Baseline prepulse inhibition dependency of orexin A and REM sleep deprivation.

RATIONALE: Prepulse inhibition (PPI) impairment reflects sensorimotor gating problems, i.e. in schizophrenia. This study aims to enlighten the role of orexinergic regulation on PPI in a psychosis-like model. OBJECTIVES: In order to understand the impact of orexinergic innervation on PPI and how it is modulated by age and baseline PPI (bPPI), chronic orexin A (OXA) injections was carried on non-sleep-deprived and sleep-deprived rats that are grouped by their bPPI. METHODS: bPPI measurements were carried on male Wistar rats on P45 or P90 followed by grouping into low-PPI and high-PPI rats. The rats were injected with OXA twice per day for four consecutive days starting on P49 or P94, while the control groups received saline injections. 72 h REMSD was carried on via modified multiple platform technique on P94 and either OXA or saline was injected during REMSD. PPI tests were carried out 30 min. after the last injection. RESULTS: Our previous study with acute OXA injection after REMSD without bPPI grouping revealed that low OXA doses might improve REMSD-induced PPI impairment. Our current results present three important conclusions: (1) The effect of OXA on PPI is bPPI-dependent and age-dependent. (2) The effect of REMSD is bPPI-dependent. (3) The effect of OXA on PPI after REMSD also depends on bPPI. CONCLUSION: Orexinergic regulation of PPI response with and without REMSD can be predicted by bPPI levels. Our findings provide potential insights into the regulation of sensorimotor gating by sleep/wakefulness systems and present potential therapeutic targets for the disorders, where PPI is disturbed.

Intranasal orexin A modulates sympathetic vascular tone: a pilot study in healthy male humans.

Previous research suggests that the neuropeptide orexin A contributes to sympathetic blood pressure (BP) control inasmuch as hypothalamic injection of orexin A increases sympathetic vasomotor tone and arterial BP in rodents. In humans with narcolepsy, a disorder associated with loss of orexin-producing neurons, vasoconstrictive muscle sympathetic nerve activity (MSNA) is reduced. Since intranasally administered oligopeptides like orexin are known to modulate brain function, we investigated the effect of intranasal orexin A on vascular sympathetic baroreflex function in healthy humans. In a balanced, double-blind crossover study, orexin A (500 nmol) and placebo, respectively, were intranasally administered to 10 lean healthy males (age 25.8 ± 4.6 yr). MSNA was assessed microneurographically before and 30-45 min after either substance administration. Additionally, baroreflex was challenged via graded infusions of vasoactive drugs before and after substance administration. Baroreflex function was defined as the correlation of BP with MSNA and heart rate. Intranasal orexin A compared with placebo induced a significant increase in resting MSNA from pre-to postadministration [Δburst rate, orexin A vs. placebo: +5.8 ± 0.8 vs. +2.1 ± 0.6 bursts/min, P = 0.007; total activity 169 ± 11.5% vs. 115 ± 5.0%; P = 0.002]. BP, heart rate, and sympathovagal balance to the heart, as represented by heart rate variability (HRV), as well as baroreflex sensitivity during the vasoactive challenge were not altered. Intranasally administered orexin A acutely induced vasoconstrictory sympathoactivation in healthy male humans. This result suggests that orexin A mediates upward resetting of the vascular baroreflex set point at centers superordinate to the mere baroreflex feedback loop.NEW & NOTEWORTHY Our pilot study adds another important part to the complex network of neuroendocrine-sympathetic interaction. Our results demonstrate that intranasal orexin A elicits an excitatory effect on sympathetic vascular tone superordinate to mere baroreflex feedback regulation. This resetting of the baroreflex set point suggests an activation of hypothalamic core centers such as the paraventricular nucleus (PVN). The role of the orexinergic system in the development of neurogenic arterial hypertension warrants further investigations.

The Potential Therapeutic Effect of Orexin-Treated versus Orexin-Untreated Adipose Tissue-Derived Mesenchymal Stem Cell Therapy on Insulin Resistance in Type 2 Diabetic Rats.

