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Orexin-producing cells in the lateral hypothalamic area have been shown to be involved in a wide variety of behavioral and cognitive functions, including the recall of appetitive associations and a variety of social behaviors. Here, we investigated the role of orexin in the acquisition and recall of socially transmitted food preferences in the rat. Rats were euthanized following either acquisition, short-term recall, or long-term recall of a socially transmitted food preference and their brains were processed for orexin-A and c-Fos expression. We found that while there were no significant differences in c-Fos expression between control and experimental subjects at any of the tested timepoints, females displayed significantly more activity in both orexinergic and non-orexinergic cells in the lateral hypothalamus. In the infralimbic cortex, we found that social behavior was significantly predictive of c-Fos expression, with social behaviors related to olfactory exploration appearing to be particularly influential. We additionally found that appetitive behavior was significantly predictive of orexin-A activity in a sex-dependent matter, with the total amount eaten correlating negatively with orexin-A/c-Fos colocalization in male rats but not female rats. These findings suggest a potential sex-specific role for the orexin system in balancing the stimulation of feeding behavior with the sleep/wake cycle.

PURPOSE The orexigenic peptides, ghrelin, galanin, and orexin-A, have an important role in food intake and energy homeostasis and regulate the higher brain functions including the sleep-wake state. Although the interactions of these neuropeptides affect neuroendocrine systems resulting in obesity, a major risk factor for obstructive sleep apnea syndrome (OSAS), the mechanism has not been fully elucidated. The objective of this study was to evaluate the association of serum ghrelin, galanin, and orexin-A levels with OSAS. METHODS In this cross-sectional study, patients who underwent one-night polysomnography and conformed to the inclusion criteria were asked to participate. A blood sample was obtained from all participants on the morning of the sleep test to evaluate the serum levels of ghrelin, galanin, and orexin-A using the enzyme-linked immunosorbent assay (ELISA) method. Demographic characteristics, polysomnography data, and serum levels of the participants were recorded and analyzed. Comparison between the OSAS groups was performed by independent sample t-test, Mann-Whitney U test, and Kruskal-Wallis test with post hoc K-W test using SPSS 20.0. RESULTS Of 272 patients, those in the OSAS group (n=210) were older than patients in the non-OSAS group (n=62), p < 0.003, and had increased BMI, p < 0.006. Patients with, serum ghrelin, galanin, and orexin-A levels were significantly elevated in patients with OSAS (635.9 pg/mL vs. 420.7 pg/mL, 91.0 pg/mL vs. 60.0 pg/mL, 600.3 pg/mL vs. 485.6 pg/mL, respectively) and found to be higher in patients with severe OSAS than mild and moderate cases (p

The mammalian brain stores and distinguishes among episodic memories, i.e. memories formed during the personal experience, through a mechanism of pattern separation computed in the hippocampal dentate gyrus. Decision-making for food-related behaviors, such as the choice and intake of food, might be affected in obese subjects by alterations in the retrieval of episodic memories. Adult neurogenesis in the dentate gyrus regulates the pattern separation. Several molecular factors affect adult neurogenesis and exert a critical role in the development and plasticity of newborn neurons. Orexin-A/hypocretin-1 and downstream endocannabinoid 2-arachidonoylglycerol signaling are altered in obese mice. Here, we show that excessive orexin-A/2-arachidonoylglycerol/cannabinoid receptor type-1 signaling leads to the dysfunction of adult hippocampal neurogenesis and the subsequent inhibition of plasticity and impairment of pattern separation. By inhibiting orexin-A action at orexin-1 receptors we rescued both plasticity and pattern separation impairment in obese mice, thus providing a molecular and functional mechanism to explain alterations in episodic memory in obesity., The authors show that adult hippocampal neurogenesis is altered in the dentate gyrus of obese mice with subsequent inhibition of long-term potentiation and impairment of pattern separation. Inhibition of orexin-A action at orexin-1 receptors rescued both impairments in obese mice.

Brain orexin system hyperactivity contributes to neurogenic hypertension. We previously reported upregulated neuronal kinin B1 receptor (B1R) expression in hypertension. However, the role of central B1R activation on the orexin system in neurogenic hypertension has not been examined. We hypothesized that kinin B1R contributes to hypertension via upregulation of brain orexin-arginine vasopressin signaling. We utilized deoxycorticosterone acetate (DOCA)-salt hypertension model in wild-type (WT) and B1R knockout (B1RKO) mice. In WT mice, DOCA-salt-treatment increased gene and protein expression of orexin A, orexin receptor 1, and orexin receptor 2 in the hypothalamic paraventricular nucleus and these effects were attenuated in B1RKO mice. Furthermore, DOCA-salt- treatment increased plasma arginine vasopressin levels in WT mice, but not in B1RKO mice. Cultured primary hypothalamic neurons expressed orexin A and orexin receptor 1. B1R specific agonist (LDABK) stimulation of primary neurons increased B1R protein expression, which was abrogated by B1R selective antagonist R715 but not by the dual orexin receptor antagonist, ACT 462206, suggesting that B1R is upstream of the orexin system. These data provide novel evidence that B1R blockade blunts orexin hyperactivity and constitutes a potential therapeutic target for the treatment of salt-sensitive hypertension.

Cerebral ischaemia is accompanied by infectious complications due to immunosuppression, known as stroke-induced immunodepression (SIID). Orexin-A (OXA), a neuropeptide produced in the hypothalamus, has been reported to have neuroprotective properties after stroke and is known to modulate inflammatory processes in peripheral tissues. The aim of this study was to determine the effects of orexin-A (OXA) on cerebral ischaemic inflammatory injury and SIID following experimental stroke. Cerebral ischaemia was induced in C57/BL6 mice by middle cerebral artery occlusion (MCAO). A mouse model of pneumonia and poststroke pneumococcal pneumonia was established by intratracheal inoculation with S. pneumoniae in a normal mouse or MCAO mouse model on the third day. We found that OXA postconditioning inhibited cerebral ischaemic inflammatory injury. The mechanism involved downregulation of the NF-κB signalling pathway. In addition, OXA may serve as a potential treatment target for attenuating stroke-induced immunodepression in mice.

Objective(s): To investigate the regulatory effects of the nucleus accumbens (NAcSh)-lateral hypothalamus (LHA) GABAergic neural pathway on palatable food (PF) intake via orexin-A expression in diet-induced obesity (DIO) rats. Materials and Methods: NAcSh-LHA GABAergic pathways were observed by fluorogold retrograde tracing combined with fluorescence immunohistochemistry, and the regulatory effects of this neural pathway on PF intake were detected after 1) microinjection of GABA-A receptor agonist muscimol (MUS) or antagonist bicuculine (BIC) into LHA, 2) electrical stimulation NAcSh, and 3) blocking the orexin-A receptor by icv SB334867. Results: Compared with rats on a normal diet (ND), NAcSh-LHA GABAergic neurons in the DIO rats were significantly decreased, and orexin-A expression in LHA significantly increased (p 0.05). After NAcSh electrical stimulation or SB334867 ICV injection, the PF intake was significantly decreased in the DIO rats (p 0.05). Conclusion: This GABAergic pathway could regulate the expression of orexin-A in LHA and PF intake. Orexin-A neurons in LHA of DIO rats might be less sensitive to GABAergic signals and may consequently lead to more hedonic food intake.

Purpose Time-restricted feeding (TRF) reverses obesity and insulin resistance, yet the central mechanisms underlying its beneficial effects are not fully understood. Recent studies suggest a critical role of hypothalamic galanin and its receptors in the regulation of energy balance. It is yet unclear whether TRF could regulate the expression of galanin and its receptors in the hypothalamus of mice fed a high-fat diet. Methods To test this effect, we subjected mice to either ad lib or TRF of a high-fat diet for 8 h per day. After 4 weeks, galanin and many neuropeptides associated with the function of metabolism were examined. Results The present findings showed that mice under TRF consume equivalent calories from a high-fat diet as those with ad lib access, yet are protected against obesity and have improved glucose metabolism. Plasma galanin, orexin A, irisin and adropin levels were significantly reversed by TRF regimen. Besides, TRF regimen reversed the progression of metabolic disorders in mice by increasing GLUT4 and PGC-1α expression in skeletal muscles. Moreover, the levels of galanin and GALR1 expression were severely diminished in the hypothalamus of the TRF mice, whereas GALR2 was highly expressed. Conclusions TRF diminished galanin and GALR1 expression, and increased GALR2 expression in the hypothalamus of mice fed a high-fat diet. The current studies provide additional evidence that TRF is effective in improving HFD-induced hyperglycemia and insulin resistance in mice, and this effect could be associated with TRF-induced changes of the galanin systems in the hypothalamus. Level of evidence No level of evidence, animal studies.

