Orexins promote survival of rat cortical neurons

Orexin A and B (hypocretin-1 and -2) are hypothalamic peptides that exert their biological functions by stimulation of two specific, membrane-bound receptors, OX₁R and OX₂R. Recently, we have demonstrated the expression of both types of orexin receptors in rat cortical neurons, with the OX₂R level being markedly higher compared to OX₁R. In the present study we investigated the receptor-mediated effects of orexin A, an agonist of OX₁R and OX₂ R, orexin B and [Ala¹¹-D-Leu¹⁵]orexin B, preferential agonists of OX₂R, on survival of cultured neurons derived from rat cerebral cortex. The three tested peptides markedly increased neuronal viability in a concentration-dependent manner. The pro-survival properties of orexins were associated with an attenuation of caspase-3 activity. Comparable potency of orexin A, orexin B and [Ala¹¹-D-Leu¹⁵]orexin B suggests a predominant role of OX₂R in the studied phenomenon. Our findings provide new insights into the role of orexins in CNS as potential neuroprotective factors.     

Orexin receptor subtype activation and locomotor behaviour in the rat

Aim: Orexin-producing neurones, located primarily in the perifornical region of the lateral hypothalamus, project to a wide spectrum of brain sites where they influence numerous behaviours as well as modulating the neuroendocrine and autonomic responses to stress. While some of the actions of orexin appear to be mediated via the type 1 receptor, some are not, including its action on the release of one stress hormone, prolactin. We describe here the ability of orexin to increase locomotor behaviours and identify the importance of both receptor subtypes in these actions. Methods: Rats were tested for their behavioural responses to the central activation of both the type 1 (OX₁R) and type 2 (OX₂R) receptor (ICV orexin A), compared to OX₂R activation using a relatively selective OX₂R agonist in the absence or presence of an orexin receptor antagonist that possesses highest affinity for OX₁R. Results: Increases in locomotor activity were observed, effects which were expressed by not only orexin A, which binds to both the OX₁R and the OX₂R receptors, but also by the relatively selective OX₂R agonist [(Ala¹¹, Leu¹⁵)-orexin B]. Furthermore, the OX₁R selective antagonist only partially blocked the action of orexin A on most locomotor behaviours and did not block the actions of [(Ala¹¹, Leu¹⁵)-orexin B]. Conclusion: We conclude that orexin A exerts its effects on locomotor behaviour via both the OX₁R and OX₂R and that agonism or antagonism of only one of these receptors for therapeutic purposes (i.e. sleep disorders) would not provide selectivity in terms of associated behavioural side effects.     

Promotion of Sleep by Suvorexant—A Novel Dual Orexin Receptor Antagonist

Abstract: Orexins/hypocretins are key neuropeptides responsible for regulating central arousal and reward circuits. Two receptors respond to orexin signaling, orexin 1 receptor (OX₁R) and orexin 2 receptor (OX₂R) with partially overlapping nervous system distributions. Genetic studies suggest orexin receptor antagonists could be therapeutic for insomnia and other disorders with disruptions of sleep and wake. Suvorexant (MK-4305) is a potent, selective, and orally bioavailable antagonist of OX₁R and OX₂R currently under clinical investigation as a novel therapy for insomnia. Examination of Suvorexant in radioligand binding assays using tissue from transgenic rats expressing the human OX₂R found nearly full receptor occupancy (>90%) at plasma exposures of 1.1 μM. Dosed orally Suvorexant significantly and dose-dependently reduced locomotor activity and promoted sleep in rats (10, 30, and 100 mg/kg), dogs (1 and 3 mg/kg), and rhesus monkeys (10 mg/kg). Consistent cross-species sleep/wake architecture changes produced by Suvorexant highlight a unique opportunity to develop dual orexin antagonists as a novel therapy for insomnia.     

Pharmacological evaluation of orexin receptor antagonists in preclinical animal models of pain