Type 2 diabetes mellitus is a chronic metabolic disease characterized by resistance to peripheral insulin actions. Mesenchymal stem cells have been studied for years in T2DM therapy, including adipose tissue-derived mesenchymal stem cells (AD-MSCs). Orexin neuropeptides (A and B) are well-known regulators of appetite and physical activity. The aim of this work was to elucidate the possible therapeutic effect of AD-MSC preconditioning with orexin A (OXA) on insulin resistance in rats. Twenty-eight adult male albino rats were divided into 4 equal groups: a normal control group and 3 diabetic groups (a control T2DM group, diabetic rats treated by an AD-MSCs group, and diabetic rats treated by AD-MSCs preconditioned with OXA). We noticed that the treated groups showed a significant alleviation of insulin resistance parameters as shown in lowering the serum levels of glucose, insulin, total cholesterol, inflammatory markers, and HOMA-IR as compared to the control diabetic group with more significant reduction observed in the OXA-pretreated AD-MSCs-administrated group. More improvement was also noted in the glucose uptake and GLUT-4 gene expression in the skeletal muscle and adipose tissue in the OXA-pretreated AD-MSCs-administrated group compared to the untreated diabetic group. Conclusion. Preconditioning of AD-MSCs with OXA can significantly increase their potential to reduce the insulin resistance in the rat model of T2DM.

Orexin-A alleviates astrocytic apoptosis and inflammation via inhibiting OX1R-mediated NF-κB and MAPK signaling pathways in cerebral ischemia/reperfusion injury.

Orexin-A (OXA) is a neuropeptide with neuroprotective effect by reducing cerebral ischemia/reperfusion injury (CIRI). Inflammation and apoptosis mediated by astrocyte activation are the key pathological mechanisms for CIRI. We thus attempted to confirm neuroprotective effects of OXA on astrocytic inflammation and apoptosis in CIRI and clarify the relative mechanisms. A middle cerebral artery occlusion and reperfusion (MCAO/R) rat model and U251 glioma cells model subjected to oxygen glucose deprivation and reperfusion (OGD/R) were established, with or without OXA treatment. Neurological deficit score was determined, and cerebral infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Western Blot was used to detect the expressions of NF-κB p65, p-p65, p-ERK, p-p38, GFAP, OX1R, IL-1ß, TNF-α, IL-6, iNOS, Bcl-2, Bax, CytC, cleaved caspase-9 and cleaved caspase-3 in vivo and in vitro. Pro-inflammatory cytokines in cell supernatant IL-1ß, TNF-α and IL-6 were determined by ELISA. Hoechst 33342 staining was used to detect the apoptosis of astrocyte. Immunofluorescent staining was performed to assess the nuclear translocation of p65 and the expression of GFAP. The results showed that OXA significantly improved neurological deficit score and decreased the volume of infarct area in brain. OXA decreased inflammatory mediators, inhibited astrocyte activation and nuclear translocation of NF-κB and phosphorylation of NF-κB, MAPK/ERK and MAPK/p38. Besides, OXA suppressed apoptosis via upregulating the ratio of Bcl-2/Bax and downregulating cytochrome C, cleaved-caspase-9 and cleaved caspase-3. Overall, it was concluded that OXA exerts neuroprotective effect during CIRI through attenuating astrocytes apoptosis, astrocytes activation and pro-inflammatory cytokines production, by Inhibiting OX1R-mediated NF-κB, MAPK/ERK and MAPK/p38 signaling pathways. The progress in our study is helpful to elucidate the molecular mechanisms of OXA neuroprotection, which could lead to the development of new treatment strategies for ischemic stroke.

Behavioural and cardiovascular effects of orexin-A infused into the central amygdala under basal and fear conditions in rats.

The neuropeptide orexin-A (OX-A) has diverse functions, including maintaining arousal, autonomic control, motor activity and stress responses. These functions are regulated at different terminal regions where OX-A is released. The current study examined the physiological and behavioural effects of OX-A microinjections into the central amygdala (CeA) under basal and stressed conditions in rats. When OX-A was microinjected into the CeA and the animals returned to the home-cage, heart rate and mean arterial pressure were increased compared to vehicle-injected controls. General activity of the animal was also increased, indicating that OX-A activity in CeA contributes to increased arousal. This outcome is similar to the effects of central intracerebroventricular infusions of OX-A, as well as the cardiovascular effects previously demonstrated at many of OX's efferent hypothalamic and brainstem structures. In a second study, animals were fear-conditioned to a context by delivery of electric footshocks and then animals were re-exposed to the conditioned context at test. When OX-A was microinjected at test, freezing behaviour was reduced and there was a corresponding increase in the animal's activity but no impact on the pressor and cardiac responses (i.e, blood pressure and heart rate were unchanged). This reduction in freezing suggests that OX-A activates amygdala neurons that inhibit freezing, which is similar to the actions of other neuropeptides in the CeA that modulate the appropriate defence response to fearful stimuli. Overall, these data indicate that the CeA is an important site of OX-A modulation of cardiovascular and motor activity, as well as conditioned freezing responses.