Introduction: Orexin-A is a neuropeptide synthesized in the lateral hypothalamus. Orexin-A immunoreactive fibres overlap distribution with GnRH neurons. In adult rats, orexin A is known to affect LH secretion via GnRH release modulation. Because data concerning the impact of orexin-A on the hypothalamo-pituitary axis activity are limited, we focused on the involvement of orexin-A and receptors of NPY in the modulation of LH release and LH subunit b (Lhb) mRNA expression in prepubertal female rats. Material and methods: Forty immature female Wistar rats were divided into 4 groups and received 2 intracerebroventricular (icv) microinjections of: 1 — artificial cerebrospinal fluid (CSF) (controls); 2 — CSF followed by orexin A; 3 — selective NPY receptor antagonist (BIBP) followed by CSF; 4 — BIBP followed by orexin A. One hour after the last microinjection, all rats were decapitated. Trunk blood was collected, and serum was stored at –20°C for the LH RIA examination. The adenohypophysis was immediately excised, flash-frozen, and kept at –80°C for RNA extraction. Real-time PCR amplification was carried out, and relative Lhb gene expression was calculated. Results: In comparison to the CSF-treated controls with a mean LH serum concentration of 0.40 ± 0.02 ng/mL, the mean LH serum level was diminished both after orexin-A (0.27 ± 0.01 ng/mL) and after BIBP (0.30 ± 0.02 ng/mL) icv microinjections. In the presence of BIBP, orexin-A more effectively inhibited LH release (0.20 ± 0.01 ng/mL) when compared to the BIBP-treated group. Orexin-A and BIBP exerted a consistent inhibitory effect on Lhb mRNA expression levels in the anterior pituitary gland. In comparison to the CSF-treated controls, orexin-A, and BIBP-treated females responded with, respectively, 35% and 40% reduction of Lhb mRNA expression. Orexin-A and BIBP co-administration evoked a further reduction of Lhb gene transcriptional activity. Conclusions: Orexin-A exerts a down-regulatory effect on LH synthesis and release in immature female rats. Considering that Y1R-oriented down-regulation of endogenous NPY activity did not reverse the suppressive effect of exogenous orexin-A, it might be suggested that NPY and orexin A systems can operate independently to affect gonadotropin activity in the anterior pituitary of the immature female rats.

Study Objectives The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cutoff of 110 pg/mL, and the inter- and intra-assay variability. Methods Raw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established and defined as the lowest converted concentration with a CV Results The mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n = 41, range 1.6%–17.0%), which was significantly higher than in RIAs with higher specific activity (n = 36; median 3.2%, range 0.4%–11.6%, p = .013). The CV around the 110 pg/mL cutoff was Conclusions Hypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cut-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as “undetectable” when measured with the Phoenix Pharmaceuticals RIA. Clinical Trial Information This study is not registered in a clinical trial register, as it has a retrospective database design

The neuropeptide orexin/hypocretin is implicated in sleep and arousal, energy expenditure, reward, affective state and cognition. Our previous work using diurnal Nile grass rats (Arvicanthis niloticus) found that orexin mediates the effects of environmental light, particularly daytime light intensity, on affective and cognitive behaviours. The present study further investigated how daytime light intensity affects the central orexin system in male and female grass rats. Subjects were housed for 4 weeks in 12:12 hr dim light:dark (50 lux, dimLD) or in 12:12 hr bright light:dark cycle (1000 lux, brightLD). Day/night fluctuations in some orexin measures were also assessed. Despite similar hypothalamic prepro-orexin mRNA expression across all conditions, there were significantly more orexin-immunoreactive neurons, larger somata, greater optical density or higher orexin A content at night (ZT14) than during the day (ZT2), and/or in animals housed in brightLD compared to dimLD. Grass rats in brightLD also had higher cisternal CSF levels of orexin A. Furthermore, orexin receptor OX1R and OX2R proteins in the medial prefrontal cortex were higher in brightLD than dimLD males, but lower in brightLD than dimLD females. In the CA1 and dorsal raphe nucleus, females had higher OX1R than males without any significant effects of light condition, and OX2R levels were unaffected by sex or light. These results reveal that daytime light intensity alters the central orexin system of both male and female diurnal grass rats, sometimes sex-specifically, and provides insight into the mechanisms underlying how daytime light intensity impacts orexin-regulated functions.

This research includes the isolation and purification of Orexin-A from the plasma of healthy human via various biochemical techniques, it was proposed the therapeutic role of orexin on hyperlipidemia and lipid peroxidation and it has been suggested to study the effect of isolated orexin A on the metabolism of lipids and glucose in normal and hyperlipidemic rats, a high level of orexin-A had been found only in second peak (B) isolated by gel filtration chromatography (using Sephadex G-50) and showed (34.5) fold of purification, also, the effect of isolated orexin-A on some clinical parameters had been studied in normal and hyperlipidemic male rats. The rats were injected intraperitoneally with orexin-A at a dose of 1μmol/kg of body weight/day for one month. The results, obtained before treatment and after two and four weeks of treatment, had been showing a significant decrease in the concentration of total cholesterol, triglycerides, low and very low-density lipoprotein cholesterol, glucose, malondialdehyde and hyperinsulinemia, while there was a significant increase in the concentration of high-density lipoprotein cholesterol in normal and hyperlipidemic rats. It was concluded that orexin-A had an important role in regulating the metabolism of glucose and lipids, treatment of hyperinsulinemia and insulin resistance, and decreasing lipid peroxidation in normal and hyperlipidemic rats.

Wen Ye,1 Yan Yan,2 Yunliang Tang,1 Xiaoyang Dong,1 Gengfa Chen,1 Junwei Kang,1 Lianghua Huang,1 Qi Xiong,1 Zhen Feng1 1Department of Rehabilitation, The First Affiliated Hospital of Nanchang University, Nanchang City, Jiangxi Province, People’s Republic of China; 2Department of Nephrology, The First Affiliated Hospital of Nanchang University, Nanchang City, Jiangxi Province, People’s Republic of ChinaCorrespondence: Zhen FengDepartment of Rehabilitation, The First Affiliated Hospital of Nanchang University, Nanchang City, Jiangxi Province, People’s Republic of ChinaEmail medstu@126.comBackground: Neuronal damage is the main cause of neurological diseases. Neural stem cells (NSCs) have the functions of cell repair and replacement of neurons, secretion of neurotrophic factors, and immune regulation of the neural microenvironment.Objective: Previous study found that Orexin-A had a protective effect on neurons in the central nervous system, but it is lacking in making great efforts on the function of Orexin-A on NSCs. This study aimed to investigate the anti-inflammatory responses and signaling mechanisms of Orexin-A on lipopolysaccharide (LPS)-induced NSCs.Methods: Quantitative real-time polymerase chain reaction was used to detect the mRNA level. Signaling pathway-related protein expression was detected by Western blot. The proliferation and migration of NSCs were investigated by Cell Counting Kit-8 (CCK-8) detection kit and transwell assay. Besides, the staining of hematoxylin and eosin (HE) was performed to study the morphology of cell.Results: Orexin-A decreased the pro-inflammatory cytokines of IL-1β, TNF-α, and IL-6 induced by LPS by regulating nuclear factor-k-gene binding (NF-kB) and phosphorylation of P38/Erk-mitogen-activated protein kinases (MAPKs) pathways, but not p-JNK signaling.Conclusion: Our findings indicate that Orexin-A can alleviate the inflammatory response of NSC. It can provide beneficial help in neural stem cell therapy applications.Keywords: NSCs, Orexin-A, NF-kB, MAPK

