Blockade Of Orexin Receptors Research Articles

Alteration of Phospholipase C Expression in Rat Visual Cortical Neurons by Chronic Blockade of Orexin Receptor 1

Orexin-A well-known as hypocretin-1 is a neuropeptide that is produced in the lateral hypothalamus area (LHA). It promotes widespread cortical activation. In this study, the effect of SB-334867 as OX1R antagonist on phospholipase C (PLC) level was investigated in rat cerebral cortex slice preparations adopting immunohistochemistry approach. Nine Wistar rats PND12 were grouped (n = 3 for each group) as follows: group 1 contained native rats (Control); animals in group 2 and 3 received the SB-334867 vehicle and SB-334867 (10 mg/kg, i.p) daily from PND12 to PND30. The results showed that orexin antagonist decreased PLC levels in layers II, III (P < 0.01), I and V/VI (P < 0.05) of visual cortex. Therefore, it can be concluded that orexin receptor antagonist may decrease the level of PLC in most layers of the visual cortex.

Hypoventilation and autonomic dysfunction in infant rats following orexin receptor blockade

Orexin (hypocretin) is a neuropeptide expressed by neurons in the lateral and perifornical hypothalamus that project widely to respiratory and autonomic regions of the brainstem. The activity of orexin neurons depends on vigilance state; they are most active in wakefulness, less active in quiet sleep, and silent during active sleep. Although there are well‐described facilitatory effects of orexin in adult animals on the control of breathing and autonomic response to stress, its role in infancy has not been studied. This is an important issue because there is accumulating pathological evidence of orexinergic dysfunction in some cases of the Sudden Infant Death Syndrome (SIDS), a leading cause of death in infancy that is highly associated with abnormal respiratory and autonomic control during periods of sleep. We hypothesized that in infant (~2 week old) rat pups, orexin receptor blockade would: 1) lead to respiratory dysfunction, more so in wakefulness and quiet sleep than in active sleep, and 2) compromise the thermogenic response to mild environmental cooling. To test these hypotheses we used whole‐body plethysmography to monitor breathing and metabolic O2 consumption in rat pups treated with suvorexant, a selective orexin 1 and 2 receptor antagonist. Vigilance state was determined using high‐definition video to monitor and confirm standard behavioral criteria associated with quiet sleep, active sleep, and arousal in infant rat pups. Experiment 1: Pups cycled through wakefulness, quiet and active sleep for 1 hr at thermoneutral ambient temperature (TA=31°C), at which point suvorexant (1mg/kg in 50% DMSO; n=3) or vehicle alone (n=2) was injected via an intra‐abdominal cannula, and pups cycled between wakefulness and sleep for another 1 hr. The two groups were compared with respect to respiratory frequency (f), tidal volume (VT), ventilation (VE) and metabolic O2 consumption (VO2). Experiment 2: Pups were kept at thermoneutral TA for 1 hr, then exposed to a ~2°C drop in TA over the following 15 min, and then returned to baseline TA. Pups were then injected with either suvorexant (1mg/kg; n=8) or vehicle alone (n=8), and after another 1 hr, the TA challenge was repeated. In each animal, the change in metabolic O2 consumption in response to cooling was measured before and after drug or vehicle injection. In wakefulness and quiet sleep, suvorexant reduced respiratory frequency by 48 ± 4 breaths/min (~30%; p=0.01), and VE by 770 ± 254 ml/min/kg (~40%; p&lt;0.001), with no effect on VO2. These effects were not consistently observed during active sleep, and were absent in pups given vehicle alone. Suvorexant nearly abolished the metabolic response to cooling (p=0.01). These data suggest that orexin dysfunction can severely compromise breathing and autonomic function in infancy during wakefulness and quiet sleep.Support or Funding InformationFunding for this project provided by NHLBI grant 1R01HL136710‐01A1 (PI Cummings)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blockade of orexin receptors in the bed nucleus of stria terminalis increases serotonin level only in the left hypothalamus