Blockade of corticotropin-releasing factor receptor 1 in the central amygdala prevents cocaine-seeking behaviour induced by orexin-A administered to the posterior paraventricular nucleus of the thalamus in male rats.

Background: Orexin-A (OrxA) administration in the posterior paraventricular nucleus of the thalamus (pPVT) reinstates extinguished cocaine-seeking behaviour following extended access to the drug (a model of dependence). The pPVT receives and integrates information associated with emotionally salient events and sends excitatory inputs to brain regions involved in the expression of emotional states, such as those driving cocaine-seeking behaviour (i.e., the nucleus accumbens, the central nucleus of the amygdala [CeA], the basolateral amygdala, the bed nucleus of the stria terminalis [BNST] and the prefrontal cortex). Methods: We monitored the activation pattern of these regions (measured by Fos) during cocaine-seeking induced by OrxA administered to the pPVT. The BNST and CeA emerged as being selectively activated. To test whether the functionality of these regions was pivotal during OrxA-induced cocaine-seeking behaviour, we transiently inactivated these regions concomitantly with OrxA administration to the pPVT. We then tested the participation of corticotropin-releasing factor receptors (CRF1) in the CeA during OrxA-induced cocaine-seeking using the CRF1 antagonist CP154526. Results: We observed selective activation of the CeA and BNST during cocaine-seeking induced by OrxA administered to the pPVT, but only transient inactivation of the CeA prevented cocaine-seeking behaviour. Administration of CP154526 to the CeA prevented OrxAinduced cocaine-seeking behaviour. Limitations: The use of only male rats could have been a limitation. Other limitations could have been the use of an indirect approach to test the hypothesis that administration of OrxA to the pPVT drives cocaine-seeking via CRF1 signalling in the CeA, and a lack of analysis of the participation of CeA subregions. Conclusion: Cocaine-seeking behaviour induced by OrxA administered to the pPVT is driven by activation of the CeA via CRF1 signalling.

Orexin-A Intensifies Mouse Pupillary Light Response by Modulating Intrinsically Photosensitive Retinal Ganglion Cells.

We show for the first time that the neuropeptide orexin modulates pupillary light response, a non-image-forming visual function, in mice of either sex. Intravitreal injection of the orexin receptor (OXR) antagonist TCS1102 and orexin-A reduced and enhanced pupillary constriction in response to light, respectively. Orexin-A activated OX1Rs on M2-type intrinsically photosensitive retinal ganglion cells (M2 cells), and caused membrane depolarization of these cells by modulating inward rectifier potassium channels and nonselective cation channels, thus resulting in an increase in intrinsic excitability. The increased intrinsic excitability could account for the orexin-A-evoked increase in spontaneous discharges and light-induced spiking rates of M2 cells, leading to an intensification of pupillary constriction. Orexin-A did not alter the light response of M1 cells, which could be because of no or weak expression of OX1Rs on them, as revealed by RNAscope in situ hybridization. In sum, orexin-A is likely to decrease the pupil size of mice by influencing M2 cells, thereby improving visual performance in awake mice via enhancing the focal depth of the eye's refractive system.SIGNIFICANCE STATEMENT This study reveals the role of the neuropeptide orexin in mouse pupillary light response, a non-image-forming visual function. Intravitreal orexin-A administration intensifies light-induced pupillary constriction via increasing the excitability of M2 intrinsically photosensitive retinal ganglion cells by activating the orexin receptor subtype OX1R. Modulation of inward rectifier potassium channels and nonselective cation channels were both involved in the ionic mechanisms underlying such intensification. Orexin could improve visual performance in awake mice by reducing the pupil size and thereby enhancing the focal depth of the eye's refractive system.

Orexin Modulation of VTA Dopamine Neuron Activity: Relevance to Schizophrenia.