Wen Ye,1 Yan Yan,2 Yunliang Tang,1 Xiaoyang Dong,1 Gengfa Chen,1 Junwei Kang,1 Lianghua Huang,1 Qi Xiong,1 Zhen Feng1 1Department of Rehabilitation, The First Affiliated Hospital of Nanchang University, Nanchang City, Jiangxi Province, People’s Republic of China; 2Department of Nephrology, The First Affiliated Hospital of Nanchang University, Nanchang City, Jiangxi Province, People’s Republic of ChinaCorrespondence: Zhen FengDepartment of Rehabilitation, The First Affiliated Hospital of Nanchang University, Nanchang City, Jiangxi Province, People’s Republic of ChinaEmail medstu@126.comBackground: Neuronal damage is the main cause of neurological diseases. Neural stem cells (NSCs) have the functions of cell repair and replacement of neurons, secretion of neurotrophic factors, and immune regulation of the neural microenvironment.Objective: Previous study found that Orexin-A had a protective effect on neurons in the central nervous system, but it is lacking in making great efforts on the function of Orexin-A on NSCs. This study aimed to investigate the anti-inflammatory responses and signaling mechanisms of Orexin-A on lipopolysaccharide (LPS)-induced NSCs.Methods: Quantitative real-time polymerase chain reaction was used to detect the mRNA level. Signaling pathway-related protein expression was detected by Western blot. The proliferation and migration of NSCs were investigated by Cell Counting Kit-8 (CCK-8) detection kit and transwell assay. Besides, the staining of hematoxylin and eosin (HE) was performed to study the morphology of cell.Results: Orexin-A decreased the pro-inflammatory cytokines of IL-1β, TNF-α, and IL-6 induced by LPS by regulating nuclear factor-k-gene binding (NF-kB) and phosphorylation of P38/Erk-mitogen-activated protein kinases (MAPKs) pathways, but not p-JNK signaling.Conclusion: Our findings indicate that Orexin-A can alleviate the inflammatory response of NSC. It can provide beneficial help in neural stem cell therapy applications.Keywords: NSCs, Orexin-A, NF-kB, MAPK

SummaryTo study the role of orexin A in the reproductive regulation of Mongolian sheep, ovine ovarian granulosa cells were cultured in vitro. The cells were divided into groups after luteinization, the experimental group was given orexin A and the transcriptome was sequenced together with that of the control group. The different genes related to reproduction were screened out. qRT-PCR, western blot and enzyme-linked immunosorbent assay (ELISA) were used to verify the selected genes and detect the effect on progesterone secretion. In total, 123 differentially expressed genes were obtained by sequencing. Six genes with high expression related to reproduction (PRRT2, ABCG1, SOX4, TBX3, ID1 and ATP8) were screened. The results of qRT-PCR were consistent with those of sequencing; western blot and ELISA were used to verify the protein levels of steroidogenic acute regulatory protein (StAR) and its related PRRT2 and ABCG1, and to detect their effect on progesterone secretion. Validation results were consistent with those of qRT-PCR and sequencing. The experimental group was given orexin A and compared with the control group. Expression of PRRT2 protein was significantly increased (P < 0.05), ABCG1 protein expression was significantly decreased (P < 0.05), StAR expression was significantly increased (P < 0.05), and progesterone secretion was significantly increased (P < 0.05). The results showed that orexin A promoted the expression of StAR by upregulating PRRT2 and downregulating ABCG1, therefore affecting secretion of progesterone. Gene expression characteristics of orexin A affecting progesterone secretion were preliminarily explored; this study provides a theoretical basis for further study on signalling pathways and reproductive regulation in Mongolian sheep.

Objective(s): Nausea and vomiting are perennial problems in cancer patients undergoing chemotherapy. Orexin-A (OXA) has been shown to regulate feeding and gastric motility. Seabuckthorn (Hippophae rhamnoides L.) seed oil (SSO) has been proved to promote digestion and bowel movements. We investigated whether SSO alleviated cisplatin-induced vomiting and its possible mechanism involved in OXA. Materials and Methods: Rats were randomly divided into normal control group (NCG), cisplatin group (CG), SSO low-dose group (SLG), SSO middle-dose group (SMG) SSO high-dose group (SHG), and ondansetron group (OG). Rats were pretreated respectively with SSO (0.850, 1.675, and 3.350 g/kg·BW) and ondansetron (2 mg/kg·BW) in SLG, SMG, SHG, and OG for 6 days, and the same volume of saline in NCG and CG groups. On the 6th day, cisplatin (6 mg/kg, IP) was administered in all groups except NCG. The cumulative food and kaolin intake, gastric emptying, plasma OXA level, OX1R mRNA and protein expression in the hypothalamus and brainstem, and OXA expression in the lateral hypothalamic area (LHA) were observed, and the HPLC method was used to analyze the composition of SSO. Results: Kaolin intake in cisplatin-induced vomiting rats was significantly reduced (p

Orexin is a neuropeptide that is primarily synthesized and secreted by the lateral hypothalamus (LH) and includes two substances derived from the same precursor (orexin A [OXA] and orexin B [OXB]). Studies have shown that orexin is not only involved in the regulation of eating, the sleep–wake cycle, and energy metabolism, but also closely associated with various physiological functions, such as cardiovascular control, reproduction, stress, reward, addiction, and the modulation of pain transmission. At present, studies that have been performed both domestically and abroad have confirmed that orexin and its receptors are closely associated with pain regulation. In this article, the research progress on acute pain regulation involving orexin is reviewed.

Study Objectives The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (

Study Objectives: The diagnosis of narcolepsy type 1 (NT1) is based upon the presence of cataplexy and/or a cerebrospinal fluid (CSF) hypocretin-1/orexin-A level 200 pg/mL), a higher percentage of individuals with intermediate hypocretin-1 levels had typical cataplexy (75% or 12/16 vs 9% or 8/88, p < .05), and/or met the diagnostic polysomnographic (PSG) and Multiple Sleep Latency Test (MSLT) criteria for narcolepsy (50 vs 6%, p < .001). Of those with typical cataplexy, 88% had low, 7% intermediate, and 5% normal hypocretin-1 levels (p < .001). Atypical cataplexy was also associated with hypocretin deficiency but to a lesser extent. A hypocretin-1 cutoff of 150 pg/mL best predicted the presence of typical cataplexy and/or positive PSG and MSLT findings. Conclusion: Individuals with intermediate hypocretin-1 levels or typical cataplexy more often have outcomes fitting the PSG and MSLT criteria for narcolepsy than those with normal levels or atypical cataplexy. In addition, typical cataplexy has a much stronger association with hypocretin-1 deficiency than atypical cataplexy. We suggest increasing the NT1 diagnostic hypocretin-1 cutoff and adding the presence of clearly defined typical cataplexy to the diagnostic criteria of NT1.

Study Objectives The diagnosis of narcolepsy type 1 (NT1) is based upon the presence of cataplexy and/or a cerebrospinal fluid (CSF) hypocretin-1/orexin-A level ≤ 110 pg/mL. We determined the clinical and diagnostic characteristics of patients with intermediate hypocretin-1 levels (111–200 pg/mL) and the diagnostic value of cataplexy characteristics in individuals with central disorders of hypersomnolence. Methods Retrospective cross-sectional study of 355 people with known CSF hypocretin-1 levels who visited specialized Sleep-Wake Centers in the Netherlands. For n = 271, we had full data on cataplexy type (“typical” or “atypical” cataplexy). Results Compared to those with normal hypocretin-1 levels (>200 pg/mL), a higher percentage of individuals with intermediate hypocretin-1 levels had typical cataplexy (75% or 12/16 vs 9% or 8/88, p Conclusion Individuals with intermediate hypocretin-1 levels or typical cataplexy more often have outcomes fitting the PSG and MSLT criteria for narcolepsy than those with normal levels or atypical cataplexy. In addition, typical cataplexy has a much stronger association with hypocretin-1 deficiency than atypical cataplexy. We suggest increasing the NT1 diagnostic hypocretin-1 cutoff and adding the presence of clearly defined typical cataplexy to the diagnostic criteria of NT1. Clinical trial information: This study is not registered in a clinical trial register, as it has a retrospective database design.