The aim of investigation was to study the role of the hypothalamic serotoninergic system in mechanisms of blocking action of the orexin receptor antagonist on amphetamine-induced self-stimulation in rats. Methods. 27 male Wistar rats weighing 250-300 g were the object of the investigation. One week before the experiment, the microelectrodes were implanted into the lateral hypothalamus for self-stimulation and microcannulas into the bad nucleus of stria terminalis (BNST) for microinjections. The electrodes were implanted bilaterally and cannulas unilaterally (right or left). Intact rats and rats treated with amphetamine were controls. On the day of the experiment, amphetamine 1 mg/kg i.p. was injected, and then SB-408124 1 µg in 1 µl was administered into the BNST in 10 min. The rats were decapitated in 15 min after microinjection of SB-408124. The level of serotonin (Ser) and its metabolite 5-HIAA was determined in the right and the left hypothalamus by HPLC with electrochemical detection. Results. In intact rats, the 5-HIAA level in the left hypothalamus was higher than in the right one (p &lt; 0.05). After systemic administration of amphetamine the ratio of 5-HIAA/Ser was significantly decreased both in the left (p &lt; 0.05) and the right (p &lt; 0.05) hypothalamus. The levels of Ser and 5-HIAA stated symmetrical. It is important that effects of orexin antagonist after amphetamine administration appeared only on the side contralateral to microinjection being significantly only in the left hypothalamus. After administration of orexin antagonist into the right BNST, the level of Ser in the left hypothalamus increased compared both to intact rats (p &lt; 0.05) and rats treated with amphetamine (p &lt; 0.05). Conclusion. The results suggest that the left hypothalamus plays a leading role in developing of self-stimulation reaction and support the hypothesis that the serotoninergic system is involved in mechanism of blocking action of orexin receptors antagonist on self-stimulation activated with amphetamine. (For citation: Karpova IV, Bychkov ER, Lebedev AA, Shabanov PD. Blockade of orexin receptors in the bed nucleus of stria terminalis increases serotonin level only in the left hypothalamus. Reviews on Clinical Pharmacology and Drug Therapy. 2018;16(2):33-36. doi: 10.17816/RCF16233-36).

Hippocampal orexin receptor blocking prevented the stress induced social learning and memory deficits.

Stress as a homeostatic challenge leads to the malfunction of learning and memory processes, namely social learning and memory. The orexin system is involved in stress responses through connections to the hypothalamic-pituitary axis (HPA). In addition, the hippocampus, a structure vulnerable to stress-induced changes, expresses orexin receptors 1 and 2 (OXr1 and OXr2) in various sub-regions. The present study is aimed at assessing the effects of hippocampal orexin receptor blockade on social learning and memory impairments and anxiety development following stress. Male Wistar rats (220-250 g) underwent cannula implantation in the hippocampus. Acute (two mild electric shocks, 5.5 mA) and chronic stresses (ten days of restraint, 6 h daily) were applied with or without injection of orexin receptor antagonists (SB-334867 or TCS OX 29). Sociability and social novelty in animals were assessed in a three-chamber social maze at the end of stress application. Anxiety and exploratory behavior of animals were then examined, with 20 min intervals, using the open field (OF) and elevated plus maze (EPM) tests, respectively. Cisterna Magna cerebro-spinal fluid (CSF) was drained, before sacrifice, for orexin (OX) assay and trunk blood was collected to measure the plasma corticosterone (CRT). Neither the acute nor the chronic stress could affect the sociability. The acute but not chronic stress prevented the animal from sniffing the familiar caged rat in the novelty session, a response which was reversed following the blockade of both OXRs. Furthermore, acute but not chronic stress, led to increased anxiety and immobility behavior which were both impeded by blocking the orexin receptor (OXR). Conversely, OX content in CSF increased due to chronic restraint stress, an effect that was reversed by orexin blockade. Finally, elevated plasma CRT was recorded in response to both acute and chronic stresses. The observed increase in plasma CRT in chronically-stressed rats was abolished following inhibition of OXRs, however a similar effect was not seen in the acute-stress group. Our results identify hippocampal OXRs as potential candidates capable of preventing acute stress-induced impairments of social novelty and anxiety behavior, and chronic stress-induced plasma CRT and CSF orexin, changes. OXR manipulation may improve adaptation to stress pathophysiology.

