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.     

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.     

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.

     

下丘脑食欲素在药物成瘾中的作用

下丘脑是调控自然奖赏的重要脑区,它能特异性地表达一种神经肽——食欲素(orexin),这种神经肽在药物奖赏中的作用受到广泛关注。在成瘾研究中,发现不同脑区中的食欲素神经元对奖赏和动机行为的调节作用是不相同的:围穹窿区(PFA)和背内侧下丘脑区(DMH)的食欲素神经元主要参与激活应激系统,而外侧下丘脑(LH)的食欲素神经元主要通过激活与奖赏学习相关的大脑环路参与奖赏行为的调控。提示食欲素系统可在延长戒断防止复吸发生中成为新的研究目标,食欲素受体可以作为治疗药物成瘾的一种新的治疗靶标。     

State-dependent effects of orexins on the serotonergic dorsal raphe neurons in the rat

The serotonergic dorsal raphe (DR) neurons play an important role in sleep-wakefulness regulation. Orexinergic neurons in the lateral hypothalamus densely project to the brainstem sites including the DR. To test the effects of orexins on the serotonergic DR neurons, we applied orexin A (0.1 mM) by pressure to these neurons in unanesthetized and urethane anesthetized rats. Orexin A caused excitation in 10 of 15 neurons under unanesthetized condition. The excitation was characterized by slow onset (0-18 s), long lasting duration (15-150 s) and state-dependency. Orexin A applied during REM sleep or slow wave sleep induced significant excitation while during wakefulness, the similar amount of orexin A did not increase the firing rate any more. In the anesthetized animals, orexin A induced excitation in four of eight neurons. The excitation had slow onset and was long lasting. These results suggest that orexinergic neurons exert excitatory influence on the serotonergic DR neurons to maintain tonic activity of them, thereby participating in regulation of sleep-wakefulness cycles and other functions.     

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.     

Hypothalamic orexin neurons regulate arousal according to energy balance in mice

Mammals respond to reduced food availability by becoming more wakeful and active, yet the central pathways regulating arousal and instinctual motor programs (such as food seeking) according to homeostatic need are not well understood. We demonstrate that hypothalamic orexin neurons monitor indicators of energy balance and mediate adaptive augmentation of arousal in response to fasting. Activity of isolated orexin neurons is inhibited by glucose and leptin and stimulated by ghrelin. Orexin expression of normal and ob/ob mice correlates negatively with changes in blood glucose, leptin, and food intake. Transgenic mice, in which orexin neurons are ablated, fail to respond to fasting with increased wakefulness and activity. These findings indicate that orexin neurons provide a crucial link between energy balance and arousal.     

Lesions of orexin neurons block conditioned place preference for sexual behavior in male rats

The hypothalamic neuropeptide orexin (hypocretin) mediates reward related to drugs of abuse and food intake. However, a role for orexin in sexual reward has yet to be investigated. Orexin neurons are activated by sexual behavior, but endogenous orexin does not appear to be essential for sexual performance and motivation in male rats. Therefore, the goal of the current study was to test the hypothesis that orexin is critically involved in processing of sexual reward in male rats. First, it was demonstrated following exposure to conditioned contextual cues associated with sexual behavior in a conditioned place preference paradigm that cFos expression is induced in orexin neurons, indicating activation of orexin neurons by cues predicting sexual reward. Next, orexin-cell specific lesions were utilized to determine the functional role of orexin in sexual reward processing. Hypothalami of adult male rats were infused with orexin-B-conjugated saporin, resulting in greater than 80% loss of orexin neurons in the perifornical-dorsomedial and lateral hypothalamus. Orexin lesions did not affect expression of sexual behavior, but prevented formation of conditioned place preference for a sexual behavior paired chamber. In contrast, intact sham-treated males or males with partial lesions developed a conditioned place preference for mating. Orexin lesioned males maintained the ability to form a conditioned place aversion to lithium chloride-induced visceral illness, indicating that orexin lesions did not disrupt associative contextual memory. Overall, these findings suggest that orexin is not essential for sexual performance or motivation, but is critical for reward processing and conditioned cue-induced seeking of sexual behavior.     

The SK-N-MC cell line expresses an orexin binding site different from recombinant orexin 1-type receptor.