Key points Rhythmic processes in living organisms are controlled by biological clocks. The orexinergic system of the lateral hypothalamus carries circadian information to provide arousal for the brain during the active phase.Here, we show that orexins exert an excitatory action in three parts of the lateral geniculate nucleus (LGN), in particular upon directly retinorecipient neurons in the non‐image forming visual structures.We provide evidence for the high nocturnal levels of orexins with stable circadian expression of predominant orexin receptor 2 in the LGN.Our data additionally establish the convergence of orexinergic and pituitary adenylate cyclase (PAC)‐activating peptide/PAC1 receptor systems (used by melanopsin‐expressing retinal ganglion cells), which directly regulates responses to the retinal input.These results help us better understand circadian orexinergic control over the non‐image forming subcortical visual system, forming the animal's preparedness for the behaviourally active night. Abstract The orexinergic system of the lateral hypothalamus is tightly interlinked with the master circadian clock and displays daily variation in activity to provide arousal‐related excitation for the plethora of brain structures in a circadian manner. Here, using a combination of electrophysiological, optogenetic, histological, molecular and neuronal tracing methods, we explore a particular link between orexinergic and visual systems in rat. The results of the present study demonstrate that orexinergic fibre density at the area of subcortical visual system exerts a clear day to night variability, reaching a maximum at behaviourally active night. We also show pronounced electrophysiological activations of neurons in the lateral geniculate nucleus by orexin A through 24 h, via identified distinct orexin receptors, with the ventrolateral geniculate displaying a daily cycle of responsiveness. In addition, for the first time, we provide a direct evidence for orexins to act on retinorecipient neurons with a high convergence of orexinergic and putatively retinal pituitary adenylate cyclase (PAC)‐activating peptide/PAC1 receptor systems. Altogether, the present study ties orexins to non‐image forming visual structures with implications for circadian orexinergic modulation of neurons, which process information on ambient light levels., Key points Rhythmic processes in living organisms are controlled by biological clocks. The orexinergic system of the lateral hypothalamus carries circadian information to provide arousal for the brain during the active phase.Here, we show that orexins exert an excitatory action in three parts of the lateral geniculate nucleus (LGN), in particular upon directly retinorecipient neurons in the non‐image forming visual structures.We provide evidence for the high nocturnal levels of orexins with stable circadian expression of predominant orexin receptor 2 in the LGN.Our data additionally establish the convergence of orexinergic and pituitary adenylate cyclase (PAC)‐activating peptide/PAC1 receptor systems (used by melanopsin‐expressing retinal ganglion cells), which directly regulates responses to the retinal input.These results help us better understand circadian orexinergic control over the non‐image forming subcortical visual system, forming the animal's preparedness for the behaviourally active night.

Summary Purpose The response of orexin-A and brain-derived neurotrophic factor (BDNF) to exercise and diet are not fully understood. We examined the effect of acute aerobic exercise 2 h after the consumption of four meals on serum levels of orexin-A and BDNF in overweight men. Methods We used a quasi-experimental and single-blinded crossover study in 16 overweight men (age 23 ± 6.1 y) who voluntarily participated in four sessions separated by 7d, consisting of 30-min of treadmill running (60–70% of maximum heart rate) after consuming one of four selected meals: Normal (Normal), High carbohydrate (HCHO), High protein (HP) and High-fat (HF) meals. Blood samples were taken after fasting, two hours after meal consumption (before exercise), and after exercise test. Results The results indicated that orexin-A and BDNF levels significantly increased after acute exercise test following HCHO (18.24 ± 25.06% and 13.38 ± 4.93%, respectively, P 0.05), and HF meals (−0.43 ± 8.97% and −0.06 ± 4.01%, respectively, P > 0.05). When the four conditions were compared, between group comparisons demonstrated that orexin-A was significantly higher in HCHO (P ≤ 0.019) and HP meal (P ≤ 0.033) vs. HF meal after exercise. Further, BDNF was significantly higher after exercise in HCHO (P ≤ 0.001) and HP (P ≤ 0.001), and Normal (P ≤ 0.039) meals vs. HF meal. Conclusions It appears that the exercise-induced increase in orexin-A and BDNF following caloric reduction is attenuated by HF meals in overweight men.

Objective To analyze the correlation between serum orexin-A (OXA) and early postoperative cognitive function in elderly patients undergoing lumbar spinal surgery under general anesthesia. Methods A total of 76 elderly patients (age ≥65 years) underwent lumbar spine surgery under general anesthesia from December 2018 to December 2019 in the Affiliated Hospital of Inner Mongolia Medical University were collected. All the enrolled patients were evaluated by the same doctor with the Montreal cognitive assessment scale (MoCA) on one day before the surgery and one to three days after the surgery, and venous blood was extracted from the patient on the operation day and one day after the operation, and the serum levels of OXA and S100β were measured by ELISA. According to the results of cognitive function assessment, the patients were divided into postoperative cognitive dysfunction (POCD) group and non-postoperative cognitive dysfunction (NPOCD) group. The differences in serum OXA and S100β protein levels between the two groups and their correlation with MoCA scores were statistically analyzed. Results There was no statistically significant difference in the MoCA score, serum OXA level, and S100β protein level before surgery, and heart rate (HR), mean artery pressure (MAP), bispectral index (BIS) and pulse oxygen saturation (SpO2) levels before anesthesia induction (T0), at the start of surgery (T1), at 1h after the start of surgery (T2), at the withdrawal time (T3), and at 15 minutes after extubation (T4) between the two groups (P>0.05). At the first, second, and third day after operation, the MoCA scores of the POCD group were lower than those before the operation, and they were both lower than those of the NPOCD group, the difference was statistically significant (P

Background Aerobic exercise can greatly assist in reducing collateral effects of metabolic syndrome (MetS). Moreover, aerobic exercise is associated with sympathetic activation and adaptive responses to sustain muscle engagement, changes in the release of Orexin A, a pleiotropic neuropeptide. Aim The aim of this study was to analyze the beneficial effects of aerobic exercise without dietary changes, in a cohort of MetS subjects, focusing on the role of sympathetic and orexinergic activity. Several blood parameters linked to MetS ROS production, heart rate, galvanic skin response, d-ROM test, and Orexin A serum levels were evaluated in ten males with MetS (BMI 30-34.9) before and after a period of 6 months of aerobic exercise compared to ten healthy subjects. Methods Ten male subjects (aged 54 ± 4.16) with MetS (MetS group) and ten healthy males (aged 49.7 ± 2.79, Healthy group) were told about the study protocol and possible risks, signed the informed consent, and voluntarily participated in the study. Several blood parameters were evaluated in the two tested groups and were re-evaluated in the MetS group after 6 months of training (MetS6M group). The training protocol consisted of more than 30 min/day of walking (average speed of 4.5 km/h) and 3 days/week of aerobic activities (jogging under heart rate control - 120-140 bpm for 45 min). Results The results showed that exercise induced a significant increase in GSR and plasma Orexin A but no significant increase in d-ROM values. Significant decreases in the serum ALT enzyme, triglycerides, and total cholesterol were found, while the HDL levels were significantly higher. Finally, a significant reduction of BMI and resting HR were reported. Conclusion The results of this study confirm that physical activity is associated with sympathetic activation, having a pivotal role against adverse effects linked to MetS. Moreover, this study demonstrates that, in patients with MetS, Orexin A is involved in hormonal adaptations to exercise.

Backgrounds Endocannabinoids especially anadamide (AEA) and 2‑arachidonoylglycerol (2-AG) together with appetite modulators have recently been of great importance in body weight regulation and obesity incidence. The present study was carried out to investigate AEA and 2-AG levels and their association with leptin, insulin, orexin – A, and anthropometric indices in obese women. Methods The demographic and anthropometric data of 180 overweight/ obese women with mean age 34.2 ± 8.27 years old, and mean BMI 32.54 ± 3.73 kg/m2 were evaluated. The plasma levels of anadamide and 2‑arachidonoylglycerol levels and also serum levels of leptin, insulin and orexin- A concentrations were measured. Pearson and spearmen correlation tests along with hieratical regression test were used to assess the association of endocannabinoids levels with anthropometric indices and appetite modulators. Results Significant correlations were revealed between AEA and 2-AG with leptin, BMI, waist circumference (WC) and body fat percent (BF%) (P Conclusion The findings showed that levels of AEA and 2-AG were associated with BMI, WC, BF%, and leptin and insulin levels. Also, BMI, WC, BF%, leptin and, insulin levels can have predictive value for determining AEA and 2-AG.

The effects of prenatal stress (PNS) on the formation of the orexinergic and dopaminergic systems of the brain were studied in 14- and 30-day-old rat pups born to females subjected to sleep deprivation (6 h/day) using the “small areas” method from day 13 to day 19 of pregnancy. The open fi eld test showed impairments to motor development in 14-day-old PNS rat pups. Immunohistochemical studies of brain sections showed that the hypothalamus contained a signifi cantly larger quantity of orexin A in neurons in the periforniceal area, while high-performance liquid chromatography demonstrated increases in the dopamine level and its rate of metabolism as compared with control rat pups born to intact females. Western blotting results provided evidence of a decrease in the striatal level of the GABA-sythesizing enzyme (GAD65) and an increase in the level of tyrosine hydroxylase (an enzyme involved in dopamine synthesis) phosphorylated at serine-40 as compared with controls. By day 30 of life measures of motor activity in PNS rat pups were no different from those in controls, though PNS rat pups had higher levels of anxiety and lower levels of exploratory activity. The results of morphological and biochemical studies also provided evidence that there were no differences between the study parameters in the hypothalamus and striatum in 30-day-old rat pups as compared with controls, which was confi rmed by results from electrophysiological studies indicating that there were no differences in the organization of the sleep-waking cycle in 30-day-old PNS and control rat pups. We discuss here the morphofunctional interactions of the orexinergic and dopaminergic systems of the brain in the early postnatal period of development of the body and the role of orexins in the compensatory mechanisms of the brain.