Sex- and Age-dependent Effects of Orexin 1 Receptor Blockade on Open-Field Behavior and Neuronal Activity

Adolescence is a sensitive and critical period in brain development where psychiatric disorders such as anxiety, depression and post-traumatic stress disorder are more likely to emerge following a stressful life event. Females are two times more likely to suffer from psychiatric disorders than males. Patients with these disorders show alterations in orexins (also called hypocretins), important neuropeptides that regulate arousal, wakefulness and the hypothalamic–pituitary–adrenal axis activity. Little is known on the role of orexins in mediating arousal behaviors in male and female rats during adolescence or adulthood. Here, we examine the influence of orexin 1 receptor blockade by SB334867 in open-field behavior in male and female rats during early adolescence (PND 31–33) or adulthood (PND 75–77). Animals were injected with 0 (vehicle), 1, 10, or 30 mg/kg SB334867 (i.p.). Thirty minutes later, they were placed in an open field, and behavior and neuronal activity (c-Fos) were assessed. In adolescent males, SB334867 significantly increased immobility in the 10 mg/kg group compared to vehicle. However, this increase in immobility in adolescent males was not observed in adolescent females. In contrast to adolescent males, adult males in the 10 mg/kg dose group showed the opposite effect on immobility compared to vehicle. These results indicate that 10 mg/kg dose of SB334867 has opposing effects in adolescent and adult males, but few effects in adolescent and adult females. Differences in functional networks between limbic regions may underlie these effects of orexin receptor blockade that are sex- and age-dependent in rats.

Effects of Suvorexant, a Dual Orexin/Hypocretin Receptor Antagonist, on Impulsive Behavior Associated with Cocaine

Hypothalamic hypocretin (orexin) peptides mediate arousal, attention, and reward processing. Fibers containing orexins project to brain structures that govern motivated behavior, including the ventral tegmental area (VTA). A number of psychiatric conditions, including attention deficit hyperactivity disorder (ADHD) and substance use disorders, are characterized by deficits in impulse control, however the relationship between orexin and impulsive behavior is incompletely characterized. The effects of systemic or centrally administered orexin receptor (OXR) antagonists on measures of impulsive-like behavior in rats were evaluated using the five-choice serial reaction time task (5-CSRTT) and delay discounting procedures. These paradigms were also used to test the capacity of OXR antagonists to attenuate acute cocaine-evoked impulsivity. Finally, immunohistochemistry and calcium imaging were used to assess potential cellular mechanisms by which OXR blockade may influence motor impulsivity. Suvorexant, a dual (OX1/2R) orexin receptor antagonist, reduced cocaine-evoked premature responses in 5-CSRTT when administered systemically or directly into VTA. Neither suvorexant nor OX1R- or OX2R-selective compounds (SB334867 or TCS-OX2-29, respectively) altered delay discounting. Finally, suvorexant did not alter Fos-immunoreactivity within tyrosine hydroxylase-immunolabeled neurons of VTA, but did attenuate cocaine- and orexin-induced increases in calcium transient amplitude within neurons of VTA. Results from the present studies suggest potential therapeutic utility of OXR antagonists in reducing psychostimulant-induced motor impulsivity. These findings also support the view that orexin transmission is closely involved in executive function in normal and pathological conditions.

The Orexin System and Hypertension.

In this review, we focus on the role of orexin signaling in blood pressure control and its potential link to hypertension by summarizing evidence from several experimental animal models of hypertension. Studies using the spontaneously hypertensive rat (SHR) animal model of human essential hypertension show that pharmacological blockade of orexin receptors reduces blood pressure in SHRs but not in Wistar-Kyoto rats. In addition, increased activity of the orexin system contributes to elevated blood pressure and sympathetic nerve activity (SNA) in dark-active period Schlager hypertensive (BPH/2J) mice, another genetic model of neurogenic hypertension. Similar to these two models, Sprague-Dawley rats with stress-induced hypertension display an overactive central orexin system. Furthermore, upregulation of the orexin receptor 1 increases firing of hypothalamic paraventricular nucleus neurons, augments SNA, and contributes to hypertension in the obese Zucker rat, an animal model of obesity-related hypertension. Finally, we propose a hypothesis for the implication of the orexin system in salt-sensitive hypertension. All of this evidence, coupled with the important role of elevated SNA in increasing blood pressure, strongly suggests that hyperactivity of the orexin system contributes to hypertension.

Blockade of orexin receptors attenuates the cardiovascular response to air-jet stress in spontaneously hypertensive rats.