Orexin signaling, known to modulate arousal and vigilance, is also involved in nociception as orexin neurons project to regions of the brain and spinal cord involved in pain processing, and the administration of orexin peptides can alter pain response in a wide range of preclinical models. Pharmacological treatment with the potent, selective and structurally distinct dual orexin receptor antagonists (ORAs) DORA-12 and DORA-2 significantly reduced pain responses during both phases I and II of the mouse formalin pain model and significantly reversed hyperalgesia in the rat complete Freund’s adjuvant pain model, respectively. Significant antinociceptive effects of DORA-12 in the formalin model were also observed in orexin 1 receptor (OX₁R) knockout mice, but not orexin 2 receptor (OX₂R) or OX₁R/OX₂R double knockout mice. Mechanical hypersensitivity was significantly reduced with a series of structurally distinct, potent and highly selective ORAs (DORA-2, DORA-12 and DORA-22) in the rat spinal nerve ligation (SNL) injury model of neuropathic pain. Selective pharmacological targeting of OX₂R with 2-SORA-7 also reduced pain responses in acute inflammatory (complete Freund’s adjuvant) and neuropathic (SNL) rat pain models. Performance on the rotarod test of psychomotor performance and baseline thermal sensitivity were not affected in OX₁R/OX₂R knockout mice or ORA-treated mice, indicating that the observed pain-reducing effects were not due to sedation or motor deficits. These findings indicate that ORAs have pain-reducing effects across a number of acute and chronic neuropathic preclinical mouse and rat pain models. Further studies on the potential pain-relieving effects of orexin receptor antagonism are warranted.     

Histamine-1 receptor is not required as a downstream effector of orexin-2 receptor in maintenance of basal sleep/wake states

Aim: The effect of orexin on wakefulness has been suggested to be largely mediated by activation of histaminergic neurones in the tuberomammillary nucleus (TMN) via orexin receptor-2 (OX₂R). However, orexin receptors in other regions of the brain might also play important roles in maintenance of wakefulness. To dissect the role of the histaminergic system as a downstream mediator of the orexin system in the regulation of sleep/wake states without compensation by the orexin receptor-1 (OX₁R) mediated pathways, we analysed the phenotype of Histamine-1 receptor (H₁R) and OX₁R double-deficient (H₁R^−/−;OX₁R^−/−) mice. These mice lack OX₁R-mediated pathways in addition to deficiency of H₁R, which is thought to be the most important system in downstream of OX₂R. Methods: We used H₁R deficient (H₁R^−/−) mice, H₁R^−/−;OX₁R^−/− mice, OX₁R and OX₂R double-deficient (OX₁R^−/−;OX₂R^−/−) mice, and wild type controls. Rapid eye movement (REM) sleep, non-REM (NREM) sleep and awake states were determined by polygraphic electroencephalographic/electromyographic recording. Results: No abnormality in sleep/wake states was observed in H₁R^−/− mice, consistent with previous studies. H₁R^−/−;OX₁R^−/− mice also showed a sleep/wake phenotype comparable to that of wild type mice, while OX₁R^−/−; OX₂R^−/− mice showed severe fragmentation of sleep/wake states. Conclusion: Our observations showed that regulation of the sleep/wake states is completely achieved by OX₂R-expressing neurones without involving H₁R-mediated pathways. The maintenance of basal physiological sleep/wake states is fully achieved without both H₁ and OX₁ receptors. Downstream pathways of OX₂R other than the histaminergic system might play an important role in the maintenance of sleep/wake states.     

Expression of orexin A and its receptor 1 in the human prostate

The peptides orexin A (OXA) and orexin B, deriving from the cleavage of the precursor molecule prepro‐orexin, bind two G‐coupled transmembrane receptors, named as receptor 1 (OX1R) and receptor 2 for orexin, showing different affinity‐binding properties. First discovered in the rat hypothalamus, orexins and their receptors have been also found in many peripheral tissues where they exert neuroendocrine, autocrine and paracrine functions. Because inconclusive data on their localization in the mammalian prostate are reported, the aim of this study was to investigate the presence of prepro‐orexin, OXA and OX1R in the human normal and hyperplastic gland. Immunohistochemistry revealed the localization of both OXA and OX1R in the cytoplasm of the follicular exocrine epithelium of all tested normal and hyperplastic prostates. Positive immunostaining was mainly observed in the basal cells of the stratified epithelium, and only rarely in the apical cells. The expression of mRNAs coding for prepro‐orexin and OX1R and of proteins in the tissues was also ascertained by polymerase chain reaction and Western blotting analysis, respectively. In order to gain insights into the functional activity of OXA in the prostate, we administered different concentrations of OXA to cultured prostatic epithelial cells PNT1A. We first demonstrated that PNT1A cells express OX1R. The addition of OXA did not affect PNT1A cell proliferation, while it enhanced cAMP synthesis and Ca²⁺ release from intracellular storage. Overall, our results definitely demonstrate the expression of OXA and OX1R in the human prostate, and suggest an active role for them in the metabolism of the gland.     