Aim. To conduct a comparative analysis of serum orexin A levels in women of different age periods with and without sleep disorder and vasomotor symptoms. To evaluate the dynamics of orexin A levels under menopausal hormone therapy. Materials and methods. The study included 50 postmenopausal women and 30 women of reproductive age with a regular menstrual cycle. Using block randomization, patients are divided into 3 groups: group 1 (main group), n=25, -STRAW+ 10 (+1b and +1c), patients with sleep disorder and vasomotor symptoms; group 2 (comparison group), n=25, STRAW+ 10 (+1b and +1c), patients with vasomotor symptoms without sleep disorder; group 3 (control group), n=30, STRAW+ 10 (-4), women of reproductive age without sleep disorder. Group 1 patients were given menopausal hormone therapy. A comparative analysis was carried out using the questionnaire for assessing menopausal symptoms severity by the Greene Scale (the Greene Climacteric Scale) and Rating Scale for subjective sleep characteristics. After 12 weeks of treatment, a control examination was performed. Results. In group 1 women, the serum orexin A levels were significantly higher compared to the women without the symptoms. The link between the orexin A levels and menopause syndrome severity was established. A significant decrease in the menopausal symptoms severity after 12 weeks of menopausal hormone therapy was shown. It was accompanied by a 1,3-fold decrease in orexin A levels. Conclusions. The obtained data indicate the possible role of orexin A and the orexin neuropeptide system in the pathogenesis of sleep disorder and vasomotor symptoms in postmenopausal women.

Post-traumatic stress disorder (PTSD) patients exhibit abnormal learning and memory. Axons from orexin neurons in the lateral hypothalamus innervate the hippocampus, modulating learning and memory via the orexin 1 and 2 receptors (OX1R and OX2R). However, the role of the orexin system in the learning and memory dysfunction observed in PTSD is unknown. This was investigated in the present study using PTSD animal model-single prolonged stress (SPS) rats. Spatial learning and memory in the rats were evaluated with the Morris water maze (MWM) test; changes in body weight and food intake were recorded to assess changes in appetite; and the expression of orexin-A and its receptors in the hypothalamus and hippocampus was examined and quantified by immunohistochemistry, western blotting and real-time PCR. The results showed that spatial memory was impaired and food intake was decreased in SPS rats; this was accompanied by downregulation of orexin-A in the hypothalamus and upregulation of OX1R and OX2R in the hippocampus and of OX1R in the hypothalamus. Intracerebroventricular administration of orexin-A improved spatial memory and enhanced appetite in SPS rats and partly reversed the increases in OX1R and OX2R levels in the hippocampus and hypothalamus. These results suggest that the orexin system plays a critical role in the memory and appetite dysfunction observed in PTSD.

Sleep biomarkers in brain such as melatonin, BDNF (Brain-derived neurotrophic factor), PGD2 (Prostaglandin D2), leptin, orexin-A, and acetylcholine were evaluated in the unpredictable chronic stress (UCS) rodent model in the presence of Consciousness Energy Healing Treated (the Trivedi Effect®) novel test formulation in male Sprague Dawley (SD) rats using ELISA assay. The test formulation was consisted of minerals (Zn, Fe, Cu, Se, Ca, Mg), vitamins (C, E, B6, B12, D3), β-carotene, ginseng, and cannabidiol isolate. The test formulation constituents were divided into two parts, one part of each ingredient was distinct as the untreated test formulation, while the other portion of the test formulation and a group of animals received Biofield Energy Healing Treatment by a renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi. The level of melatonin in groups viz. G5 (Biofield Energy Treated Test formulation) and G7 (15-days pre-treatment of Biofield Energy Treated Test formulation) was significantly increased by 17.6% (p≤0.01) and 16%, respectively as compared with the disease control group (G2). Brain-derived neurotrophic factor (BDNF) level in brain was increased by 5.2% in G7 group as compared with the G4. Prostaglandins D2 (PGD2) level was significantly (p≤0.001) increased by 12.7%, 18.1%, 23.7%, and 30.7% in the G6, G7, G8 (15 days pre-treatment of Biofield Energy Treated Test formulation to the Biofield Energy Treatment per se rats), and G9 (untreated test formulation to the Biofield Energy Treatment per se to the rats) groups, respectively as compared with the G2. The level of leptin after Biofield Energy Treatment and with the test formulation was altered. However, orexin-A level was significantly decreased by 37.1% (p≤0.05), 32.6%, 40.5% (p≤0.05), 44.4% (p≤0.05), and 28.2% in the G5, G6, G7, G8, and G9 groups respectively, as compared with the G2. Similarly, acetylcholine (Ach) level was significantly (p≤0.001) decreased by 42.5%, 49.2%, 40.1%, 47.9%, and 45% in the G5, G6, G7, G8, and G9 groups, respectively as compared with the G2. Overall, the results showed the significant slowdown the stress-related disease progression and its complications/symptoms in the preventive in the Biofield Energy Treatment group per se and/or Biofield Energy Treated Test formulation groups (viz. G6, G7, G8, and G9) comparatively with the disease group. The Trivedi Effect® showed increased level of melatonin and decreased levels of insomnia related brain biomarkers which might be helpful to induce better sleep in human.

Objectives: The relationships between orexins and stress-related conditions have been well documented in animal studies. However, human studies confirming this relationship are limited. The aim of this study was to investigate the association between orexin-A and anxiety disorders in adolescents. Additionally, we aimed to examine the relationship between orexin-A and cortisol levels in those with anxiety disorders.Methods: A total of 56 medication-free adolescents diagnosed with any anxiety disorder, except for specific phobias, and 32 healthy controls were included in this study. Depression, state and trait anxiety levels of the participants were measured using self-report scales. Orexin-A and cortisol levels were measured by an enzyme-linked immunosorbent assay (ELISA).Results: Analysis of covariance (ANCOVA) indicated that serum orexin-A levels were significantly higher in the anxiety disorder group than in the control group while controlling for age, sex and depression levels. After controlling for age and sex, orexin-A levels were positively and negatively correlated to depression and cortisol levels, respectively. In addition, a positive correlation trend between trait anxiety and orexin-A was found.Conclusions: Orexin-A levels are higher in adolescents with anxiety disorder; however, depressive symptoms should be considered when investigating this relationship.

Background Recently, many studies have investigated the association between orexin A and Alzheimer's disease (AD). However, it remains to be determined whether the observed changes in orexin A levels are associated with pathological changes underlying AD, or cognitive function. In particular, a direct association between cerebrospinal fluid (CSF) orexin A levels and cognitive function has not been reported to date. Objective The aim of this study was to identify whether there is a direct association between the orexinergic system and cognitive function in AD. Methods For this study, we included 22 patients with AD and 25 control subjects who underwent general physical, neurological, and psychiatric examinations, neuroimaging, and CSF collection by lumbar puncture were enrolled. Correlations between CSF orexin A levels and CSF AD biomarker levels (i.e., levels of phosphorylated tau [p-tau], Aβ42, and Aβ42/Aβ40) were assessed to confirm the results of previous studies. Moreover, the correlation between CSF orexin A levels and Mini-Mental State Examination (MMSE) and Japanese version of the Montreal Cognitive Assessment (MoCA-J) scores were analyzed. Results There was a significant positive correlation between CSF orexin-A levels and cognitive function (MMSE scores: r = 0.591, p = 0.04, MoCA score: r = 0.571, p = 0.006) in AD patients. Conclusion This is the first study to our knowledge demonstrating an association between cognitive function and CSF orexin A levels in AD. Our results suggest the possibility that orexinergic system overexpression is not always a negative factor for cognitive function In AD.