This study tested the hypothesis that orexin plays a role in the elevated pressor response to acute stress in the spontaneously hypertensive rat (SHR). The pressor response to air jet stress (AJS) (n=11/group) was 2.5 times greater in vehicle treated SHR versus Wistar (WIS) rats. Systemic delivery of 30mg/kg of the dual orexin receptor antagonist almorexant did not significantly change resting mean arterial pressure (MAP) but did attenuate the pressor response elicited by AJS to a greater extent in the SHR compared to the Wistar rats (~65% versus ~33% reduction respectively; n=6/group). Alternatively 100mg/kg almorexant reduced resting MAP in the SHR (~25mm Hg drop) and attenuated both the heart rate (HR; ~50% reduction) and MAP (~62% reduction) response to AJS in both strains (n=6/group). Systemic application of SB-334867 (3mg/kg), an orexin receptor type 1 antagonist (n=5/group), selectively reduced resting MAP and attenuated the HR response to AJS in the SHR but had no effect on the pressor response in either strain. The potential role of endogenous orexin release in cardiovascular control in the SHR was linked to a significant increase in brain-derived neurotrophic factor mRNA expression in the hypothalamus and elevated orexin receptor expression (type 2 only) in the dorsal pons when compared to WIS (n=4/group). These results demonstrate that the exaggerated pressor response in the SHR to stress is linked to increased orexin receptor activation and possibly altered orexin receptor expression in the dorsal pons and BDNF expression in the hypothalamus.

Functional interaction between orexin-1 and CB1 receptors in the periaqueductal gray matter during antinociception induced by chemical stimulation of the lateral hypothalamus in rats.

Chemical stimulation of the lateral hypothalamus (LH) with carbachol induces antinociception which is antagonized by blockade of orexin receptors in some pain modulatory sites in the tail-flick test. In this study, we evaluated the role of orexin-1 and CB1 receptors in the periaqueductal gray matter (PAG), a critical pain modulatory site, in mediation of antinociceptive responses induced by LH stimulation in rats. One hundred thirty-two adult male albino Wistar rats weighing 180-250g were unilaterally implanted with two separate cannulae into the LH and ventrolateral PAG (vlPAG). Intra-vlPAG administration of SB334867, as a selective orexin-1 receptor antagonist (0.5, 1.5, 5, 15 and 50nM), or AM251, as a selective CB1 receptor antagonist (1, 3, 10, 30 and 100nM), was performed just 5min before carbachol (125nM) microinjection into the LH. Our findings showed that SB334867 or AM251 administration dose dependently prevented the development of LH-induced antinociception in rats. Treatment with two antagonists at the same time could not intensify their effects in comparison with separate administration of antagonists. It seems that antinociceptive effect of intra-LH administration of carbachol is mediated, at least partially, through the activation of orexin-1 and CB1 receptors in the vlPAG. This work demonstrates a pain modulatory role of the orexinergic system via the PAG in hypothalamic-mediated analgesia suggesting that orexins can be advantageously targeted to achieve analgesia. WHAT DOES THIS STUDY ADD?: OX1 receptor antagonist (SB334867) administration into the ventrolateral periaqueductal gray matter (vlPAG) dose dependently blocked the carbachol-induced antinociception. CB1 receptor antagonist (AM251) microinjection in the vlPAG prevented carbachol-induced antinociception in a dose-dependent manner. Concurrent administration of SB334867 and AM251 into the vlPAG did not reinforce the antinociceptive responses.

An augmented CO2 chemoreflex and overactive orexin system are linked with hypertension in young and adult spontaneously hypertensive rats.

