OX1R mRNA Research Articles

Long-term intermittent hypoxia induces anxiety-like behavior and affects expression of orexin and its receptors differently in the mouse brain.

Studies have revealed a possible connection between orexin, narcolepsy, and obstructive sleep apnea (OSA). Orexin has an important role in the maintenance of arousal and wakefulness/sleeping states. To better understand the pathophysiological mechanism of OSA, we used a chronic intermittent hypoxia (CIH) model in mice to mimic OSA. In this way, we explored the effect of CIH on the locomotor activity and orexin system in the hypothalamus, cerebral cortex, and brainstem of mice. Male C57BL/6J mice (8weeks) in the CIH group were exposed in a hypoxia chamber for 8h/day for 28weeks. The re-oxygenation groups comprised the W2 group and W4 group, which were exposed to 28weeks of CIH followed by 2weeks and 4weeks of re-oxygenation, respectively. The open field test was undertaken to observe locomotor activity. mRNA expression of orexin, orexin receptor type 1 (OX1R), and OX2R mRNA was evaluated by real-time reverse transcription-quantitative polymerase chain reaction. Mice subjected to long-term CIH exhibited significant anxiety-like behavior during the light period, and this behavior lasted until 4weeks of re-oxygenation. mRNA expression of orexin was upregulated in the hypothalamus. mRNA expression of OX1R mRNA in the cerebral cortex and brainstem was downregulated by CIH. Two weeks and 4weeks of re-oxygenation could not reverse these alternations. Long-term CIH may induce anxiety-like behavior and re-oxygenation cannot reverse these behavior. Moreover, OX1R has a significant role in the anxiety-related symptoms observed in long-term CIH.

Effect of Andrographolide and Resveratrol on OX1R and Prepro-orexin mRNA expression in CIPN-induced hypothalamus of mice with oxaliplatin

Chemotherapy-induced peripheral neuropathy (CIPN) is a one of side effect in cancer patients that receive antineoplastic agent, like oxaliplatin. Orexinergic system in the hypothalamus is the one of system that modulate nociceptive and neuropathy. Because there is flavonoids such as resveratrol and andrographolide that may prevent chemotherapy induced peripheral neuropathy, this study analyzed effects of andrographolide and resveratrol treatment on PPOrx and OX1R mRNA expression in hypothalamic oxaliplatin-induced mice. Materials and Methods This study was conducted for 22 days in mice. Mice injected with oxaliplatin followed by andrographolide or resveratrol. Chemotherapy induced peripheral neuropathic pain was assessed based on withdrawal threshold, mRNA PPOrx expression, and mRNA OX1R expression. Results The results showed that intraperitoneal injection of 100mg/kg resveratrol and 20mg/kg andrographolide increased the withdrawal threshold after oxaliplatin induction. Resveratrol administration also increased the relative expression of PPOrx mRNA significantly, but not the OX1R mRNA relative expression. On the other hand, administration of andrographolide did not cause a change in the expression of PPOrx and OX1R in the hypothalamus. Conclusions Intraperitoneal injection of andrographolide and resveratrol reduces the mechanical allodynia response in oxaliplatin-induced mice significantly. The mechanism of andrographolide increases the withdrawal threshold does not via the orexinergic system, but the mechanism of resveratrol via the orexinergic system.

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Gonadotropin Expression by Mouse Gonadotrope Cells.