Orexin A and B (also known as hypocretins), two recently discovered neuropeptides, play an important role in food intake, sleep/wake cycle and neuroendocrine functions. Orexins are endogenous ligands of two G-protein-coupled receptors, termed OX1 and OX2. This work presents the first short orexin A and B analogues, orexin A 23-33 and orexin B 18-28, with high affinity (119 +/- 49 and 49 +/- 23 nm) for OX1 receptors expressed on SK-N-MC cells and indicates the importance of the C-terminal part of the orexin peptides for this ligand-receptor interaction. However, these C-terminal fragments of orexin did not displace the 125I-labelled orexin B from the recombinant orexin 1 receptor stably expressed in Chinese hamster ovary cells. To examine the role of the shortened orexin A 23-33 in feeding, its effects in mimicking or antagonizing the effects of orexin A were studied in rats after administration via the lateral hypothalamus. In contrast with orexin A, which potently induced feeding up to 4 h after administration, orexin A 23-33 neither induced feeding nor inhibited orexin A-induced feeding. Modafinil (Vigil), which was shown earlier to activate orexin neurons, displayed binding neither to the orexin receptor expressed on SK-N-MC cells nor to the recombinant orexin 1 receptor, which indicates that modafinil displays its antinarcoleptic action via another yet unknown mechanism. PCR and subsequent sequencing revealed expression of the full-length orexin 1 receptor mRNA in SK-N-MC and NT-2 cells. Interestingly, sequencing of several cDNA clones derived from RNA of both SK-N-MC and NT-2 cells differed from the published nucleotide sequence at position 1375. Amino acid prediction of this A -->G change results in an isoleucine --> valine substitution at the protein level, which may provide evidence for an editing process.     

Elevated body temperature during sleep in orexin knockout mice

Core body temperature (Tb) is influenced by many physiological factors, including behavioral state, locomotor activity, and biological rhythms. To determine the relative roles of these factors, we examined Tb in orexin knockout (KO) mice, which have a narcolepsy-like phenotype with severe sleep-wake fragmentation. Because orexin is released during wakefulness and is thought to promote heat production, we hypothesized that orexin KO mice would have lower Tb while awake. Surprisingly, Tb was the same in orexin KO mice and wild-type (WT) littermates during sustained wakefulness. Orexin KO mice had normal diurnal variations in Tb, but the ultradian rhythms of Tb, locomotor activity, and wakefulness were markedly reduced. During the first 15 min of spontaneous sleep, the Tb of WT mice decreased by 1.0 degrees C, but Tb in orexin KO mice decreased only 0.4 degrees C. Even during intense recovery sleep after 8 h of sleep deprivation, the Tb of orexin KO mice remained 0.7 degrees C higher than in WT mice. This blunted fall in Tb during sleep may be due to inadequate activation of heat loss mechanisms or sustained activity in heat-generating systems. These observations reveal an unexpected role for orexin in thermoregulation. In addition, because heat loss is an essential aspect of sleep, the blunted fall in Tb of orexin KO mice may provide an explanation for the fragmented sleep of narcolepsy.     

食欲素信号系统在成瘾中的作用

成瘾是对成瘾物质的强迫性、持续性需求并缺乏控制能力的行为,它会导致大脑中枢奖赏回路的改变。下丘脑是调控自然奖赏的重要脑区,它能特异性地表达一种被称为食欲素(orexins/hypocretins)的神经肽。食欲素通过作用于食欲素受体调控睡眠、觉醒状态,同时,食欲素受体在药物成瘾和奖赏相关的行为中也有重要作用,投射到不同脑区的食欲素对不同药物导致的成瘾调节作用也不同,调控食欲素信号系统,将可能成为治疗成瘾的重要方法。本文重点总结了食欲素信号系统在不同药物成瘾过程中的作用的最新研究进展。     

Characterization of orexin A immunoreactivity in the rat area postrema

The distribution of orexin A immunoreactivity and the synaptic relationships of orexin A-positive neurons in the rat area postrema were studied using both light and electron microscopy techniques. At the light microscope level, numerous orexin A-like immunoreactive fibers were found within the area postrema. Using electron microscopy, immunoreactivity within fibers was confined primarily to the axon terminals, most of which contained dense-cored vesicles. Both axo-somatic and axo-dendritic synapses made by orexin A-like immunoreactive axon terminals were found, with these synapses being both symmetric and asymmetric in form. Orexin A-like immunoreactive axon terminals could be found presynaptic to two different immunonegative profiles including the perikarya and dendrites. Occasionally, some orexin A-like immunoreactive profiles, most likely to be dendrites, could be seen receiving synaptic inputs from immunonegative or immunopositive axon terminals. The present results suggest that the physiological function of orexin A in the area postrema depends on synaptic relationships with other immunopositive and immunonegative neurons, with the action of orexin A mediated via a self-modulation feedback mechanism.     