Activation of central chemoreceptors by CO2 increases sympathetic nerve activity (SNA), arterial blood pressure (ABP) and breathing. These effects are exaggerated in spontaneously hypertensive rats (SHRs), resulting in an augmented CO2 chemoreflex that affects both breathing and ABP. The augmented CO2 chemoreflex and the high ABP are measureable in young SHRs (postnatal day30-58) and become greater in adult SHRs. Blockade of orexin receptors can normalize the augmented CO2 chemoreflex and the high ABP in young SHRs and normalize the augmented CO2 chemoreflex and significantly lower the high ABP in adult SHRs. In the hypothalamus, SHRs have more orexin neurons, and a greater proportion of them increase their activity with CO2 . The orexin system is overactive in SHRs and contributes to the augmented CO2 chemoreflex and hypertension. Modulation of the orexin system may be beneficial in the treatment of neurogenic hypertension. Activation of central chemoreceptors by CO2 increases arterial blood pressure (ABP), sympathetic nerve activity and breathing. In spontaneously hypertensive rats (SHRs), high ABP is associated with enhanced sympathetic nerve activity and peripheral chemoreflexes. We hypothesized that an augmented CO2 chemoreflex and overactive orexin system are linked with high ABP in both young (postnatal day30-58) and adult SHRs (4-6 months). Our main findings are as follows. (i)An augmented CO2 chemoreflex and higher ABP in SHRs are measureable at a young age and increase in adulthood. In wakefulness, the ventilatory response to normoxic hypercapnia is higher in young SHRs (mean±SEM: 179±11% increase) than in age-matched normotensive Wistar-Kyoto rats (114±9% increase), but lower than in adult SHRs (226±10% increase; P<0.05). The resting ABP is higher in young SHRs (122±5mmHg) than in age-matched Wistar-Kyoto rats (99±5mmHg), but lower than in adult SHRs (152±4mmHg; P<0.05). (ii)Spontaneously hypertensive rats have more orexin neurons and more CO2 -activated orexin neurons in the hypothalamus. (iii)Antagonism of orexin receptors with a dual orexin receptor antagonist, almorexant, normalizes the augmented CO2 chemoreflex in young and adult SHRs and the high ABP in young SHRs and significantly lowers ABP in adult SHRs. (iv)Attenuation of peripheral chemoreflexes by hyperoxia does not abolish the augmented CO2 chemoreflex (breathing and ABP) in SHRs, which indicates an important role for the central chemoreflex. We suggest that an overactive orexin system may play an important role in the augmented central CO2 chemoreflex and in the development of hypertension in SHRs.

Blockade of orexin receptors reduces glutamatergic excitation of adrenal premotor neurons in the rostral ventrolateral medulla

Excitation of adrenal sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) elicits epinephrine release as part of the glucose counter‐regulatory response to hypoglycemia. It is known that this response is partly mediated by excitatory inputs from hypothalamic orexin neurons. Although some orexin neurons co‐express glutamate, whether orexin and glutamate interact in order to facilitate adrenal sympathetic premotor neurons is unknown. In pentobarbitone‐anesthetized male Sprague‐Dawley rats, we show tested whether glutamatergic activation of adrenal premotor neurons depends on activation of orexin receptors (Ox‐R). Microinjection of glutamate (50 mM/50 nl) into the RVLM, where adrenal premotor neurons are found, increased adrenal sympathetic nerve activity (ASNA). The increase in ASNA was reduced following non‐selective blockade of orexin receptors. RVLM orexin receptors were blocked by microinjecting a combination of SB334867 (Ox1‐R antagonist) and TCS OX2 29 (Ox2‐R antagonist) into the RVLM. The reduction in ASNA in response to glutamate microinjection into the RVLM was dose dependent (0.1–10 mM/50 nl), and the response was abolished with 1 mM of the combined antagonists (P&lt;0.05). The results suggest that the counter‐regulatory release of epinephrine during hypoglycemia depends on the interaction between glutamate and orexin receptors located on the adrenal sympathetic premotor neurons.Support or Funding InformationNHMRC AustraliaAustin Medical Research Foundation

Upregulation of Orexin in the Paraventricular Nucleus Contributes to Salt Sensitive Hypertension

