-REM sleep transitions depends critically upon OX2R activation, the profound dysregulation of REM sleep control unique to the narcolepsy-cataplexy syndrome emerges from loss of signaling through both OX2R-dependent and OX2R-independent pathways.     

A mathematical model of the sleep/wake cycle

We present a biologically-based mathematical model that accounts for several features of the human sleep/wake cycle. These features include the timing of sleep and wakefulness under normal and sleep-deprived conditions, ultradian rhythms, more frequent switching between sleep and wakefulness due to the loss of orexin and the circadian dependence of several sleep measures. The model demonstrates how these features depend on interactions between a circadian pacemaker and a sleep homeostat and provides a biological basis for the two-process model for sleep regulation. The model is based on previous “flip–flop” conceptual models for sleep/wake and REM/NREM and we explore whether the neuronal components in these flip–flop models, with the inclusion of a sleep-homeostatic process and the circadian pacemaker, are sufficient to account for the features of the sleep/wake cycle listed above. The model is minimal in the sense that, besides the sleep homeostat and constant cortical drives, the model includes only those nuclei described in the flip–flop models. Each of the cell groups is modeled by at most two differential equations for the evolution of the total population activity, and the synaptic connections are consistent with those described in the flip–flop models. A detailed analysis of the model leads to an understanding of the mathematical mechanisms, as well as insights into the biological mechanisms, underlying sleep/wake dynamics.     

Regulation of orexin neurons by the monoaminergic and cholinergic systems

Orexins are a pair of neuropeptides implicated in energy homeostasis and arousal. Here we characterize the electrophysiological properties of orexin neurons using slice preparations from transgenic mice in which orexin neurons specifically express green fluorescent protein. Orexin neurons showed high frequency firing with little adaptation by injecting a positive current. The hyperpolarization-activated current was observed in orexin neurons by a negative current injection. The neurotransmitters, which were implicated in sleep/wake regulation, affected the activity of orexin neurons; noradrenaline and serotonin hyperpolarized, while carbachol depolarized orexin neurons in either the presence or absence of tetrodotoxin. It has been reported that orexins directly or indirectly activate the nuclei that are the origin of the neurons containing these neurotransmitters. Our data suggest that orexin neurons have reciprocal neural circuitries between these nuclei for either a positive or negative feedback loop and orchestrate the activity of these neurons to regulate the vigilance states.     

Orexin neurons receive glycinergic innervations

Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Circadian regulation gene polymorphisms are associated with sleep disruption and duration,and circadian phase and rhythm in adults with HIV

Genes involved in circadian regulation, such as circadian locomotor output cycles kaput [CLOCK], cryptochrome [CRY1] and period [PER], have been associated with sleep outcomes in prior animal and human research. However, it is unclear whether polymorphisms in these genes are associated with the sleep disturbances commonly experienced by adults living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Thus, the purpose of this study was to describe polymorphisms in selected circadian genes that are associated with sleep duration or disruption as well as the sleep–wake rhythm strength and phase timing among adults living with HIV/AIDS. A convenience sample of 289 adults with HIV/AIDS was recruited from HIV clinics and community sites in the San Francisco Bay Area. A wrist actigraph was worn for 72?h on weekdays to estimate sleep duration or total sleep time (TST), sleep disruption or percentage of wake after sleep onset (WASO) and several circadian rhythm parameters: mesor, amplitude, the ratio of mesor to amplitude (circadian quotient), and 24-h autocorrelation. Circadian phase measures included clock time for peak activity (acrophase) from actigraphy movement data, and bed time and final wake time from actigraphy and self-report. Genotyping was conducted for polymorphisms in five candidate genes involved in circadian regulation: CLOCK, CRY1, PER1, PER2 and PER3. Demographic and clinical variables were evaluated as potential covariates. Interactions between genotype and HIV variables (i.e. viral load, years since HIV diagnosis) were also evaluated. Controlling for potentially confounding variables (e.g. race, gender, CD4+ T-cell count, waist circumference, medication use, smoking and depressive symptoms), CLOCK was associated with WASO, 24-h autocorrelation and objectively-measured bed time; CRY1 was associated with circadian quotient; PER1 was associated with mesor and self-reported habitual wake time; PER2 was associated with TST, mesor, circadian quotient, 24-h autocorrelation and bed and wake times; PER3 was associated with amplitude, 24-h autocorrelation, acrophase and bed and wake times. Most of the observed associations involved a significant interaction between genotype and HIV. In this chronic illness population, polymorphisms in several circadian genes were associated with measures of sleep disruption and timing. These findings extend the evidence for an association between genetic variability in circadian regulation and sleep outcomes to include the sleep–wake patterns experienced by adults living with HIV/AIDS. These results provide direction for future intervention research related to circadian sleep–wake behavior patterns.     