Orexins/hypocretins control bistability of hippocampal long-term synaptic plasticity through co-activation of multiple kinases

Aim: Orexins/hypocretins (OX/Hcrt) are hypothalamic neuropeptides linking sleep–wakefulness, appetite and neuroendocrine control. Their role and mechanisms of action on higher brain functions, such as learning and memory, are not clear. Methods: We used field recordings of excitatory post-synaptic potentials (fEPSP) in acute mouse brain slice preparations to study the effects of orexins and pharmacological inhibitors of multiple kinases on long-term synaptic plasticity in the hippocampus. Results: Orexin-A (OX-A) but not orexin-B (OX-B) induces a state-dependent long-term potentiation of synaptic transmission (LTP_OX) at Schaffer collateral-CA1 synapses in hippocampal slices from adult (8- to 12-week-old) mice. In contrast, OX-A applied to slices from juvenile (3- to 4-week-old) animals causes a long-term depression (LTD_OX) in the same pathway. LTP_OX is blocked by pharmacological inhibition of orexin receptor-1 (OX1R) and plasticity-related kinases, including serine/threonine- (CaMKII, PKC, PKA, MAPK), lipid- (PI3K), and receptor tyrosine kinases (Trk). Inhibition of OX1R, CaMKII, PKC, PKA and Trk unmasks LTD_OX in adult animals. Conclusion: Orexins control not only the bistability of arousal states and threshold for appetitive behaviours but, in an age- and kinase-dependent manner, also bidirectional long-term synaptic plasticity in the hippocampus, providing a possible link between behavioural state and memory functions.     

Mapping of the binding sites for the OX1 orexin receptor antagonist, SB-334867, using orexin/hypocretin receptor chimaeras

The binding sites for agonists and antagonist of orexin receptors are not know, hampering progressive drug design approaches. In the current study, we utilized chimaeric orexin receptor approach to map the receptor areas contributing to the selectivity of the classical antagonist, SB-334867, for OX₁ receptors. Altogether ten chimaeras between OX₁ and OX₂ orexin receptors were utilized. The receptors were transiently expressed in HEK-293 cells. The ability (K_B) of SB-334867 to inhibit orexin-A-induced inositol phosphate release (phospholipase C activity) was measured. The results, in synthesis, suggest that there are several possible interactions contributing to the high affinity binding, all of which are not required simultaneously. This is indicated by the fact that most of the chimaeras display affinity (at least somewhat) higher than OX₂. As previously shown for the agonist distinction, the second quarter of the receptor, from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 seems to be most central also for SB-334867 binding, but also the third quarter, from the transmembrane helix 4 to the transmembrane helix 6 is able to contribute (and compensate for loss of other sites). A previous study has suggested that amino acids conserved between OX₁ and OX₂ receptors would somehow confer selectivity for subtype-selective antagonists. In contrast to previous findings, our results indicate that the amino acids distinct between the receptor subtypes are in key position.     

Localization of orexin B and orexin-2 receptor in the rat epididymis

The peptides orexin A (OXA) and orexin B (OXB) derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin, were originally described in the rat hypothalamus. Successively, they have been found in many other brain regions as well as in peripheral organs of mammals and other less evolved animals. The widespread localization of orexins accounts for the multiple activities that they exert in the body, including the regulation of energy homeostasis, feeding, metabolism, sleep and arousal, stress, addiction, and cardiovascular and endocrine functions. Both OXA and OXB peptides bind to two G-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R) receptor, though with different binding affinity. Altered expression/activity of orexins and their receptors has been associated with a large number of human diseases. Though at present evidence highlighted a role for orexins and cognate receptors in mammalian reproduction, their central and/or local effects on gonadal functions remain poorly known. Here, we investigated the localization of OXB and OX2R in the rat epididymis. Immunohistochemical staining of sections from caput, corpus and cauda segments of the organ showed intense signals for both OXB and OX2R in the principal cells of the lining epithelium, while no staining was detected in the other cell types. Negative results were obtained from immunohistochemical analysis of hypothalamic and testicular tissues from OX2R knock-out mice (OX2R^?/?) and OX1R/OX2R double knock-out (OX1R^?/?; OX2R^?/?) mice, thus demonstrating the specificity of the rabbit polyclonal anti-OX2R antibody used in our study. On contrary, the same antibody clearly showed the presence of OX2R in sections from hypothalamus and testis of normal mice and rats which are well known to express the receptor. Thus, our results provide the first definite evidence for the immunohistochemical localization of OXB and OX2R in the principal cells of rat epididymis.     