Orexin and orexin receptors are extensively expressed in the hypothalamic paraventricular nucleus (PVN) and the hyperactivity of PVN orexin contributes to increased sympathetic nerve activity (SNA) and elevated blood pressure in several animal models. However, the role of PVN orexin in the development of salt sensitive hypertension (SSHTN) is unknown. In this study, we tested the hypothesis that PVN orexin expression is increased in SSHTN and contributes to the development of SSHTN via regulating vasopressin secretion. Eight‐week‐old Dahl salt sensitive (DS) rats were divided into two groups and were fed either a high salt (HS, 8% NaCl) or normal salt (NS, 0.4% NaCl) diet. Six weeks following diet treatment, rats were anesthetized and their mean arterial pressure (MAP) and sympathetic nerve activity (SNA) response to orexin receptor blockade were recorded. In a separate group of rats PVN orexin mRNA levels were measured. The results showed that HS intake induced significant increases in MAP (HS: 146±2 vs. NS: 112±5; n=5; P&lt;0.05) and PVN orexin mRNA levels (HS: 3.8±0.37 vs. NS: 1.0±0.68; n=3; P&lt;0.05). Bilateral PVN microinjection of orexin receptor 1 (OX1R) antagonist SB408124 (30 pmol/50nl) resulted in a greater reduction in arterial blood pressure in DS rats with a HS intake compared to their cohorts with a NS diet (HS: −16±5 vs. NS: −4±4 mmHg; n=5–6; P&lt;0.05). OX1R blockade also increased splanchnic SNA (HS: 48.2±12.6%) vs. NS: 35.82±9%; P=0.2) and renal SNA (HS: 26.7±7.5% vs. NS: 17.4±9.5%; P=0.2) in DS rats with either HS or NS diet. Incubation of orexin A resulted in a dose‐dependent increase in vasopressin mRNA levels (100nM: 2.9‐fold; 1μM; 5.6 fold; 10μM: 18‐fold) in primary cultured neurons from the hypothalamus containing the PVN of neonatal Sprague Dawley rats. These orexin A‐induced increases of vasopressin mRNA were abolished by OX1R blockade. These results coupled with the evidence that plasma vasopressin is upregulated in DS rats under the condition of HS‐induced hypertension suggest that increased PVN orexin expression may be involved in the development of SSHTN through regulating vasopressin secretion.Support or Funding InformationThis study is supported by AHA 11SDG7420029 and NIH R15‐HL122952.

Orexin and Central Modulation of Cardiovascular and Respiratory Function.

Orexin makes an important contribution to the regulation of cardiorespiratory function. When injected centrally under anesthesia, orexin increases blood pressure, heart rate, sympathetic nerve activity, and the amplitude and frequency of respiration. This is consistent with the location of orexin neurons in the hypothalamus and the distribution of orexin terminals at all levels of the central autonomic and respiratory network. These cardiorespiratory responses are components of arousal and are necessary to allow the expression of motivated behaviors. Thus, orexin contributes to the cardiorespiratory response to acute stressors, especially those of a psychogenic nature. Consequently, upregulation of orexin signaling, whether it is spontaneous or environmentally induced, can increase blood pressure and lead to hypertension, as is the case for the spontaneously hypertensive rat and the hypertensive BPH/2J Schlager mouse. Blockade of orexin receptors will reduce blood pressure in these animals, which could be a new pharmacological approach for the treatment of some forms of hypertension. Orexin can also magnify the respiratory reflex to hypercapnia in order to maintain respiratory homeostasis, and this may be in part why it is upregulated during obstructive sleep apnea. In this pathological condition, blockade of orexin receptors would make the apnea worse. To summarize, orexin is an important modulator of cardiorespiratory function. Acting on orexin signaling may help in the treatment of some cardiovascular and respiratory disorders.

Decreased plasma orexin levels in subjects with impaired sleep quality

The orexin system has been identified to play an important role in controlling arousal and regulation of sleep and wakefulness. An orexin deficit has been identified as the most important part of the pathophysiology of sleepiness in narcolepsy. Although orexin receptor blockade has been identified as a successful new treatment approach to insomnia, a disorder of hyper-arousal, no study has investigated whether orexin plasma levels are elevated in this disorder. In this investigation we analyzed the association between the orexin plasma levels and Sleep Quality (PSQI) scores in a sample of 259 subjects (18–55 [M = 33.4] years) from the general population and hypothesized poor sleepers to show higher orexin levels. Opposite to the hypothesis the global PSQI score correlates negatively with orexin levels (r = −.199, p = .002). Nearly one quarter (24.1%) of the sample was classified as poor sleepers (global PSQI score > 5). In poor sleepers a significantly lower plasma orexin level was found compared to good sleepers (p = .014). Further analysis revealed significant negative relationships between the plasma orexin levels and the PSQI component scores “subjective sleep quality” (r = −.140, p = .03) and “daytime dysfunction” (r = −.194, p = .002). Although a high percentage of the poor sleepers was most likely suffering from insomnia their probable hyperarousal did not appear to be associated with an increase in plasma orexin levels.