BACKGROUND: The hippocampus is a region consistently implicated in schizophrenia and has been advanced as a therapeutic target for positive, negative, and cognitive deficits associated with the disease. Recently, we reported that the paraventricular nucleus of the thalamus (PVT) works in concert with the ventral hippocampus to regulate dopamine system function; however, the PVT has yet to be investigated as target for the treatment of the disease. Given the dense expression of orexin receptors in the thalamus, we believe these to be a possible target for pharmacological regulation of PVT activity. METHODS: Here we used the methylazoxymethanol acetate (MAM) rodent model, which displays pathological alterations consistent with schizophrenia to determine whether orexin receptor blockade can restore ventral tegmental area dopamine system function. We measured dopamine neuron population activity, using in vivo electrophysiology, following administration of the dual orexin antagonist, TCS 1102 (both intraperitoneal and intracranial into the PVT in MAM- and saline-treated rats), and orexin A and B peptides (intracranial into the PVT in naïve rats). RESULTS: Aberrant dopamine system function in MAM-treated rats was normalized by the systemic administration of TCS 1102. To investigate the potential site of action, the orexin peptides A and B were administered directly into the PVT, where they significantly increased ventral tegmental area dopamine neuron population activity in control rats. In addition, the direct administration of TCS 1102 into the PVT reproduced the beneficial effects seen with the systemic administration in MAM-treated rats. CONCLUSION: Taken together, these data suggest the orexin system may represent a novel site of therapeutic intervention for psychosis via an action in the PVT.

Orexin-A alleviates cerebral ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress-mediated apoptosis.

Orexin­A (OXA) protects neurons against cerebral ischemia­reperfusion injury (CIRI). Endoplasmic reticulum stress (ERS) induces apoptosis after CIRI by activating caspase­12 and the CHOP pathway. The present study aimed to determine whether OXA mitigates CIRI by inhibiting ERS­induced neuronal apoptosis. A model of CIRI was established, in which rats were subjected to middle cerebral artery occlusion with ischemic intervention for 2 h, followed by reperfusion for 24 h. Neurological deficit examination and 2,3,5­triphenyltetrazolium chloride staining were performed to assess the level of CIRI and neuroprotection by OXA. Expression levels of ERS­related proteins and cleaved caspase­3 were measured via western blotting, while the rate of neuronal apoptosis in the cortex was determined using a TUNEL assay. OXA treatment decreased the infarct volume of rats after CIRI and attenuated neuron apoptosis. Furthermore, administration of OXA decreased the expression levels of GRP78, phosphorylated (p)­PERK, p­eukaryotic initiation factor­2α, p­inositol requiring enzyme 1α, p­JNK, cleaved caspase­12, CHOP and cleaved caspase­3, all of which were induced by CIRI. Collectively, these findings suggested that OXA attenuated CIRI by inhibiting ERS­mediated apoptosis, thus clarifying the mechanism underlying its neuroprotective effect and providing a novel therapeutic direction for the treatment of CIRI.

Involvement of the tuberomammillary nucleus of the hypothalamus in the modulation of nociception and joint edema in a model of monoarthritis.

AIMS: Investigate the involvement of the histaminergic projections from tuberomammillary nucleus (TMN) to the spinal cord in the modulation of nociception and peripheral edema in a model of monoarthritis. MAIN METHODS: Subacute monoarthritis was induced by an intraarticular injection of carrageenan followed by LPS 72 h later. Disability and joint edema were assessed at the 3rd hour after LPS and at every hour up to 6 h. KEY FINDINGS: Intrathecal administration of histamine potentiated joint incapacitation and edema, while the H1R antagonist cetirizine decreased both. The H3R agonist immepip decreased both incapacitation and edema, while the H3R antagonist thioperamide had the opposite effect. The microinjection of glutamate into the ventral TMN (vTMN) caused an increase of incapacitation and articular edema, whereas the blockade of this nucleus by cobalt chloride inhibited both parameters. Intrathecal administration of cetirizine prevented the increase of incapacitation and joint edema caused by glutamate microinjection into the vTMN. Similarly, an intrathecal injection of the NKCC1 cotransporter inhibitor bumetanide prevented the effects of glutamate microinjection into the vTMN, whereas coadministration of histamine with bumetanide only inhibited the potentiation of joint edema. A microinjection of orexin B into the vTMN potentiated incapacitation and joint edema, while coadministration of the OX1/2 receptor antagonist almorexant with orexin B did not. SIGNIFICANCE: These data support the notion that TMN participates in the modulation of a peripheral inflammatory process by means of histaminergic projections to the spinal cord, and the hypothalamus may trigger TMN activation by means of glutamate and orexin.