Relevance. The study of sleep disorders mechanisms after traumatic brain injury is complicated and poorly understood. Traumatic damage to the structures that are responsible for the sleep-wake cycle regulation is a common cause of sleep disorders after traumatic brain injury. The number of hypothalamic neurotransmitter systems, which are involved in the sleep-wake cycle regulation, could change its functional activity after trauma that suggests their key role in the development of disturbances of this process. The aim of the study was to assess the morphological alterations of the hypothalamus neurons that is involved in the regulation of sleep and wakefulness after traumatic brain injury in an experiment. Methods. For a combined analysis of posttraumatic disturbances of the sleep-wake cycle and morphofunctional changes in the neurotransmitter systems which are involved in the regulation of the sleep-wake cycle, we used a polysomnography in rats during a month and then an immunohistochemical method for estimating the quantify the orexin A, melanin-concentrating hormone, histamine and tyrosine hydroxylase. Results. The number of histamine-containing cells in the tuberomammillary nuclei of the hypothalamus is obviously decreased after traumatic brain injury in animals. This alteration of the degree of immunoreactivity of histamine-containing cells after traumatic brain injury correlated with sleep duration changes in animals. The number of noradrenergic and orexinergic neurons was compare with control animal group. Conclusion. These results suggest that a change in the functional activity of histamine-containing neurons after traumatic brain injury may be the cause of post-traumatic sleep and wakefulness disorders. Our results may lead to a creating of a new approach for a therapy for posttraumatic sleep-wake disturbances.

AIMS Orexin-A is known to induce anti-nociceptive effects in animal models of chronic pain. We have found that orexin-A inhibits KCl loading-induced increases in the intracellular calcium ion levels ([Ca2+ ]i ) in C-fiber-like neurons of rats showing inflammatory nociceptive behavior. Here, we examined the effects of orexin-A on the depolarization of C-fiber-like neurons derived from a rat model for another type of chronic pain, namely neuropathic pain. Thus, we analyzed the effects of orexin-A on KCl-induced increases in [Ca2+ ]i in C-fiber-like neurons of rats with sciatic nerve ligation. METHODS Paw withdrawal and threshold force in response to tactile stimuli were evaluated using von Frey filaments. Sham-operated rats served as controls. [Ca2+ ]i in neurons were visualized by calcium fluorescent probe. Changes in [Ca2+ ]i were assessed using relative fluorescence intensity. RESULTS Seven days after sciatic nerve ligation, paw withdrawal and threshold force for tactile stimuli were increased and reduced, respectively. KCl loading to neurons from either sciatic nerve-ligated or control rats increased relative fluorescence intensity. The KCl-induced increase in relative fluorescence intensity in sciatic nerve-ligated, but not that of control, rats was inhibited by orexin-A. The OX1 and OX2 receptor antagonist MK-4305 and OX2 receptor antagonist EMPA, but not the OX1 receptor antagonist SB 334867, each counteracted orexin-A-induced inhibition of KCl-provoked increases in relative fluorescence intensity. CONCLUSION The present findings constitute neuropharmacological evidence that OX2 but not OX1 receptors mediate the inhibitory effects of orexin-A on KCl-induced increases in [Ca2+ ]i in C-fiber-like neurons of rats showing hyperalgesia provoked by sciatic nerve ligation.

Background The orexin system regulates various functions, including sleep/wake cycles, feeding, and cognition. Orexin A and orexin B are endogenous neuropeptides for both orexin 1 (OX1) and orexin 2 (OX2) receptors. Orexin A has a potent agonistic activity for both the receptors and is known to increase locomotor activity in rats. However, it has not been elucidated how each receptor contributes to orexin A-induced hyperlocomotion. Methods We examined the effects of an OX1 receptor antagonist, SB 334867, and an OX2 receptor antagonist, EMPA, as well as an OX1 and OX2 receptor antagonist on hyperlocomotion caused by intracerebroventricular administration of orexin A or an OX2 receptor agonist, ADL-OXB ([Ala11, d -Leu15]-orexin B), in rats. Results EMPA (100 mg/kg, ip) but not SB 334867 (3–10 mg/kg, ip) showed antagonistic effects on ADL-OXB-induced hyperlocomotion without affecting the spontaneous locomotor activity. Both EMPA (100 mg/kg, ip) and the OX1 and OX2 receptor antagonist (3–30 mg/kg, po) antagonized orexin A-induced hyperlocomotion, while SB 334867 (3‒–10 mg/kg, ip) showed no effects. Conclusions Our results suggest that orexin A-induced hyperlocomotion is mainly mediated by the activation of the OX2 receptor.

Background: Multiple sclerosis (MS) is a chronic inflammatory and autoimmune disease affecting various inflammatory and nutritional parameters. Therefore, this study aimed to investigate the relationship between the Body Mass Index (BMI) of MS patients and the serum levels of leptin, orexin-A, and Transforming Growth Factor beta (TGF-beta). Methods: This cross-sectional study included 25 patients suffering from MS and 40 healthy individuals as the case and control groups, respectively. The serum levels of leptin, orexin-A, and TGF-beta were assessed in the participants using the Enzyme-Linked Immunosorbent Assay methods. Moreover, data were analyzed using the descriptive statistical indices, t-test, chi-square test, and linear regression test. Results: According to our results, the participants' mean age was 38.04 +/- 7.53 and 40.23 +/- 5.88 in the case and control groups, respectively. Also, the groups were riot significantly different in gender, age, alcohol consumption, and smoking (p > 0.05). It was found that the mean serum levels of orexin-A and TGF-beta were significantly lower in the MS patients compared to the control group, while the mean serum leptin levels were significantly higher (42.8 vs. 18.9 ng/ml, p < 0.001). Moreover, there was no significant relationship between the BMI of the MS patients and their serum levels of orexin-A, TGF-beta, and leptin (p > 0.05). Conclusions: In conclusion, we found significantly lower levels of orexin-A and TGF-beta and a significantly higher level of leptin in the MS patients compared to the control group. In addition, there was no significant relationship between the BMI and the serum levels of orexin-A, TGF-beta, and leptin in MS patients., Tabriz University of Medical Sciences [ID.: IR T.B.Z.M.E.D.REC.1398.527], The study was supported and sponsored by the Tabriz University of Medical Sciences (ID.: IR T.B.Z.M.E.D.REC.1398.527).

The orexinergic system delivers excitation for multiple brain centers to facilitate behavioral arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its tar get subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecu lar approaches across 24 h, together with the neuronal tract-tracing methods to investigate the daily coordination between the orexin system and the rodent SC. Higher orexinergic input was found to occur nocturnally in the superficial layers of the SC, in time for nocturnal silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day–night changes. Additionally, we establish the lateral hypothalamic origin of the orexin innervation to the SC and that the SC neurons robustly respond to orexin A via $OX_{2}$ receptor in both excitatory and $GABA_{A}$ receptor-dependent inhibitory manners. Together, our evidence eluci dates the combination of intrinsic and extrinsic clock mechanisms that shape the daily function of the visual layers of the SC.

Sleep-wakefulness is disrupted in most neurological and psychiatric disorders. Although clinical data implicate orexin (hypocretin), a crucial sleep/wake regulatory neuropeptide, in such disorders, limited sample volumes effectively prevent quantification of cerebrospinal fluid (CSF) levels of orexin A in mouse models of brain disorders. Current enzyme- and radio-immunoassays for orexin A generally require 50–100 µL CSF, whereas typical CSF sample volumes from mice are ~5–10 µL/mouse. We therefore aimed to develop and validate a liquid chromatography (LC) targeted mass spectrometry (MS) method for the absolute quantification of orexin A in the CSF of individual mice. LC coupled to tandem MS (LC-MS/MS) and a triple quadrupole (QQQ) mass spectrometer were used to develop a LC electrospray ionization multiple-reaction monitoring (LC-ESI-MRM) method. CSF orexin A levels of C57BL/6JARC mice were quantified using this method at the predicted peak and trough of diurnal orexin A release and following sleep deprivation. The LC-ESI-MRM assay was robust and sensitive, with an intra-assay variation

Orexin neurons play an important role in stress-related mental disorders including post-traumatic stress disorder (PTSD). Anxiety- and depression-related symptoms commonly occur in combination with PTSD. However, the role of the orexin system in mediating alterations in these affective symptoms remains unclear. The medial prefrontal cortex (mPFC) is implicated in both cognitive and emotional processing. In the present study, we investigated anxiety- and depression-related behavioral changes using the elevated plus maze, the sucrose preference test, and the open field test in male rats with single prolonged stress (SPS) induced-PTSD. The expression of orexin-A in the hypothalamus and orexin receptors (OX1R and OX2R) in the mPFC was detected and quantified by immunohistochemistry, western blotting, and real-time polymerase chain reaction. We found that the SPS rats exhibited enhanced levels of anxiety, reduced exploratory activities, and anhedonia. Furthermore, SPS resulted in reductions in the expression of orexin-A in the hypothalamus and the increased the expression of OX1R in the mPFC. The intracerebroventricular administration of orexin-A alleviated behavioral changes in SPS rats and partly restored the increased levels of OX1R in the mPFC. These results suggest that the orexin system plays a role in the anxiety- and depression-related symptoms observed in PTSD.