Spontaneously hypertensive rats have more orexin neurons in their medial hypothalamus than normotensive rats.

What is the central question of this study? Blockade of orexin receptors reduces blood pressure in spontaneously hypertensive rats (SHRs) but not in normotensive Wistar-Kyoto (WKY) rats, suggesting that upregulation of orexin signalling underlies the hypertensive phenotype of the SHR. However, it is not known what causes this upregulation. What is the main finding and its importance? Using orexin immunolabelling, we show that SHRs have 20% more orexin neurons than normotensive WKY and Wistar rats in the medial hypothalamus, which is a good match to their blood pressure phenotype. In contrast, there is no such match for the orexin neurons of the lateral hypothalamus. Essential hypertension may be linked to an increase in orexin neurons in the medial hypothalamus. The neuropeptide orexin contributes to the regulation of blood pressure as part of its role in the control of arousal during wakefulness and motivated behaviour (including responses to psychological stress). Recent work shows that pharmacological blockade of orexin receptors reduces blood pressure in spontaneously hypertensive rats (SHRs) but not in normotensive Wistar-Kyoto (WKY) rats. It is not clear why orexin signalling is upregulated in the SHR, but one possibility is that these animals have more orexin neurons than their normotensive WKY and Wistar relatives. To test this possibility, SHRs, WKY and Wistar male rats (6-16 weeks old) were killed, perfused and their brains sectioned and immunolabelled for orexin A. Labelled neurons were plotted and counted in the six best labelled hemisections (120 μm apart) of each brain. There were significantly more orexin neurons (+20%) in the medial hypothalamus (medial to fornix) of SHRs compared with WKY and Wistar rats (126 ± 4 versus 106 ± 5 and 104 ± 5 per hemisection, respectively, P < 0.05), which matches well the blood pressure phenotypes. In contrast, counts in the lateral hypothalamus did not match the blood pressure phenotypes (69 ± 2 versus 50 ± 3 and 76 ± 4, respectively). The results support the idea that orexin signalling is upregulated in the SHR and suggest that this is due, at least in part, to a greater number of orexin neurons in the medial hypothalamus. These medial orexin neurons, which are also involved in hyperarousal and stress responses, may contribute to the development of essential hypertension.

The electrical activity of hippocampal pyramidal neuron is subjected to descending control by the brain orexin/hypocretin system

The hippocampus receives sparse orexinergic innervation from the lateral hypothalamus and expresses a high level of orexin receptor. The function of orexin receptor in the regulation of hippocampal neural activity has never been investigated. In this study, in vivo single unit recording was performed in urethane-anesthetized rats. After 15min of baseline recording from pyramidal neuron within the CA1 region of the dorsal hippocampus, i.c.v. injection of orexin-A 0.5nmol, SB334867 400nmol, a selective orexin receptor 1 antagonist, saline, or DMSO, or microinjection of carbachol 250nmol or saline into the ipsilateral lateral hypothalamus were performed using a Hamilton microsyringe, and the spontaneous firing activity continued to be recorded for 25min. Results showed that orexin administration into the lateral cerebral ventricle excited 6 out of 8 neurons and inhibited 1 neuron. Chemical stimulation of the lateral hypothalamus by carbachol excited 9 out of 13 hippocampal neurons and inhibited 3 neurons. On the other hand, i.c.v. injection of the SB334867, caused reductions in the firing activity of 6 out of 10 neurons and increases in 4 additional neurons. It seems that orexin neurotransmission in the hippocampus mostly elicits an excitatory response, whereas blockade of orexin receptor has an inhibitory effect. Further studies need to be done to elucidate the underlying mechanism of orexin action on hippocampal neurons.

Orexin neuropeptides contribute to the development and persistence of generalized avoidance behavior in the rat.