Orexin plays a key role in the regulation of sleep and wakefulness and in feeding behavior in the central nervous system, but its receptors are expressed in various peripheral tissues including endocrine tissues. In the present study, we elucidated the effects of orexin on pituitary gonadotropin regulation by focusing on the functional involvement of bone morphogenetic proteins (BMPs) and clock genes using mouse gonadotrope LβT2 cells that express orexin type 1 (OX1R) and type 2 (OX2R) receptors. Treatments with orexin A enhanced LHβ and FSHβ mRNA expression in a dose-dependent manner in the absence of GnRH, whereas orexin A in turn suppressed GnRH-induced gonadotropin expression in LβT2 cells. Orexin A downregulated GnRH receptor expression, while GnRH enhanced OX1R and OX2R mRNA expression. Treatments with orexin A as well as GnRH increased the mRNA levels of Bmal1 and Clock, which are oscillational regulators for gonadotropin expression. Of note, treatments with BMP-6 and -15 enhanced OX1R and OX2R mRNA expression with upregulation of clock gene expression. On the other hand, orexin A enhanced BMP receptor signaling of Smad1/5/9 phosphorylation through upregulation of ALK-2/BMPRII among the BMP receptors expressed in LβT2 cells. Collectively, the results indicate that orexin regulates gonadotropin expression via clock gene expression by mutually interacting with GnRH action and the pituitary BMP system in gonadotrope cells.

Level of mRNA of orexin receptors of second type (ORXR2) in conditions of chronic alcoholation in structures of the rat brain

BACKGROUND: Orexin and its receptors are involved in the mechanisms of pathological craving for alcohol, but there is no unambiguous answer about the expression of the OX2R gene in the rat brain under conditions of alcohol addiction.
 AIM: The aim of this study was to determine the level of OX2R mRNA in brain structures in rats under conditions of prolonged alcoholization and during the period of abstinence.
 MATERIALS AND METHODS: Adult male Wistar rats were alcoholized with 15% ethanol as the only source of liquid for 6 months. The rats were decapitated after chronic alcoholization, as well as on the 1st and 7th days of alcohol withdrawal. Samples of brain structures (prefrontal cortex, hippocampus, ventral tegmental area) were frozen in liquid nitrogen and stored at 80C. Determination of the level of OX2R mRNA was carried out using the method of PCR-analysis.
 RESULTS: The level of ORXR2 gene expression significantly increased in the hippocampus on the 7th day of abstinence in relation to the group of intact animals and in relation to the group of chronically alcoholized rats. The level of OX2R mRNA in the prefrontal cortex and ventral tegmental area in animal groups of abstinence (1st and 7th days) did not change in relation to both alcohol and the intact animals.
 CONCLUSIONS: It is concluded that the orexin brain system may represent a new potential therapeutic target for the prevention of relapse in alcohol withdrawal. Together with the previously obtained data it was concluded that orexin receptor antagonists can be used to reduce the pathological craving for alcohol.

Single Prolonged Stress Alters Vasopressin and Orexin System Expression in Sprague Dawley Rats