Backgrounds: Multiple Sclerosis (MS) is a chronic inflammatory and autoimmune disease linked to several inflammatory and dietary parameters. This study was carried out to determine the relationship between serum leptin, orexin-A, and TGF-β levels with BMI in MS patients.Methods and results: In this cross-sectional study, 25 relapsing-remitting multiple sclerosis (RRMS) patients and 40 healthy controls were enrolled. The serum level of Leptin, Orexin-A, and TGF- were measured by the Enzyme-linked immunosorbent assay (ELISA). The data was analyzed using descriptive statistics, t-test, Chi-square test, and Linear regression test. A total of 65 volunteers, including 25 MS patients and 40 healthy, were enrolled in the study. The mean age of individuals in the case and control groups was 38.04 ± 7.53 and 40.23 ± 5.88. There were no statistically significant differences between the case and control groups regarding gender, age, alcohol, and cigarette use (P>0.05). The mean serum levels of Orexin-A and TGF-ß were lower among multiple sclerosis patients than in healthy controls, but leptin was higher (42.8 vs. 18.9 ng/ml, P 0.05).Conclusion: Our results showed that the serum levels of Orexin-A and TGF-β were significantly lower. The serum level of leptin was higher among multiple sclerosis patients than among healthy controls. Also, there was no statistically significant relationship between BMI and serum levels of Orexin-A, TGF-ß, and Leptin among multiple sclerosis patients.

Background: Ciji-Hua’ai-Baosheng II Formula (CHB-II-F) is a traditional Chinese medicine formula, which specifically targets different aspects of chemotherapy-induced adverse effects in patients with cancer. In our clinical application, CHB-II-F significantly alleviated chemotherapy-induced anorexia (loss of appetite) and improved the quality of life for patients with tumor during and after chemotherapy. However, the mechanism of CHB-II-F in alleviation of chemotherapy-induced anorexia remains to be further investigated.Aim of Study: To explore the therapeutic effect and mechanism of CHB-II-F on chemotherapy-induced anorexia in the mice model of H22 hepatoma.Materials and Methods: A total of 72 Kunming mice of SPF grade were inoculated subcutaneously with H22 hepatoma cells into the right anterior armpit of the mice. After 1 week of seeding, mice were injected intraperitoneally with a high dose of 5-fluorouracil (200 mg/kg 5-FU) to establish the model of chemotherapy. The mice were randomly divided into six groups: untreated group, 5-FU group, 5-FU plus Yangzheng Xiaoji capsule (YZXJC) group, and three groups of 5-FU plus different concentrations of CHB-II-F. All the mice in each group were treated for 14 days. The body weight, food intake, tumor volume, and tumor weight of mice were measured, and pathological examinations of tumor tissue, stomach, and duodenum were carried out. Expressions of serum Leptin, Neuropeptide Y (NPY), epidermal cell growth factor (EGF), Motilin (MTL), Orexin A (OXA), Gastrin (GAS), Ghrelin, Prostaglandin E2 (PGE2), and jejunum superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined. The protein and mRNA levels of proopiomelanocortin (POMC), Orexin receptor 1 (OX1R), neuropeptide Y (NPY), cocaine and amphetamine regulated transcript peptide (CART), Agouti gene-related protein (AgRP), Leptin receptor (Ob-R), and Ghrelin receptor (GHSR) were examined in hypothalamus, and the protein levels of substance P (SP) and 5-hydroxytryptamine (5-HT) in duodenum were measured.Results: The combination of CHB-II-F and 5-FU could enhance the inhibitory effect of 5-FU on tumor. The tumor inhibition rates of 5-FU group, YZXJC group, CHB-II-F(H) group, CHB-II-F(M) group, and CHB-II-F(L) group were 58.88, 28.08, 54.96, 37.69, and 28.61%, respectively. Compared with untreated group and 5-FU group, CHB-II-F significantly increased the body weight and food intake of tumor-bearing mice; increased the content of NPY, Orexin A, Ghrelin, GAS, MTL, EGF, and PGE2 in serum and the activity of SOD in jejunum; and decreased the content of Leptin in serum and the content of MDA in jejunum. Compared with untreated group and 5-FU group, CHB-II-F also enhanced the expression of OX1R, GHSR, NPY, and AgRP protein and gene and decreased the expression of Ob-R, POMC, and CART protein and gene in hypothalamus of mice, and the gene expression was consistent with the protein expression. In addition, CHB-II-F decreased the expression of 5-HT and SP protein in duodenum.Conclusion: In the murine model of H22 hepatocellular carcinoma (HCC) receiving chemotherapy, CHB-II-F enhances the inhibitory effect of 5-FU on tumor, significantly improves the pathological injury of gastrointestinal tract caused by chemotherapy, and regulates the secretion of gastrointestinal hormones. It may alleviate chemotherapy-induced anorexia by affecting appetite regulatory factors in the feeding area of hypothalamus central nervous system and peripheral appetite regulatory factors.

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 OrxA-induced 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.

Aim: Modified Suanzaoren Decoction (MSZRD) is obtained by improving Suanzaoren Decoction (SZRT), a traditional Chinese herbal prescription that has been used to treat insomnia for more than thousands of years. Our previous study showed that MSZRD can improve the gastrointestinal discomfort related insomnia by regulating Orexin-A. This study is the first study to evaluate the effects and possible mechanisms of MSZRD in mice with insomnia caused by p-chlorophenylalanine (PCPA) combined with multifactor random stimulation. Methods: After 14 days of multifactor stimulation to ICR mice, a PCPA suspension (30 mg/mL) was injected intraperitoneally for two consecutive days to establish an insomnia model. Three different doses of MSZRD (3.6, 7.2, and 14.4 g/kg/day) were given to ICR mice for 24 days. The food intake and back temperature were measured, and behavioral tests and pentobarbital sodium-induced sleep tests were conducted. The levels of Orexin-A, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and adrenocortical hormones (CORT) in the serum and 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) in hypothalamus were measured using enzyme-linked immunosorbent assay (ELISA) kits. The levels of γ-aminobutyric acid (GABA) and glutamic acid (Glu) were measured by high-performance liquid chromatography (HPLC). The expression of 5HT1A receptor (5-HTRIA) and orexin receptor 2 antibody (OX2R) was measured by Western blot (WB) and immunohistochemical staining (ICH). Hematoxylin and eosin (H&E) staining and Nissl staining were used to assess the histological changes in hypothalamus tissue. Results: Of note, MSZRD can shorten the sleep latency of insomnia mice (P

Sleep disturbance is common in patients with Alzheimer’s disease (AD), and orexin A is a pivotal neurotransmitter for bidirectional regulating the amyloid-β (Aβ) deposition of AD brain and poor sleep. In the present study, we examined the characteristic of sleep-wake architecture in APPswe/PSldE9 (APP/PS1) and Aβ-treated mice using electroencephalogram (EEG) and electromyographic (EMG) analysis. We compared the expression of orexin A, distribution, and morphology of the corresponding orexin A neurons using innovative methods including three-dimensional reconstruction and brain tissue clearing between Wild type (WT) and APP/PS1 mice. Results from our study demonstrated that increased wakefulness and reduced NREM sleep were seen in APP/PS1 and Aβ treated mice while the expression of orexin A was significantly upregulated. Higher density and distribution of orexin A activated neurons were seen in APP/PS1 mice, with a location of 1.06 mm to 2.30 mm away from the anterior fontanelle compared to 1.34 mm to 2.18 mm away from the anterior fontanelle in WT mice. These results suggested that the population and distribution of orexin A may play an important role in the progression of AD.

Background Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. The purpose of this study was to examine neuroprotective effects of Hepad S1, an herbal medicine used for the treatment of PD, in in vitro and in vivo models of PD. Methods Differentiated neuronal PC12 cells underwent a cytotoxicity assay and oxidative stress analysis including DCF-DA staining, glutathione, and malondialdehyde, after exposure to 1-methyl-4-phenylpyridium (MPP+). Male Sprague–Dawley rats were used as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD models. After 4-week oral administration of Hepad S1 (200, 300, 400, and 500 mg/kg/day), the levels of complex enzyme I activity and dopamine, and dopaminergic neuronal cell number in substantia nigra were measured by enzyme linked immune-sorbent assay (ELISA) and microscopic observation, respectively. Circulating serotonin and orexin A were also examined by ELISA. Results Hepad S1 pretreatment prevented the ability of MPP+ challenge to decrease glutathione and increase lipid peroxidation in cells, indicating antioxidant activity. Hepad S1 recovered MPTP-induced decreases in complex I enzyme activity and enhanced dopamine availability in substantia nigra. Serum levels of serotonin and orexin A were increased by Hepad S1 treatment in model animals. Hepad S1 treatment was associated with the preservation of tyrosine hydroxylase-positive cells in the substantia nigra of MPTP-treated rats. Conclusions Hepad S1 exerts antioxidant and neuroprotective effects on neurons of the substantia nigra in a rodent model of PD.