Radioimmunoassay for orexin A

A radioimmunoassay for orexin A has been developed. Anti-orexin A antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic orexin A with bovine serum albumin. This antibody did not crossreact with orexin B, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. Radioiodination of orexin A was performed with the chloramin T method, followed by purification of radioiodinated material on Sephadex G-25 column. Orexin A was extracted from tissues using acid-acetone. The assay was performed with a double antibody system. The dilution curve of acid-acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue orexin A was about 80%,and the intra-assay and inter-assay variations were 5.2% and 7.8%, respectively. Orexin A was found in the hypothalamus, cerebrum and testis. These data suggest that this assay system is suitable for the measurement of tissue orexin A and that orexin A is found in the central nervous system and testis.     

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.     

Reactivation of hyperglycemia-induced hypocretin (HCRT) gene silencing by N-acetyl-d-mannosamine in the orexin neurons derived from human iPS cells

Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward that end, we generated human orexin neurons from induced pluripotent stem cells (hiPSCs) by treatment with N-acetyl-d-mannosamine (ManNAc) and its derivatives. The generation of orexin neurons was associated with DNA hypomethylation, histone H3/H4 hyperacetylation, and hypo-O-GlcNAcylation on the HCRT gene locus, and, thereby, the treatment of inhibitors of SIRT1 and OGT were effective at inducing orexin neurons from hiPSCs. The prolonged exposure of orexin neurons to high glucose in culture caused irreversible silencing of the HCRT gene, which was characterized by H3/H4 hypoacetylation and hyper-O-GlcNAcylation. The DNA hypomethylation status, once established in orexin neurogenesis, was maintained in the HCRT-silenced orexin neurons, indicating that histone modifications, but not DNA methylation, were responsible for the HCRT silencing. Thus, the epigenetic status of the HCRT gene is unique to the hyperglycemia-induced silencing. Intriguingly, treatment of ManNAc and its derivatives reactivated HCRT gene expression, while inhibitors SIRT1 and the OGT did not. The present study revealed that the HCRT gene was silenced by the hyperglycemia condition, and ManNAc and its derivatives were useful for restoring the orexin neurons.     

Modelling Feedback Excitation,Pacemaker Properties and Sensory Switching of Electrically Coupled Brainstem Neurons Controlling Rhythmic Activity

What cellular and network properties allow reliable neuronal rhythm generation or firing that can be started and stopped by brief synaptic inputs? We investigate rhythmic activity in an electrically-coupled population of brainstem neurons driving swimming locomotion in young frog tadpoles, and how activity is switched on and off by brief sensory stimulation. We build a computational model of 30 electrically-coupled conditional pacemaker neurons on one side of the tadpole hindbrain and spinal cord. Based on experimental estimates for neuron properties, population sizes, synapse strengths and connections, we show that: long-lasting, mutual, glutamatergic excitation between the neurons allows the network to sustain rhythmic pacemaker firing at swimming frequencies following brief synaptic excitation; activity persists but rhythm breaks down without electrical coupling; NMDA voltage-dependency doubles the range of synaptic feedback strengths generating sustained rhythm. The network can be switched on and off at short latency by brief synaptic excitation and inhibition. We demonstrate that a population of generic Hodgkin-Huxley type neurons coupled by glutamatergic excitatory feedback can generate sustained asynchronous firing switched on and off synaptically. We conclude that networks of neurons with NMDAR mediated feedback excitation can generate self-sustained activity following brief synaptic excitation. The frequency of activity is limited by the kinetics of the neuron membrane channels and can be stopped by brief inhibitory input. Network activity can be rhythmic at lower frequencies if the neurons are electrically coupled. Our key finding is that excitatory synaptic feedback within a population of neurons can produce switchable, stable, sustained firing without synaptic inhibition.     