Posttraumatic stress disorder (PTSD) is a complex psychiatric disorder triggered by a terrifying event, causing severe anxiety and nightmares. Studies showed that patients with PTSD have increased risk of cardiovascular disease. However, the effect of PTSD on blood pressure remains unraveled. In this study, we test the hypothesis that PTSD alters expression of arginine vasopressin (AVP) and orexin system components and therefore increases blood pressure. Single prolonged stress (SPS) protocol including 2 hours of restraint, a 20‐min forced swimming and exposure to diethyl ether until loss of consciousness was used to create a PTSD model. Eight‐week‐old male Sprague Dawley (SD) rats were divided into two groups and treated with or without SPS, then all rats were single‐housed and remained undisturbed for 7 days. Their blood pressure (BP) and heart rates (HR) were recorded by radio telemetry transmitter system (Data Science International) prior to the treatment as baseline, and 1‐ day, and 7‐day post the treatment. The data showed that SPS significantly increased BP (ΔBP: 8.384 ± 1.187 mmHg, N=6 P<0.05) and HR (ΔHR: 32.88 ± 9.149 beats/min, N=6 P<0.05) compared to control rats (ΔBP: 2.346 ± 0.9324 mmHg, ΔHR: ‐1.503 ± 5.960 beats/min, N=8) on day 1 post treatment, but no significance difference was observed in BP and HR on day 7 compared to their own baseline and between SPS and control groups.To investigate whether AVP and orexin systems participate in PTSD development, we examined paraventricular nucleus (PVN) and adrenal gland AVP‐ and orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) expression in both SD male and female rats with or without SPS at day 7. Our data showed that PVN AVP mRNA levels were increased by 2‐fold in the SPS group compared to control in male rats (P<0.05) but this change was not observed in female rats. No changes were observed in PVN OX1R and OX2R mRNA levels between SPS and control rats in both males and females. Peripheral orexin receptors’ protein levels were altered but showed inconsistency in male and female rats: western blot data showed that adrenal gland OX1R protein levels were increased in the SPS group (control: 1.000 ± 0.07424 N=5 vs SPS: 1.531 ± 0.2016 N=5; P<0.05) in male rats, while OX2R protein levels were decreased in female rats in female rats (control: 1.000 ± 0.01025 N=5 vs SPS: 0.8840 ± 0.04533 N=6; P<0.05). Renal OX1R was also increased (control: 1.000 ± 0.02794 N=3 vs SPS: 1.374 ± 0.1385 N=3; P<0.06) in female rats under SPS. ELISA assay showed that plasma orexin A levels were increased in female rats under SPS (control: 1.000 ± 0.1600 pg/mL N=3 vs SPS: 1.561 ± 0.1262 pg/mL; N=3 P<0.05) but not in male rats.These results suggested that the central AVP system and peripheral orexin system may play a role in PTSD‐related high blood pressure development, and the peripheral orexin system may mediate stress response of female rats more than male rats. These molecular differences between SD male and female rats may also help to answer why females and males showed different responses to trauma. Further study will continue to find the PTSD‐sensitized hypertensive model and investigate specific functions of AVP and orexin system in PTSD.

Zuogui Wan improves trabecular bone microarchitecture in ovariectomy-induced osteoporosis rats by regulating orexin-A and orexin receptor.

To investigate the protective effects of Zuogui Wan (ZGW) on bone loss induced by ovariectomy (OVX) and its mechanism via orexin-A and orexin receptors in the osteoporosis rat model. Fifty Sprague-Dawley female rats were randomly divided into sham-operated (sham) group and four OVX subgroups. Rats subjected to sham and OVX were treated with the vehicle (OVX, 1 mL/100 g weight, n = 10), 17β-estradiol (E2, 50 μg*kg-1*d-1), and ZGW at the doses of 2.3 (ZGW-L) and 4.6 (ZGW-H) g/kg/day lyophilized powder daily for 3 months, respectively. The serum biochemical parameters of 17β-estrogen (17β-E2), tartrate-resistant acid phosphatase (TRACP-5b) and bone alkaline phosphatase (BALP) were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was used to detect the changes in the morphological structure in bones. Microcomputed tomography was used to evaluate the bone mineral density and microarchitecture of the distal femur. The gene or protein expression of orexin-A, orexin receptor 1 (OX1R), orexin receptor 2 (OX2R), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were assayed by either quantitative polymerase chain reaction or Western blot analysis. Compared with the OVX group, ZGW could reduce the serum level of TRACP-5b and increased the serum levels of BALP and17β-E2 (P < 0.01). Meanwhile, ZGW could prevent bone loss and improved bone trabecular microarchitecture by increasing the trabeculae structure thickness and trabecular number, and arranging the trabeculae structure properly. Compared with the OVX group, it was upregulated for the orexin-A and OX2R mRNA or protein expression from the hypothalamus and tibiae, and OPG in the tibiae of ZGW groups (P < 0.01, < 0.05), while downregulated for the OX1R mRNA and protein expression in the tibiae and hypothalamus and RANKL from the tibiae (P < 0.01). ZGW exhibited a protective effect for PMOP that may be mediated via orexin-A and orexin receptors regulation.

Induction of narcolepsy-like symptoms by orexin receptor antagonists in mice.