Centrally administered orexin prevents lipopolysaccharide and colchicine induced lethality via the vagal cholinergic pathway in a sepsis model in rats.

Orexins are neuropeptides implicated in several physiological functions. Accumulating findings suggest a relationship between orexin and sepsis. A recent study demonstrated that orexin acts centrally to improve conditions in sepsis. The present study aims to clarify the precise mechanisms by which central orexin could induce a protective action against septic conditions. We established a new septic model by treating rats with lipopolysaccharide (LPS) and colchicine and used this to examine the effect of brain orexin on survival. Observation of survival was stopped three days after the chemicals injection or at death. We established a lethal model (rats died within 24 h) by injecting subcutaneously a combination of 1 mg/kg LPS and 1 mg/kg colchicine. A Toll-like receptor 4 (TLR4) inhibitor completely blocked lethality, suggesting a vital role of LPS-TLR4 signaling in the process. Intracisternal orexin-A dose-dependently reduced lethality in the sepsis model while neither intracisternal orexin-B nor intraperitoneal orexin-A changed the mortality rate. Vagal stimulation with carbachol or 2-deoxy-D-glucose improved survival and atropine potently blocked the protection by carbachol or 2-deoxy-D-glucose. The orexin-A-induced reduction of lethality was significantly blocked by atropine or surgical vagotomy. Intracisternal injection of an OX1 receptor antagonist blocked the improvement of survival by intracisternal injection of orexin-A, carbachol, or 2-deoxy-D-glucose. These results suggest that orexin acts centrally to reduce the lethality in our septic model treated (LPS and colchicine). Activation of the vagal cholinergic pathway may mediate the action of orexin, and the OX1 receptor in the brain might play a role in the process. Since the efferent vagus nerve mediates anti-inflammatory mechanisms, we speculate that the vagal cholinergic anti-inflammatory pathway is implicated in the mechanisms of septic lethality reduction by brain orexin.

Orexin A injection into the ventral medial prefrontal cortex induces antidepressant-like effects: Possible involvement of local Orexin-1 and Trk receptors.

RATIONALE: Reduced levels of orexin-A (OXA) in the central nervous system (CNS) have been associated with the pathophysiology of depression and its exogenous administration promotes antidepressant-like effect. The mechanisms associated with these effects are, however, not yet known. Herein, we investigated the hypothesis that OXA effects could be associated with orexin 1 receptor (OX1R) and tyrosine receptor kinase B (TrkB) activation, in the ventromedial prefrontal cortex (vmPFC), a brain region that is central to depression neurobiology. OBJECTIVES: 1. To Investigate the effects induced by OXA administration into the vmPFC; 2. Evaluate the participation of OX1R and TrkB in behavioral responses induced by OXA. METHODS: Male Wistar rats received intra-vmPFC injections of OXA (10, 50 and 100 pmol/0.2 µL) and were exposed to the forced swimming test (FST) or the open field test (OFT). Independent groups received an intra-vmPFC injection of SB334867 (OX1R antagonist, 10 nmol/0.2 µL) or K252a (non-selective Trk antagonist, 10 pmol/0.2 µL), before local injection of OXA, and were exposed to the same tests. RESULTS: OXA injection (100 pmol/0.2 µL) into the vmPFC induced antidepressant-like effect, which was prevented by SB334867 and K252a pretreatments. CONCLUSION: OXA signaling in the vmPFC induces antidepressant-like effect in the FST which is dependent on OX1R and Trk receptors.

Pharmacological characterization of a novel potent, selective, and orally active orexin 2 receptor antagonist, SDM-878.