Distribution of the orexin-1 receptor (OX1R) in the mouse forebrain and rostral brainstem: A characterisation of OX1R-eGFP mice

We have utilised a transgenic reporter mouse in which green fluorescent protein (GFP) expression is driven by the orexin-1 receptor (OX₁R) promoter to systematically map the distribution of OX₁R-expressing neurons throughout the mouse forebrain and rostral brainstem. GFP labelling was observed in perikarya and fibres in an extensive range of brain loci encompassing the olfactory and cerebral cortices, dorsal and ventral pallidum, hippocampus, amygdaloid regions, septal areas, thalamic nuclei, hypothalamic nuclei and several brainstem regions, consistent with previous studies of OX₁R mRNA expression. This is the first study to systematically characterise the neuroanatomical distribution of OX₁R in the OX₁R-eGFP mouse, confirming its veracity as a faithful reporter of OX₁R expression and utility for future studies assessing the role of OX₁R in more complex behaviours.     

出生前尼古丁暴露增加新生大鼠下丘脑orexin A及延髓内orexin 1型受体表达

流行病学调查显示,出生前暴露于烟雾环境是新生儿猝死综合征发生的首位原因,尼古丁是香烟烟雾中最主要的影响胎儿神经系统发育的成分。为了观察出生前尼古丁暴露对新生大鼠下丘脑orexin A(OXA)及延髓内orexin 1型受体(OX1R)表达的影响,本实验将20只雌性成年大鼠随机均分为二组,怀孕后第5 d开始每天分别皮下注射尼古丁6 mg/kg(模型组)或等量的生理盐水(对照组),直至分娩。随机选取模型组和对照组所产的新生大鼠(1～3 d),采用免疫组织化学方法和图像分析技术,观察新生鼠下丘脑内OXA及延髓内OX1R阳性神经元的分布情况。结果显示:两组新生大鼠下丘脑内OXA免疫阳性细胞均有表达,且都主要存在于下丘脑背内侧区与穹窿周围,模型组的新生大鼠OXA免疫阳性细胞的相对光密度(ROD)值高于对照组(P<0.05)。延髓内OX1R免疫阳性细胞在两组内均有广泛分布,主要分布在腹外侧区和舌下神经核。在这两个区域,模型组新生鼠的OX1R免疫阳性细胞的ROD值均高于对照组(P<0.001)。以上结果表明,出生前尼古丁暴露的新生大鼠,下丘脑OXA及延髓内OX1R的表达均上调,提示出生前尼古丁暴露改变了新生大鼠脑内OXA系统递质的释放和突触传递,这意味着脑内orexin系统参与出生前尼古丁暴露导致的各种疾患。     

Individual differences in wheel-running rhythms are related to temporal and spatial patterns of activation of orexin A and B cells in a diurnal rodent (Arvicanthis niloticus)

This study investigated the relationship between the orexins and patterns of activity in the diurnal Nile grass rat, Arvicanthis niloticus. Some individuals of this species switch to a more nocturnal pattern when given access to a running wheel, while others continue to be most active during the day. In both day- and night-active grass rats, the percentages of orexin A (OXA) and orexin B (OXB) cells expressing Fos were highest when animals were actively running in wheels. In night-active animals, removal of the running wheel significantly decreased OXA and OXB cell Fos expression. Additionally, in night-active animals, clear regional differences were apparent. In these animals the presence of a wheel induced higher percentages of Fos in both OXA and OXB cells in medial regions of the lateral hypothalamus than in lateral regions. In night-active animals without access to wheels, this medial-lateral gradient was present only in OXA cells. No regional differences were observed in day-active animals. This study demonstrates that individual differences in the patterns of activation of OXA and OXB cell populations are related to differences in the temporal pattern of wheel running. We also present evidence that orexin cells have projections to the intergeniculate leaflet that appear to make contact with neuropeptide-Y cells. We discuss the possibility that these fibers may be involved in relaying feedback regarding the activity state of the animal to the circadian system through these projections.     

The osmotically and histamine-induced enhancement of the plasma vasopressin level is diminished by intracerebroventricularly administered orexin in rats

The effects of the centrally administered neuropeptides orexin-A on water intake and vasopressin (VP) secretion were studied in male Wistar rats (180–250 g). Different doses (10, 30, and 90 μg/10 μl) of the orexins and the specific orexin receptor-1 (OX₁) antagonist SB 408124 (30 μg/10 μl) were administered intracerebroventricularly (i.c.v.) under anaesthesia, and the water consumption was measured during 6 h. A plasma VP level elevation was induced by histamine (10 mg/kg) or 2.5% NaCl (10 ml/kg) administered intraperitoneally (i.p.). The plasma VP levels were measured by radioimmunoassay. Increased water consumption was observed after the administration of 30 μg/10 μl orexin-A. There were no changes in basal VP secretion after the administration of different doses of the orexins. A significant increase in plasma VP concentration was detected following histamine administration. After 2.5% NaCl administration, there was a moderate VP level enhancement. Intracerebroventricularly administered orexin-A (30 μg/10 μl) blocked the VP level increase induced by either histamine or 2.5% NaCl administration. The inhibitory effects were prevented by the specific OX₁ receptor antagonist. In conclusion, the orexins increased water consumption. After 30 μg/10 μl orexin-A administration, the polydipsia was more pronounced. The OX₁ receptor antagonist significantly decreased the polydipsia. Histamine or hyperosmotic VP release enhancement was blocked by previously administered orexin. This inhibition was not observed following OX₁ receptor antagonist administration. Our results suggest that the effects of the orexins on water consumption or blockade of the histamine and osmosis-induced VP level increase are mediated by the OX₁ receptor.     