Orexins/hypocretins are hypothalamic neuropeptides that promote and stabilize wakefulness by binding to the orexin receptor type-1 (OX1R) and type-2 (OX2R). Disruption of orexinergic signaling results in the sleep disorder narcolepsy in mice, rats, dogs, and humans. The orexin receptor antagonist suvorexant promotes sleep by blocking both OX1R and OX2R. Whereas suvorexant has been clinically approved for the treatment of insomnia because it is well tolerated in experimental animals as well as in human patients, a logical question remains as to why orexin receptor antagonists do not induce overt narcolepsy-like symptoms. Here we show that acute and chronic suvorexant promotes both rapid eye movement (REM) and non-rapid eye movement (NREM) sleep without inducing cataplexy in mice. Interestingly, chronic suvorexant increases OX2R mRNA and decreases orexin mRNA and peptide levels, which remain low long after termination of suvorexant administration. When mice are chronically treated with suvorexant and then re-challenged with the antagonist after a 1-week washout, however, cataplexy and sleep-onset REM (SOREM) are observed, which are exacerbated by chocolate administration. Heterozygous orexin knockout mice, with lower brain orexin levels, show cataplexy and SOREM after acute suvorexant administration. Furthermore, we find that acute suvorexant can induce cataplexy and SOREM in wild-type mice when co-administered with chocolate under stress-free (temporally anesthetized) conditions. Taken together, these results suggest that suvorexant can inhibit orexin synthesis resulting in susceptibility to narcolepsy-like symptoms in mice under certain conditions.

Prevention of cisplatin-induced nausea and vomiting by seabuckthorn (Hippophae rhamnoides L.) seed oil: Insights at the level of orexin-A in rats.

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<0.05) and gastric emptying delayed by cisplatin was improved (P<0.05-0.01) by pretreatment with SSO. Plasma OXA concentration, OX1R expression in the hypothalamus and brainstem increased significantly (P<0.05–0.01). Furthermore, OXA expression in LHA also increased significantly (P<0.05). Conclusion:SSO prevents cisplatin-induced vomiting in rats, which is possibly involved in increasing peripheral and central OXA and the expression of OX1R in the hypothalamus and brainstem.

Increase in the level of orexin receptor 1 (OX1R) mRNA in the brain structures of rats prone to impulsivity in behavior

Orexin and its receptors are involved in the mechanisms of pathological craving for alcohol and psychoactive drugs. The orexin system is also involved in the mechanisms of non-chemical forms of addiction: binge eating and gambling. The aim of this work was to study the level of orexin receptor mRNA in the hypothalamus, hippocampus, and prefrontal cortex of rats prone to impulsivity in behavior in a model for studying the elements of gambling addiction (a variant of the Iowa Gambling Task test). Brain structures were isolated on the 22nd day of the experiment. The expression of the OX1R gene was higher in the hypothalamus by 122% and in the hippocampus by 149% in rats that preferred to receive a high reward, but with a low probability as compared with a group of animals that preferred a low level of reinforcement, but with a 100% probability. In the prefrontal cortex, on the contrary, no significant changes were observed in the level of OX1R mRNA. The level of OX2R mRNA insignificantly changed in the hypothalamus, hippocampus, and prefrontal cortex of rats prone to impulsivity in behavior. The data indicate involvement of OX1R in the hypothalamus and hippocampus in mechanisms mediating impulsive behavior and the choice of the significance of positive reinforcement in terms of its varying strength and probability.

Interaction of Orexin A and Vasopressin in the Brain Plays a Role in Blood Pressure Regulation in WKY and SHR Rats.