AIMS: Recently, we identified a novel orexin 2 (OX2 ) receptor antagonist, SDM-878 (2-(3-(2-(1H-pyrazol-1-yl)nicotinoyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-3-methoxyisonicotinonitrile). The purpose of the present study is to characterize the in vitro and in vivo pharmacological effects of SDM-878. METHODS: The in vitro potency and selectivity of SDM-878 were examined in CHO cells that exhibit stable expression of human orexin 1 (OX1 ), human orexin 2 (OX2 ), rat OX1 , and rat OX2 receptors. Then, the plasma half-life, oral bioavailability, and brain penetration of SDM-878 were examined in rats. The in vivo effect of SDM-878 in rats was tested using electroencephalography (EEG). The target engagement of SDM-878 in the rat brain was examined using the antagonistic effect against hyperlocomotion caused by the intracerebroventricular administration of the OX2 receptor agonist, ADL-OXB ([Ala11 , d-Leu15 ]-orexin B). RESULTS: SDM-878 showed potent inhibitory activities for human and rat OX2 receptors with IC values of 10.6 and 8.8 nM, respectively, and approximately 1000-fold selectivity against the OX1 receptor. In rat studies, SDM-878 exhibited a relatively short half-life in plasma, oral bioavailability, and good brain penetration. These data indicate that SDM-878 is a potent, selective, orally active, and brain-penetrable OX2 receptor antagonist. In behavioral studies using rats, SDM-878 (100 mg/kg) antagonized hyperlocomotion caused by intracerebroventricular administration of ADL-OXB. SDM-878 exhibited a potent sleep-promoting effect at the same dose (100 mg/kg) in a rat EEG study. CONCLUSION: Our results suggest that SDM-878 is likely to be a good pharmacological tool for investigating the role of the OX2 receptor and may have therapeutic potential for the treatment of insomnia.

Intranasal administration of orexin peptides: Mechanisms and therapeutic potential for age-related cognitive dysfunction.

Cognitive impairment is a core feature of several neuropsychiatric and neurological disorders, including narcolepsy and age-related dementias. Current pharmacotherapeutic approaches to cognitive enhancement are few in number and limited in efficacy. Thus, novel treatment strategies are needed. The hypothalamic orexin (hypocretin) system, a central integrator of physiological function, plays an important role in modulating cognition. Several single- and dual-orexin receptor antagonists are available for various clinical and preclinical applications, but the paucity of orexin agonists has limited the ability to research their therapeutic potential. To circumvent this hurdle, direct intranasal administration of orexin peptides is being investigated as a prospective treatment for cognitive dysfunction, narcolepsy or other disorders in which deficient orexin signaling has been implicated. Here, we describe the possible mechanisms and therapeutic potential of intranasal orexin delivery. Combined with the behavioral evidence that intranasal orexin-A administration improves cognitive function in narcoleptic and sleep-deprived subjects, our neurochemical studies in young and aged animals highlights the capacity for intranasal orexin administration to improve age-related deficits in neurotransmission. In summary, we highlight prior and original work from our lab and from others that provides a framework for the use of intranasal orexin peptides in treating cognitive dysfunction, especially as it relates to age-related cognitive disorders.

Orexin-A Exerts Equivocal Role in Atherosclerosis Process Depending on the Duration of Exposure: In Vitro Study.

Orexin-A is a peptide hormone that plays a crucial role in feeding regulation and energy homeostasis. Diurnal intermittent fasting (DIF) has been found to increase orexin-A plasma levels during fasting hours, while Ramadan fasting which resembles DIF, has led to beneficial effects on endothelial function. Herein, we aimed to investigate the effects of orexin-A on the expression of molecules involved in the atherogenesis process: Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), in human aortic endothelial cells (HAECs). HAECs were incubated with orexin-A at concentrations of 40 ng/mL, 200 ng/mL and 400 ng/mL for 6, 12 and 24 h. The mRNA levels of MCP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 and orexin-1 receptor were measured by real-time qPCR. We also evaluated the MMP-2, p38, phospho-p38, NF-κΒ/p65 as well as TIMP-1 protein levels by Western blot and ELISA, respectively. MMP-2 activity was measured by gelatin zymography. Short-term 6-h incubation of HAECs with orexin-A at a high concentration (400 ng/mL) decreased MCP-1, MMP-2 expression, MMP-2/TIMP-1 ratio (p < 0.05), and MMP-2 activity, while incubation for 24 h increased MCP-1, MMP-2 expression (p < 0.05), MMP-2/TIMP-1 and MMP-2/TIMP-2 ratio (p < 0.01 and p < 0.05, respectively) as well as MMP-2 activity. The dual effects of orexin-A are mediated, at least in part, via regulation of p38 and NF-κΒ pathway. Orexin-A may have an equivocal role in atherosclerosis process with its effects depending on the duration of exposure.

Orexin activated emergence from isoflurane anaesthesia involves excitation of ventral tegmental area dopaminergic neurones in rats.