The Hypocretin/Orexin System: Implications for Drug Reward and Relapse

Hypocretins (also known as orexins) are hypothalamic neuropeptides involved in the regulation of sleep/wake states and feeding behavior. Recent studies have also demonstrated an important role for the hypocretin/orexin system in the addictive properties of drugs of abuse, consistent with the reciprocal innervations between hypocretin neurons and brain areas involved in reward processing. This system participates in the primary reinforcing effects of opioids, nicotine, and alcohol. Hypocretins are also involved in the neurobiological mechanisms underlying relapse to drug-seeking behavior induced by drug-related environmental stimuli and stress, as mainly described in the case of psychostimulants. Based on these preclinical studies, the use of selective ligands targeting hypocretin receptors could represent a new therapeutical strategy for the treatment of substance abuse disorders. In this review, we discuss and update the current knowledge about the participation of the hypocretin system in drug addiction and the possible neurobiological mechanisms involved in these processes regulated by hypocretin transmission.     

Molecular genetic studies on sleep-wake regulation, with special emphasis on the prostaglandin D(2) system.

To elucidate the exact role of the PGD(2) system in sleep-wake regulation in vivo, the sleep behavior of knockout mice, generated in the author's and other laboratories, was examined for lipocalin-type PGD synthase (L-PGDS), PGD receptor, adenosine A(2A) receptor, and histamine H(1) receptor; transgenic mice overexpressing the human L-PGDS gene, generated in the author's laboratory, were also examined. The circadian profiles of sleep patterns of wild-type and the genetically manipulated mice were essentially identical, indicating the possibility that the deficiency of one system may be effectively compensated by some other systems during development. Available evidence indicated that the PGD(2) system is involved in the homeostatic regulation of non-rapid eye movement sleep and that the arousal effect of orexin A is mediated by the histamine H(1) receptor system.     

Reverse pharmacology of orexin: from an orphan GPCR to integrative physiology

Orexins, which were initially identified as endogenous peptide ligands for two orphan G-protein coupled receptors (GPCRs), have been shown to have an important role in the regulation of energy homeostasis. Furthermore, the discovery of orexin deficiency in narcolepsy patients indicated that orexins are highly important factors for the sleep/wakefulness regulation. The efferent and afferent systems of orexin-producing neurons suggest interactions between these cells and arousal centers in the brainstem as well as important feeding centers in the hypothalamus. Electrophysiological studies have shown that orexin neurons are regulated by humoral factors, including leptin, glucose, and ghrelin as well as monoamines and acetylcholin. Thus, orexin neurons have functional interactions with hypothalamic feeding pathways and monoaminergic/cholinergic centers to provide a link between peripheral energy balance and the CNS mechanisms that coordinate sleep/wakefulness states and motivated behavior such as food seeking.     

Orexins and adipoinsular axis function in the rat

Orexin A and B are recently identified as peptides that are derived from the same precursor and their expression is highly specifically localized in neurons located in the lateral hypothalamic area, a region implicated in the feeding behaviour. These peptides appear to be a part of a complex circuit that integrates the aspects of energy metabolism, cardiovascular function, hormone homeostasis and sleep/wake behaviours. The functional linking of orexins with leptin and insulin suggests the possibility of its involvement in the regulation of the adipoinsular axis, and the present investigation was designed to examine the potential role of orexins in this axis regulation. In all the tested doses (8, 16 and 40 nmol/kg body weight (b.w.)), subcutaneous (s.c.) injections of orexin A caused the significant increase in insulin and leptin blood levels. These elevations were observed 60 and 120 min after peptide administration. On the other hand, after the orexin B administration, elevated insulin and leptin blood concentrations were found only at 60 min of the experiment, and in that time point, the increases were comparable to that evoked by orexin A. In comparison with the control animals, the administration of orexins for 7 days resulted in a significant gain in body weight. Prolonged administration of either orexin A or orexin B significantly elevated insulin and leptin blood concentrations. Under these conditions, the orexin A effect on the leptin secretion was more marked than on the insulin secretion, and this difference is reflected by the lowered insulin/leptin molar ratio. These results suggest that orexins play an important role in the adipoinsular axis function and may be a significant regulator of both insulin and leptin secretion. In this regard, we suggest the updated functional model of Kieffer and Habener [Am. J. Physiol.: Endocrinol. Metab. 27 (2000) E1] that proposed the adipoinsular axis. Our model is extended by the probable humoral links between orexins and leptin and orexins and insulin and points on the dependence of the effects evoked by orexins, leptin and insulin on the blood glucose levels.     