BackgroundOrexin A (OXA) and vasopressin (AVP) exert a central hypertensive effect due to an increase in sympathetic nerve activity. To date, little is known about the interaction of these 2 neuropeptides in the central regulation of blood pressure. The present study compared the consequences of infusion into the left cerebral ventricle (ICV) of OXA on mean arterial blood pressure (MABP) in normotensive (WKY) and spontaneously hypertensive (SHR) rats, and explored whether the central pressor action of OXA in these 2 strains depends on activation of brain AVP V1a receptors (V1aR).Material/MethodsTen groups of experiments were performed on 12-week-old WKY and SHR rats implanted with ICV cannulas for infusion of OXA (3 nmol) and V1aR antagonist (V1aRANT, 500 ng), administered separately and together. Levels of V1aR and OXR in the medulla oblongata of WKY and SHR rats were compared in separate series.ResultsWe found that: 1) OXA significantly increased MABP only in WKY rats, 2) V1aRANT prevented an increase in MABP induced by OXA in WKY rats and decreased MABP in SHR rats, 3) OXA abolished the hypotensive action of V1aRANT in SHR rats, and 4) SHR rats had significantly higher levels of OX1R and V1aR proteins and OX1R mRNA in the brain medulla.ConclusionsThe present study shows that OXA and AVP can interact in the brain to affect blood pressure regulation, and that this interaction differs in normotension and hypertension.

Diurnal Regulation of the Orexin/Hypocretin System in Mice

A prominent feature of the hypothalamic neuropeptides orexins/hypocretins is their role in the regulation of sleep-wake behavior. While there is strong evidence for a diurnal (i.e. 24-h) rhythmicity of the expression of prepro-orexin (PPO) and its cleavage products, orexin A and B, it is not known whether orexin receptors are also subject to diurnal regulation. Here we ask whether besides the regulation of PPO the expression of the orexin receptor subtypes OX1R and OX2R varies over 24 hours in the mouse brain. The mRNA levels of PPO, OX1R, and OX2R as well as of various clock genes were analyzed over 24 hours in the hypothalamus, cortex, and adrenal glands of male mice using qPCR. We found a significant diurnal regulation of the mRNA levels of PPO as well as both orexin receptor subtypes in the brain, while no regulation was observed in adrenal glands. While in the cortex the mRNA levels of both OX1R and OX2R showed a significant diurnal regulation, in the hypothalamus, only the OX2R mRNA expression was subject to a diurnal rhythm. The expression of both orexin receptor subtypes significantly correlated with that of clock genes. Remarkably, the expression pattern of OX2R showed a strong and highly significant correlation with that of the clock gene Bmal1 in the cortex and hypothalamus. These results suggest that the rhythmic expression of orexin receptors is linked to clock gene expression and that OX2R may potentially play a role in the timing of sleep-wake behavior.

Involvement of orexin A in nocturnal melatonin secretion into the cerebrospinal fluid and the blood plasma in seasonal sheep

In sheep, differences in orexin A (OXA) gene expression and activity are related to changes in energy demand and seasonal reproduction. However, the mechanism by which and the key place where the OXA signal is integrated with photoperiod, whose main biochemical expression is melatonin (MEL), remain unknown. We examined the effects of cisterna magna injections of OXA (0.3 μg/kg body weight) on nocturnal cerebrospinal fluid (CSF) and plasma MEL concentrations; mRNA and protein expression of two rate-limiting enzymes for MEL biosynthesis, tryptophan 5-hydroxylase-1 (TPH1) and arylalkylamine-N-acetyltransferase (AA-NAT); and OXA receptor (OX1R, OX2R) expression in the pineal gland (PG) obtained from twenty ewes during the short-day (SD) and long-day (LD) seasons. OXA increased (P < 0.001) CSF and plasma MEL concentrations regardless of the season. Plasma MEL was positively correlated (P < 0.001) with CSF MEL in the OXA-treated sheep in both seasons. OXA had no effect (P > 0.05) on TPH1 transcript or protein level but upregulated (P < 0.05) AA-NAT mRNA and protein expression in both seasons. OXA enhanced (P < 0.05) OX1R mRNA level only during the LD season. Our results show that the endocrine activity of the ovine PG is regulated by day length and non-photic signals via hypothalamic OXA. These results are important for understanding the work of the biological clock and recognizing mechanisms responsible for the adaptation of seasonal animals to the changing external environment conditions. OXA and MEL are both involved in the regulation of the sleep-wakefulness system, therefore our results can be used in the study on the circadian rhythm disorders in humans (e.g. jet lag, insomnia, seasonal depression).