Orexin A in the VTA is critical for the induction of synaptic plasticity and behavioral sensitization to cocaine

Dopamine neurons in the ventral tegmental area (VTA) represent a critical site of synaptic plasticity induced by addictive drugs. Orexin/hypocretin-containing neurons in the lateral hypothalamus project to the VTA, and behavioral studies have suggested that orexin neurons play an important role in motivation, feeding, and adaptive behaviors. However, the role of orexin signaling in neural plasticity is poorly understood. The present study shows that in vitro application of orexin A induces potentiation of N-methyl-D-aspartate receptor (NMDAR)-mediated neurotransmission via a PLC/PKC-dependent insertion of NMDARs in VTA dopamine neuron synapses. Furthermore, in vivo administration of an orexin 1 receptor antagonist blocks locomotor sensitization to cocaine and occludes cocaine-induced potentiation of excitatory currents in VTA dopamine neurons. These results provide in vitro and in vivo evidence for a critical role of orexin signaling in the VTA in neural plasticity relevant to addiction.     

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.     

Diminished feeding responsiveness to orexin A (hypocretin 1) in aged rats is accompanied by decreased neuronal activation

Orexin A is produced in caudal lateral, posterior, perifornical, and dorsomedial hypothalamic areas. Orexin A in the rostro-dorsal lateral hypothalamic area (rLHa) stimulates feeding and activates several feeding-regulatory brain areas. We hypothesized that aging diminishes feeding and c-Fos-immunoreactivity (c-Fos-ir; marker of neuronal activation) response to orexin A. Young (3 mo), middle-aged (12 mo), and old (24 mo) male Fischer 344 rLHa-cannulated rats were injected with orexin A (0.5, 1, and 2 nmol). Food intake was measured at 1, 2, and 4 h. c-Fos-ir in hypothalamic, limbic, and hindbrain regions was measured in two additional sets of rLHa-orexin A injected rats. In a separate study, orexin A effects on feeding and c-Fos-ir were measured in 6-mo-old rats. Orexin A significantly elevated feeding in rats aged 3, 6, and 12 mo in the 0-1 and 1-2- h time intervals, whereas in old rats this was significant in the 1-2 h time interval only. At 1 h, 6-8 (of 14) brain areas showed elevated c-Fos-ir in response to orexin A in 3- and 6-mo-old rats, but 24-mo-old rats exhibited attenuated or absent c-Fos-ir response in all brain regions except the hypothalamic paraventricular nucleus (PVN) and rostral nucleus of the solitary tract (rNTS). Orexin A did not elevate c-Fos-ir in 3-mo-old rats at 2 h after injection, whereas the PVN and mediodorsal thalamic nucleus (MD) showed elevated c-Fos-ir at 2 h in 24-mo-old rats. These data suggest that delayed and diminished feeding responses in old animals may be due to ineffective neural signaling and implicate the orexin A network as one feeding system affected by aging.     

Selective excitation of GABAergic neurons in the substantia nigra of the rat by orexin/hypocretin in vitro

Dysfunction of the orexin/hypocretin neurotransmitter system leads to the sleep disorder narcolepsy. Narcolepsy is characterized by excessive daytime sleepiness and the occurrence of cataplexy--a sudden loss of muscle tone triggered by emotionally arousing events. Both symptoms can be treated with drugs that act on dopaminergic systems. Here we have investigated the effect of orexins on the firing of dopaminergic and GABAergic neurons of the substantia nigra (SN) in brain slices. Surprisingly, dopaminergic neurons in pars compacta were unaffected by orexins. In contrast, bath application of orexin A (100 nM) or orexin B (5-300 nM) greatly increased the firing rate of GABAergic neurons in pars reticulata. The orexin B-mediated excitation was unaffected by blocking synaptic transmission (using low-Ca2+/high-Mg2+ solution). However, the effect of orexin B was reduced significantly by thapsigargin (1 microM) and inhibitors of protein kinase A. The presence of orexinergic fibres in the SN pars reticulata was demonstrated by immunohistochemical methods with the fibre density increasing in the rostrocaudal direction. The orexin excitation of SN reticulata cells may help to maintain their high firing rate during waking. Furthermore, the absence of orexin effects in narcolepsy may predispose affected individuals to attacks of cataplexy.