BACKGROUND: Orexin can facilitate emergence after general anaesthesia via multiple neural pathways. Dopaminergic neurones in the ventral tegmental area (VTA) participate in behavioural arousal from anaesthesia. We investigated the regulation of dopaminergic VTA neurones by orexinergic neurones during emergence from general anaesthesia. METHODS: Orexins were microinjected into the VTA to determine the effects on isoflurane anaesthesia induction, emergence, and maintenance. Orexin receptors and dopaminergic neurones in the VTA were identified using immunofluorescence. Orexinergic terminals in the VTA were optogenetically regulated to detect the endogenous orexin-mediated regulation of dopaminergic neurones during anaesthesia in Hcrtcre rats. RESULTS: Injection of orexin-A (100 pmol) into the VTA reduced emergence time [from 949 (118) to 727 (101) s; P=0.0058] and reduced the electroencephalographic burst-suppression ratio (BSR) (26.6 [10.2]% vs 44.3 [6.8]%; P=0.0027) during isoflurane anaesthesia. The percentage of dopaminergic neurones that expressed either orexin-1 receptor or orexin-2 receptor was 73.4 (5.0)% and 74.4 (62.4)%, respectively. Optogenetic activation of orexinergic projections to the VTA reduced the BSR (from 40.5 [2.7]% to 22.4 [11.8]%; P=0.0019) and facilitated emergence (915 [89] vs 685 [68] s; P=0.0026), whereas optical inhibition prolonged the time to wakefulness (from 941 [92] to 1279 [250] s; P=0.011). Dopaminergic neurones in the VTA showed increased firing frequency (387 [78]% of control, P=0.005) after bath application of orexin-A. CONCLUSIONS: Orexin promotes emergence from isoflurane anaesthesia through activation of dopaminergic neurones in the VTA.

Systemic administration of orexin A ameliorates established experimental autoimmune encephalomyelitis by diminishing neuroinflammation.

BACKGROUND: Orexins (hypocretins, Hcrt) A and B are GPCR-binding hypothalamic neuropeptides known to regulate sleep/wake states and feeding behavior. A few studies have shown that orexin A exhibits anti-inflammatory and neuroprotective properties, suggesting that it might provide therapeutic effects in inflammatory and neurodegenerative diseases like multiple sclerosis (MS). In MS, encephalitogenic Th1 and Th17 cells trigger an inflammatory response in the CNS destroying the myelin sheath. Here, we investigated the effects of peripheral orexin A administration to mice undergoing experimental autoimmune encephalomyelitis (EAE), a widely used model of MS. METHODS: Mice were subcutaneously immunized with myelin oligodendrocyte glycoprotein peptide (MOG)35-55 in CFA. Mice were treated intraperitoneally for five consecutive days with either PBS or 300 µg of orexin A starting at a moderate EAE score. Molecular, cellular, and histological analysis were performed by real-time PCR, ELISA, flow cytometry, and immunofluorescence. RESULTS: Orexin A strongly ameliorated ongoing EAE, limiting the infiltration of pathogenic CD4+ T lymphocytes, and diminishing chemokine (MCP-1/CCL2 and IP-10/CXCL10) and cytokine (IFN-γ (Th1), IL-17 (Th17), TNF-α, IL-10, and TGF-ß) expressions in the CNS. Moreover, orexin A treatment was neuroprotective, decreasing demyelination, astrogliosis, and microglial activation. Despite its strong local therapeutic effects, orexin A did not impair peripheral draining lymph node cell proliferation and Th1/Th17 cytokine production in response to MOG35-55 in vitro. CONCLUSIONS: Peripherally-administered orexin A ameliorated EAE by reducing CNS neuroinflammation. These results suggest that orexins may represent new therapeutic candidates that should be further investigated for MS treatment.

Excitation of Cortical nNOS/NK1R Neurons by Hypocretin 1 is Independent of Sleep Homeostasis.