A comparison of the orexin receptor distribution in the brain between diurnal Nile grass rats (Arvicanthis niloticus) and nocturnal mice (Mus musculus)

The neuropeptide orexin/hypocretin regulates a wide range of behaviors and physiology through its receptors OX1R and OX2R, or HCRTR-1 and HCRTR-2. Although the distributions of these receptors have been established in nocturnal rodents, their distributions in the brain of diurnal species have not been studied. In the present study, we examined spatial patterns of OX1R and OX2R mRNA expression in diurnal Nile grass rats (Arvicanthis niloticus) by in situ hybridization and compared them with those in nocturnal mice (Mus musculus). Both receptors showed similar spatial patterns between species in most brain regions. However, species-specific expression was found in several regions that are mainly implicated in regulation of sleep/wakefulness, emotion and cognition. OX1R expression was detected in the caudate putamen and ventral tuberomammillary nucleus only in grass rats, while it was detected in the bed nucleus of the stria terminalis, medial division, posteromedial part only in mice. The distribution of OX2R mRNA was mostly consistent between the two species, although it was more widely expressed in the ventral tuberomammillary nucleus in grass rats compared to mice. These results suggest that neuronal pathways of the orexin system differ between chronotypes, and these differences could underlie the distinct profiles in behaviors and physiology between diurnal and nocturnal species.

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.

Chronic alcoholism influences the mRNA level of the orexin receptor type 1 (OX1R) in emotiogenic structures of the rat brain

Orexin and its receptors were shown to be involved into mechanisms of pathological craving to alcohol. This paper demonstrates that the orexin receptor type 1 (OX1R) mRNA level significantly decreased in the prefrontal cortex of rats chronically (during 6 months) consuming ethanol compared with intact control. The same results were observed on day 1 and day 7 of alcohol withdrawal after chronic alcoholization. On the contrary, in the hippocampus, the OX1R mRNA level increased on day 1 and day 7 of alcohol withdrawal. In the ventral tegmental area, the OX1R mRNA level did not change on the day 1 and day 7 of alcohol withdrawal compared with the groups of chronic alcoholization and intact control. These findings point out involvement of the prefrontal cortex and hippocampus first of all in mechanisms mediating chronic alcohol intoxication. The ventral tegmental area is described as a typical dopaminergic structure providing the executive mechanism of emotion reactions connected with alcohol abuse in particular. It is possibe, that the modulating action of orexins on dopaminergic neurons in this structure does not provide a significant effect on control of emotion reactions in alcoholism.

Orexin/hypocretin treatment restores hippocampal-dependent memory in orexin-deficient mice

Orexin A is produced in neurons of the lateral, perifornical and dorsomedial regions of the lateral hypothalamic area, which then project widely throughout the central nervous system to regulate arousal state, sleep-wake architecture, energy homeostasis and cognitive processes. Disruption of orexin signaling leads to sleep disturbances and increased body mass index, but recent studies also indicate that orexin neuron activation improves learning and memory. We hypothesized that hippocampal orexin receptor activation improves memory. To test this idea, we obtained orexin/ataxin-3 (O/A3) mice, which become deficient in orexin neurons by about 12 weeks of age. We first measured hippocampal orexin receptor 1 (OX1R) gene expression and protein levels, then tested acquisition and consolidation of two-way active avoidance (TWAA) memory, a hippocampal-dependent learning and memory task. Finally, we determined if exogenous intra-hippocampal OXA treatment could reverse cognitive impairment (as determined by TWAA) in OA/3 mice. We showed that OX1R mRNA expression and protein levels were significantly elevated in O/A3 mice, indicating the potential for preserved orexin responsiveness. The O/A3 mice were significantly impaired in TWAA memory vs. control mice, but OXA treatment (both acute and chronic) reversed these memory deficits. These results demonstrate that orexin plays an important role in hippocampal-dependent consolidation of two-way active avoidance memory, and orexin replacement can rescue the cognitive impairment.