A role for orexin in central vestibular motor control

The absence of orexin results in narcolepsy-cataplexy. While the function of the central orexinergic system in sleep regulation has been well studied, the role of orexin in motor control is largely unknown. Here, we show that orexin-A acts via OX(1) and OX(2) receptors to directly depolarize neurons in the rat lateral vestibular nucleus (LVN), a subcortical motor center, and enhance their sensitivity. A dual ionic mechanism involving both Na+-Ca2+ exchangers and inward rectifier K+ channels underlies these effects. Furthermore, orexin-A regulates central vestibular-mediated posture, motor balance and negative geotaxis. Orexin is critical when an animal is facing a major motor challenge as opposed to during rest and general movements. Therefore, orexin participates not only in sleep and emotion (nonsomatic) but also in motor (somatic) regulation, suggesting that the central orexinergic system plays an important role in somatic-nonsomatic integration. These findings may account for why the absence of orexin results in narcolepsy-cataplexy.     

On the influence of prenatal hypoxia on formation of the orexinergic system and sleep–wake cycle in early ontogenesis of rats

The role of orexin in the organization of the sleep–wake cycle (SWC) is well known. The aim of this study was to examine the timing of the orexinergic system formation in rat postnatal ontogenesis and to assess the role of orexin A in the SWC organization under normal conditions and after prenatal hypoxia undergone on days 14 and 19 of embryogenesis. The SWC was investigated in 30-day-old rats with electrodes implanted into the somatosensory and occipital cortex. Immunoreactivity within the orexigenic structures of the lateral hypothalamus was analyzed. It was shown that in control 14-day-old animals the orexinergic structures were in their formative stage, whereas in 30-day-old rats they were already as formed as in adults. In 14-day-old rats, prenatal hypoxia evoked retarded formation of the orexinergic system. In 30-day-old animals, hypoxia undergone in the prenatal period increased the activity of the orexinergic system, which was higher in animals exposed to hypoxia on day 19 than on day 14 of gestation. In 30-day-old rats, these changes were reflected in the SWC formation in the form of shorter slow-wave sleep, more fitful sleep and increased number of transitions from slow- to fast-wave sleep. The results obtained are discussed in the light of the adaptive-compensatory role of the orexigenic system in postnatal ontogenesis after prenatal damage to the central nervous system.     

Key roles of the histaminergic system in sleep-wake regulation

Histamine plays an important role in mediating wakefulness in mammals. Based on the findings from gene-manipulated mice, we provide several lines of evidence showing the roles of the histaminergic system in the somnogenic effects of prostaglandin (PG) D2 and adenosine, and in the arousal effects of PGE2 and orexin. PGD2 activates DP1 receptors (R) to promote sleep by stimulating them to release adenosine. The released adenosine activates adenosine A2AR and subsequently excites the ventrolateral preoptic area (VLPO), one of the sleep centers in the anterior hypothalamus. VLPO neurons then send inhibitory signals to downregulate the histaminergic tuberomammillary nucleus (TMN), which contributes to arousal. A1R is expressed in histaminergic neurons of the rat TMN. Adenosine in the TMN inhibits the histaminergic system via A1R and promotes non–rapid eye movement sleep. Conversely, both endogenous PGE2 and orexin activate the histaminergic system through EP4R and OX-2R, respectively, to promote wakefulness via histamine H1R. Furthermore, the arousal effect of ciproxifan, H3R antagonist, depends on the activation of histaminergic systems. These findings indicate that VLPO and TMN regulate sleep and wakefulness by means of a “flip-flop” mechanism operating in an anti-coincident manner during sleep–wake state transitions.