Hypocretin 1 and hypocretin 2 (orexin A and B) regulate sleep, wakefulness and emotion. Tumour necrosis factor alpha (TNF‐α) is an important neuroinflammation mediator. Here, we examined the effects of TNF‐α treatment on hypocretin expression in vivo and behaviour in mice. TNF‐α decreased hypocretin 1 and hypocretin 2 expression in a dose‐dependent manner in cultured hypothalamic neurons. TNF‐α decreased mRNA stability of prepro‐hypocretin, the single precursor of hypocretin 1 and hypocretin 2. Mice challenged with TNF‐α demonstrated decreased expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in hypothalamus. In response to TNF‐α, prepro‐hypocretin mRNA decay was increased in hypothalamus. TNF‐α neutralizing antibody restored the expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in vivo in TNF‐α challenged mice, supporting hypocretin system can be impaired by increased TNF‐α through decreasing hypocretin expression. Repeated TNF‐α challenge induced muscle activity during rapid eye movement sleep and sleep fragmentation, but decreased learning, cognition and memory in mice. TNF‐α neutralizing antibody blocked the effects of TNF‐α; in contrast, hypocretin receptor antagonist enhanced the effects of TNF‐α. The data support that TNF‐α is involved in the regulation of hypocretin expression, sleep and cognition. The findings shed some lights on the role of neuroinflammation in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.

Ketamine has emerged as a major substance of abuse worldwide. Evidence suggests a role of orexin system in reward processing, withdrawal, and stress response. It also interacts with the stress mechanisms of hypothalamic–pituitary–adrenal (HPA) axis to regulate drug-taking behavior. The study aimed to explore the relevance of orexin and stress hormones to chronic ketamine abuse. We enrolled 67 ketamine-dependent (KD) patients and 64 controls. The levels of orexin-A, adrenocorticotropic hormone (ACTH), and cortisol were measured at baseline, 1 week, and 2 weeks after ketamine discontinuation. KD patients were assessed by Beck Depression Inventory, Beck Anxiety Inventory, and Visual Analogue Scale for ketamine craving at baseline. Compared with the controls, KD patients had significantly lower orexin-A (0.65 ± 0.12 vs. 0.74 ± 0.10 ng/mL, p

Shoko Tsuchimine,1 Kotaro Hattori,1 Miho Ota,1 Shinsuke Hidese,1 Toshiya Teraishi,1 Daimei Sasayama,1 Hiroaki Hori,1 Takamasa Noda,2 Sumiko Yoshida,2 Fuyuko Yoshida,1 Hiroshi Kunugi11Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan; 2Department of Psychiatry, National Center Hospital, National Center of Neurology and Psychiatry, Tokyo 187-8551, JapanPurpose: Orexins are hypothalamic neuropeptides involved in the regulation of sleep, appetite and arousal. An altered orexin system has been implicated in the pathophysiology of psychiatric disorders. This study aimed to examine whether plasma orexin-A levels differ in patients with schizophrenia, major depressive disorder (MDD), or bipolar disorder (BD) compared to in healthy controls. We also examined the possible correlations between plasma orexin-A levels and clinical variables.Patients and methods: All participants were Japanese. The sample consisted of 80 patients with schizophrenia (42 women, 52.5%; mean age 36.8years), 80 patients with MDD (43 women, 53.8%; 43.7years), and 40 patients with BD (24 women, 60%; 41.1years), as well as 80 healthy controls (48 women, 60%; 47.0years). Plasma orexin-A levels were quantified by an enzyme-linked immunosorbent assay.Results: Mean orexin-A levels were significantly different across the four diagnostic groups (F=4.09; df=3; p=0.007, η2=0.06). In particular, the patients with BD showed significantly lower orexin-A levels than did the controls. When the median value of the control group (109.8pg/ml) was set as a cut-off value, subjects whose orexin-A levels were below the cut-off were more common in all psychiatric groups (schizophrenia: 73.8%, x2=9.56, df=1, p=0.003, OR=2.81, 95% CI: 1.45 to 5.45, d=0.57; MDD: 78.5%, x2=14.02, df=1, p

Purpose Orexins are hypothalamic neuropeptides involved in the regulation of sleep, appetite and arousal. An altered orexin system has been implicated in the pathophysiology of psychiatric disorders. This study aimed to examine whether plasma orexin-A levels differ in patients with schizophrenia, major depressive disorder (MDD), or bipolar disorder (BD) compared to in healthy controls. We also examined the possible correlations between plasma orexin-A levels and clinical variables. Patients and methods All participants were Japanese. The sample consisted of 80 patients with schizophrenia (42 women, 52.5%; mean age 36.8 years), 80 patients with MDD (43 women, 53.8%; 43.7 years), and 40 patients with BD (24 women, 60%; 41.1 years), as well as 80 healthy controls (48 women, 60%; 47.0 years). Plasma orexin-A levels were quantified by an enzyme-linked immunosorbent assay. Results Mean orexin-A levels were significantly different across the four diagnostic groups (F=4.09; df=3; p=0.007, η2 =0.06). In particular, the patients with BD showed significantly lower orexin-A levels than did the controls. When the median value of the control group (109.8 pg/ml) was set as a cut-off value, subjects whose orexin-A levels were below the cut-off were more common in all psychiatric groups (schizophrenia: 73.8%, x2 =9.56, df=1, p=0.003, OR=2.81, 95% CI: 1.45 to 5.45, d=0.57; MDD: 78.5%, x2 =14.02, df=1, p

Prior studies, mainly on rodents demonstrated that orexin A (OXA) plays an important role in regulating the hypothalamo-pituitary-gonadal (HPG) axis. However, the results of those works have indicated that the central impact of OXA on the HPG axis may vary depending on the species or sex of the animal, the phase of the estrous cycle, or the experimental model system (in vivo vs. in vitro). The aim of the present study was to further investigate the role of OXA in the regulation of reproduction in seasonally breeding, intact, non-steroid-primed ewes during the luteal phase of the estrous cycle and during anestrus. We examined the effect of fourth-ventricle injections of OXA (0.3 μg/kg body weight) on plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone concentrations and on the level of gonadotropin-releasing hormone receptor (GnRH-R) protein in the anterior pituitary (AP). Radioimmunoassays revealed that OXA treatments inhibited (P

Shoko Tsuchimine,1 Kotaro Hattori,1 Miho Ota,1 Shinsuke Hidese,1 Toshiya Teraishi,1 Daimei Sasayama,1 Hiroaki Hori,1 Takamasa Noda,2 Sumiko Yoshida,2 Fuyuko Yoshida,1 Hiroshi Kunugi11Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan; 2Department of Psychiatry, National Center Hospital, National Center of Neurology and Psychiatry, Tokyo 187-8551, JapanPurpose: Orexins are hypothalamic neuropeptides involved in the regulation of sleep, appetite and arousal. An altered orexin system has been implicated in the pathophysiology of psychiatric disorders. This study aimed to examine whether plasma orexin-A levels differ in patients with schizophrenia, major depressive disorder (MDD), or bipolar disorder (BD) compared to in healthy controls. We also examined the possible correlations between plasma orexin-A levels and clinical variables.Patients and methods: All participants were Japanese. The sample consisted of 80 patients with schizophrenia (42 women, 52.5%; mean age 36.8years), 80 patients with MDD (43 women, 53.8%; 43.7years), and 40 patients with BD (24 women, 60%; 41.1years), as well as 80 healthy controls (48 women, 60%; 47.0years). Plasma orexin-A levels were quantified by an enzyme-linked immunosorbent assay.Results: Mean orexin-A levels were significantly different across the four diagnostic groups (F=4.09; df=3; p=0.007, η2=0.06). In particular, the patients with BD showed significantly lower orexin-A levels than did the controls. When the median value of the control group (109.8pg/ml) was set as a cut-off value, subjects whose orexin-A levels were below the cut-off were more common in all psychiatric groups (schizophrenia: 73.8%, x2=9.56, df=1, p=0.003, OR=2.81, 95% CI: 1.45 to 5.45, d=0.57; MDD: 78.5%, x2=14.02, df=1, p