Different Molecular/Behavioral Endophenotypes in C57BL/6J Mice Predict the Impact of OX1 Receptor Blockade on Binge-Like Ethanol Intake.

Ethanol (EtOH) research has focused on stages of dependence. It is of paramount importance to more deeply understand the neurobehavioral factors promoting increased risk for EtOH binge drinking during the early stages of the addiction cycle. The first objective of this study was to evaluate whether C57BL/6J mice showing high drinking in the dark (DID) exhibit neurobehavioral traits known to contribute to EtOH binge-drinking disorders. Comparing high vs. low drinkers (HD/LD), we evaluated different types of basal anxiety-like responses, EtOH preference and sensitivity to the reinforcing properties of EtOH, and basal mRNA expression of the OX1/OX2 receptors (OX1r/OX2r) within the prefrontal cortex (PFC) and the nucleus accumbens (NAcc). Additionally, we tested binge drinking by LD/HD in response to a selective OX1r antagonist following intermittent episodes of DID (iDID). We report that DID consistently segregates two neurobehavioral endophenotypes, HD vs. LD, showing differences in neophobia and/or impulsivity/compulsivity traits. Additionally, HD mice show decreased basal OX1r and OX2r mRNA expression within the NAcc and elevated OX1r within the PFC. Exposure to several intermittent episodes of EtOH DID triggered a rapid increase in EtOH intake over time in LD mice matching that observed in HD mice. Despite HD/LD endophenotypes did not show differences in EtOH intake, they still predicted the response to a pharmacological challenge with a selective OX1r antagonist. The present data underscore the relevance of HD/LD endophenotypes stemming from DID procedures for exploring neurobehavioral processes underlying the early stages of the addiction cycle and EtOH binge-drinking disorders.

Increased activity of the orexin system in the paraventricular nucleus contributes to salt-sensitive hypertension.

The orexin system is involved in arginine vasopressin (AVP) regulation, and its overactivation has been implicated in hypertension. However, its role in salt-sensitive hypertension (SSHTN) is unknown. Here, we tested the hypothesis that hyperactivity of the orexin system in the paraventricular nucleus (PVN) contributes to SSHTN via enhancing AVP signaling. Eight-week-old male Dahl salt-sensitive (Dahl S) and age- and sex-matched Sprague-Dawley (SD) rats were placed on a high-salt (HS; 8% NaCl) or normal-salt (NS; 0.4% NaCl) diet for 4 wk. HS intake did not alter mean arterial pressure (MAP), PVN mRNA levels of orexin receptor 1 (OX1R), or OX2R but slightly increased PVN AVP mRNA expression in SD rats. HS diet induced significant increases in MAP and PVN mRNA levels of OX1R, OX2R, and AVP in Dahl S rats. Intracerebroventricular infusion of orexin A (0.2 nmol) dramatically increased AVP mRNA levels and immunoreactivity in the PVN of SD rats. Incubation of cultured hypothalamus neurons from newborn SD rats with orexin A increased AVP mRNA expression, which was attenuated by OX1R blockade. In addition, increased cerebrospinal fluid Na+ concentration through intracerebroventricular infusion of NaCl solution (4 µmol) increased PVN OX1R and AVP mRNA levels and immunoreactivity in SD rats. Furthermore, bilateral PVN microinjection of the OX1R antagonist SB-408124 resulted in a greater reduction in MAP in HS intake (-16 ± 5 mmHg) compared with NS-fed (-4 ± 4 mmHg) anesthetized Dahl S rats. These results suggest that elevated PVN OX1R activation may contribute to SSHTN by enhancing AVP signaling.NEW & NOTEWORTHY To our best knowledge, this study is the first to investigate the involvement of the orexin system in salt-sensitive hypertension. Our results suggest that the orexin system may contribute to the Dahl model of salt-sensitive hypertension by enhancing vasopressin signaling in the hypothalamic paraventricular nucleus.