Immunohistochemical localization of orexin A and orexin type 2 receptor-positive cells in the placenta of dogs

The aim of the present study was to examine the presence and distribution of cells that express immunopositivity for orexin A (OXA) and its type 2 receptor (OX2R) in the dog placenta toward the end of pregnancy using immunohistochemical techniques. In the placental fetal portion, a few OXA and OX2R-positive cells were seen scattered in the outermost coating layer of chorionic villi and in the trophoblastic protrusions. Closer to the maternal portion, immunopositive labeling for both peptides was visible in the glandular epithelia and that for OXA also in the endothelium of the capillaries. These observations allow us to hypothesize that the canine placenta may be not only a source of orexin A, but also its target, and that orexin A may play an important role in controlling the function of this important organ for normal fetal development.     

Expression of Orexin A and Its Receptor 1 in the Vestibular Glands of the Cattle Genital Tract

The hypothalamic peptide orexin A (oxA) binds specifically the G‐protein–coupled orexin receptor 1 (ox1R). It is involved in many physiological functions including the regulation of food intake, sleep–wake cycle, arterial blood pressure, heart rate, and sexual behavior. The localization of oxA in adrenal glands, stomach, bowel, pancreas, and testis has recently been assessed. Here, we provide the first evidence for the expression of oxA and ox1R in the vestibular glands of mammalian genital tract. Anat Rec, 2009. copy; 2008 Wiley‐Liss, Inc.     

Immunohistochemical localization of galectin-3 in boar testis and epididymis

The presence and distribution of galectin-3, a β-galactoside-binding protein, in boar testis and epididymis was studied. Western blot analysis detected galectin-3 in boar testis and epididymis. In particular, intense galectin-3 immunoreaction was seen in the tail of the epididymis, while it was moderate in the head and body. Galectin-3 immunolabelling was detected in the connective tissues of the testis. In the testis, galectin-3 was detected in some cells (presumably peritubular myoid cells), but not in Leydig cells or the cells of the seminiferous tubules. In the epididymis, the galectin-3 immunoreactivity in the connective tissues was the same as in the testis. Intense galectin-3 immunolabelling was seen in the covering epithelium of the epididymis tail, but in very few cells in the head and body. We postulate that galectin-3, immunodetected here in the connective tissues in the male reproductive organs, serves as an extracellular matrix. Furthermore, we postulate that intracellular galectin-3 in the epithelium of the epididymis tail plays a role in either the maintenance of the epithelium or as a source of galectin-3 in the seminal fluid; here it may play a role in sperm activation in the boar reproductive system.     

Merkel cells, a new localization of prepro-orexin and orexin receptors

Orexins (OXA and OXB) are peptides derived from a common precursor called prepro-orexin. They act through G-protein receptors named orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R). Orexins were first demonstrated in neurons of the lateral hypothalamus and found to be related to the control of food intake. However, it has been shown that they are widely distributed in both the nervous system and peripheral tissues, including endocrine organs such as the pituitary and adrenal glands. Merkel cells are neuroendocrine cells situated in the epidermis, tactile hairs and oral mucosa, and act as mechanoreceptors. Up to the present, various neuropeptides have been detected in these cells. The aim of the present study was to detect the presence of prepro-orexin and orexin receptors (OX(1)R and OX(2)R) in porcine Merkel cells using immunohistochemistry. Prepro-orexin was expressed in the cytoplasm of Merkel cells in the skin of the pig snout. Immunoreactivity for prepro-orexin was more intense in the mature side of the cell, where the dense-cored granules are accumulated. Epidermal nerve terminals associated with Merkel cells and dermal nerve fibres showed no immunostaining. Both orexin receptors (OX(1)R and OX(2)R) were also demonstrated in the cytoplasm of Merkel cells of pig snout skin. The finding of orexins and their receptors in Merkel cells suggests that they have an autocrine function. Further studies are needed to ascertain the significance of this function.