We have proposed that cortical nNOS/NK1R interneurons have a role in sleep homeostasis. The hypocretins (orexins) are wake-promoting neuropeptides and hypocretin/orexin (Hcrt) neurons project to the cortex. Hcrt peptides affect deep layer cortical neurons, and Hcrt receptor 1 (Hcrtr1; Ox1r) mRNA is expressed in cortical nNOS/NK1R cells. Therefore, we investigated whether Hcrt neuron stimulation affects cingulate cortex nNOS/NK1R neurons. Bath application of HCRT1/orexin-A evoked an inward current and membrane depolarization in most nNOS/NK1R cells which persisted in tetrodotoxin; optogenetic stimulation of Hcrt terminals expressing channelrhodopsin-2 confirmed these results, and pharmacological studies determined that HCRTR1 mediated these responses. Single-cell RT-PCR found Hcrtr1 mRNA in 31% of nNOS/NK1R cells without any Hcrtr2 mRNA expression; immunohistochemical studies of Hcrtr1-EGFP mice confirmed that a minority of nNOS/NK1R cells express HCRTR1. When Hcrt neurons degenerated in orexin-tTA;TetO DTA mice, the increased EEG delta power during NREM sleep produced in response to 4 h sleep deprivation and c-FOS expression in cortical nNOS/NK1R cells during recovery sleep were indistinguishable from that of controls. We conclude that Hcrt excitatory input to these deep layer cells is mediated through HCRTR1 but is unlikely to be involved in the putative role of cortical nNOS/NK1R neurons in sleep homeostasis.

Activation of orexin-1 receptors in the ventrolateral periaqueductal grey matter (vlPAG) modulates pulpal nociception and the induction of substance P in vlPAG and trigeminal nucleus caudalis.

AIM: To characterize the role of orexin-1 receptors (OX1Rs) in ventrolateral periaqueductal grey matter (vlPAG) on modulation of capsaicin-induced pulpal nociception in rats. METHODOLOGY: Sixty-six adult male Wistar rats (2 months old) weighing between 230 and 260 g were used. The animals were cannulated for microinjection of drugs into the vlPAG matter. Pulpalgia was induced by intradental application of capsaicin solution (100 µg) into the incisor teeth of the rats. Ten min prior to capsaicin application, orexin-A (50, 100 and 150 pmol L-1 per rat) was administered. Orexin-A (150 pmol L-1 ) was also co-administrated with SB-334867 (40 nmol L-1 per rat), an OX1Rs antagonist; or bicuculline (1 µg per rat), a GABAA receptors antagonist. Moreover, treatment effects on the release of pro-nociceptive modulator substance P (SP) in vlPAG and trigeminal nucleus caudalis (Vc) of rats were explored using an immunofluorescence technique. One-way analysis of variance was used for the statistical analysis. RESULTS: Orexin-A dose-dependently decreased capsaicin-induced nociceptive behaviour. However, SB-334867 (40 nmol L-1 per rat) pretreatment (P < 0.05), but not bicuculline (1 µg per rat), attenuated the analgesic effect of orexin-A (150 pmol L-1 ). The level of SP was significantly increased in Vc and decreased in vlPAG of capsaicin-treated rats (P < 0.05). Capsaicin-induced changes in SP levels, however, were prohibited by orexin-A treatment (150 pmol L-1 ) (P < 0.05). CONCLUSIONS: Orexin-A administration into the vlPAG was associated with an inhibitory effect on capsaicin-induced pulpal nociception and bidirectional effects on the induction of SP in vlPAG and Vc of rats. Central activation of OX1Rs is a potential therapeutic tool for pulpalgia.

Co-administration of the low dose of orexin and nitrergic antagonists induces an antidepressant-like effect in mice.

It is now well-established that orexins (OXs) and their receptors are involved in the pathophysiology of depression. Considering the evidence indicating the importance of nitric oxide (NO) system in the mood modulation, this study investigated the effect of intraperitoneal (i.p.) administration of orexin 1 (OX1) receptor antagonist -SB334867- alone or in combination with NO agents on depression using the forced swimming test (FST), tail suspension test (TST) and the number of crossings in open-field test (OFT) in mice. Our results indicated that administration of SB334867 at the dose of 0.5 mg/kg decreased the immobility time in the FST without effect on locomotor activity, suggesting an antidepressant-like effect of SB334867. Moreover, l-Arginine (a NO precursor; 750 mg/kg) or L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor, 10 mg/kg) administration by itself decreased the immobility time in the FST. Interestingly, co-administration of a sub-threshold dose of L-NAME, but not l-Arginine, in combination with an ineffective dose of SB334867 produced an antidepressant-like effect in the FST and TST. It should be noted, none of the drugs elicited significant effects on the locomotor activity in the OFT. Altogether, the present data propose that a combination of the sub-effective dose of OX and NO antagonists can be evaluated as an option for the clinical treatment of depression in humans.