Influences Of Swimming On Learning And Memory And Expression Of Orexin And Ncam In Rat Hippocampus

PURPOSE: Through the different load intensity swimming exercise intervention in rats, we observed that the movement influenced on rats spatial learning and memory ability and detected the proteins expression of rat hippocampal. METHODS: 30 male SD rats were selected. After acclimatization to the laboratory for 1 week, the rats were randomly divided into 3 groups: Control Group(C), Medium load Group (M), over load Group (O).Group C were feed naturally; group M did swimming exercise intervention; group O did over load swimming exercise. The Morris Water Maze test (MWM) was performed to evaluate spatial learning and memory ability. Tissue samples were collected after the MWM test. 6 whole brain samples for immunofluorescence assay, while the bilateral hippocampus of the other 6 brains for western blot and real-time PCR analysis. RESULTS: (1) Blood testosterone detections: there was no significantly difference between group M and C (P>0.05), the blood testosterone of group O was significantly lower than group M and C (P<0.01); (2) MWM detection: during the navigation experiment, group M was easier to find the platform than group C (P<0.01). During the space exploration, rats in group M crossed where the platform was fixed more often than group C and O(p < 0.05),rats in group C crossed where the platform was fixed more often than group O(p < 0.05); (3) mRNA detection: the expression of NCAM mRNA and OXA mRNA of group M were significantly higher than group C (P<0.05), OX1R mRNA of group M was significantly lower than group C(P<0.05); (4) Protein detection: the expression of NCAM of group M was significantly higher than group C(P<0.05),the group O was significantly lower than group C(P<0.05). CONCLUSION:Medium load swimming exercise could improve the rats spatial learning and memory ability and significantly improve the expression of OXA, OX1R mRNA and OX1R, and significantly reduce the expression of OXA, OX1R mRNA, OX1R; Over load swimming exercise could damage the spatial learning and memory ability of rats, after over load swimming exercise, the expression of NCAM, OXA and OX1R was normal. Acknowledgments: Supported by the National Natural Science Foundation of China (No.31371202). Supported by the Sports Medicine key laboratory of State Administration of Sports/ Sichuan province Foundation.

Orexin receptor expression in the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axes of free-living European beavers (Castor fiber L.) in different periods of the reproductive cycle

Orexins are hypothalamic neuropeptides acting via two G protein-coupled receptors in mammals: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). In European beavers, which are seasonally breeding animals, the presence and functions of orexins and their receptors remain unknown. Our study aimed to determine the expression of OXR mRNAs and the localization of OXR proteins in hypothalamic–pituitary–adrenal/gonadal (HPA/HPG) axes in free-living beavers. The expression of OXR genes (OX1R, OX2R) and proteins was found in all analysed tissues during three periods of beavers’ reproductive cycle (April, July, November). The expression of OXR mRNAs in the beaver HPA axis varied seasonally (P<0.05). The levels of OX1R mRNA also differed between the sexes (P<0.05). In the mediobasal hypothalamus, OX1R transcript content increased in pregnant females in April (P<0.05) and OX2R expression increased in males in July (P<0.05). In the pituitary and adrenals, OX1R mRNA levels were relatively constant in females and peaked in July in males (P<0.05), whereas the OX2R was most highly expressed in males in November and in females in April (P<0.05). In gonads, OX1R expression did not fluctuate between seasons or sexes, but transcript levels were elevated in the testes in November and in the ovaries in July (P<0.05). In turn, OX2R mRNA levels varied between the sexes (P<0.05) and were higher in females (July and November) than in males (P<0.05). The circannual variations in OXR mRNA levels in HPA and HPG axes suggest that the expression of these receptors is associated with sex-specific changes in beavers’ reproductive activity and their environmental adaptations.