Avoidance of contexts directly associated with fearful experiences represents an adaptive behavioral survival strategy. Over-interpretation of contextual cues leading to generalized avoidance of situations that are only remotely similar to the original fear context represents a pathologic process that contributes to anxiety disorders. Orexin neuropeptides modulate anxiety-like behavioral and physiological responses. The objective of this paper was to investigate the impact of pharmacological orexin receptor blockade on generalized avoidance behavior. Rats received a single electric foot-shock in the dark side of a two-compartment shuttle box followed by situational context reminders. After shock, rats were treated chronically (3weeks) with the orexin receptor antagonist almorexant or with the selective serotonin reuptake inhibitor sertraline, used as positive anxiolytic control. In week 3, avoidance behavior was measured under conditions of high (dark-light (DL)-box) and low (elevated plus maze (EPM)) similarity to the original shock context. Avoidance behavior was re-assessed 5 and 17weeks after treatment termination. Avoidance in the DL box (contextual fear memory) remained unaffected by any treatment and lasted 20weeks post-shock exposure. Avoidance in the EPM (neophobic fear generalization) was partially attenuated during treatment with almorexant and sertraline at week 3. Following 5 and 17weeks of drug washout, avoidance in the EPM was significantly reduced in almorexant- but not in sertraline-treated rats. Almorexant also reduced persistent avoidance in the EPM upon treatment initiation 3weeks after shock exposure. Chronic orexin receptor blockade in rats reduces both the development and persistence of generalized avoidance in situations with low similarity to the initial shock context.

Suvorexant: first global approval.

Suvorexant (Belsomra(®)), a first-in-class, orally active dual orexin-1 receptor and orexin-2 receptor antagonist, has been developed by Merck for the treatment of insomnia. Variations in the levels of the neuropeptides orexin A and orexin B have been linked to circadian rhythms and wakefulness. Orexin-producing neurons in the lateral hypothalamus regulate wakefulness by signalling through orexin receptors. Blockade of orexin receptors is known to promote sleep. Suvorexant was approved in the US in August 2014 for the treatment of adults with sleep onset and/or sleep maintenance insomnia. The drug is also preregistration in Japan, with approval submissions planned for other countries worldwide for this indication. This article summarizes the milestones in the development of suvorexant leading to this first approval for insomnia.

Orexinergic Activation of Medullary Premotor Neurons Modulates the Adrenal Sympathoexcitation to Hypothalamic Glucoprivation

Glucoprivation activates neurons in the perifornical hypothalamus (PeH) and in the rostral ventrolateral medulla (RVLM), which results in the release of adrenaline. The current study aimed to establish 1) whether neuroglucoprivation in the PeH or in the RVLM elicits adrenaline release in vivo and 2) whether direct activation by glucoprivation or orexin release in the RVLM modulates the adrenaline release. Neuroglucoprivation in the PeH or RVLM was elicited by microinjections of 2-deoxy-D-glucose or 5-thio-D-glucose in anesthetized, euglycemic rats. Firstly, inhibition of neurons in the PeH abolished the increase in adrenal sympathetic nerve activity (ASNA) to systemic glucoprivation. Secondly, glucoprivation of neurons in the PeH increased ASNA. Thirdly, in vivo or in vitro glucoprivation did not affect the activity of RVLM adrenal premotor neurons. Finally, blockade of orexin receptors in the RVLM abolished the increase in ASNA to neuroglucoprivation in the PeH. The evoked changes in ASNA were directly correlated to levels of plasma metanephrine but not to normetanephrine. These findings suggest that orexin release modulates the activation of adrenal presympathetic neurons in the RVLM.

Ghrelin acts as an interface between physiological state and phasic dopamine signaling.

Brief, high-concentration (phasic) spikes in nucleus accumbens dopamine critically participate in aspects of food reward. Although physiological state (e.g., hunger, satiety) and associated hormones are known to affect dopamine tone in general, whether they modulate food-evoked, phasic dopamine specifically is unknown. Here, we used fast-scan cyclic voltammetry in awake, behaving rats to record dopamine spikes evoked by delivery of sugar pellets while pharmacologically manipulating central receptors for the gut "hunger" hormone ghrelin. Lateral ventricular (LV) ghrelin increased, while LV ghrelin receptor antagonism suppressed the magnitude of dopamine spikes evoked by food. Ghrelin was effective when infused directly into the lateral hypothalamus (LH), but not the ventral tegmental area (VTA). LH infusions were made in close proximity to orexin neurons, which are regulated by ghrelin and project to the VTA. Thus, we also investigated and found potentiation of food-evoked dopamine spikes by intra-VTA orexin-A. Importantly, intra-VTA blockade of orexin receptors attenuated food intake induced by LV ghrelin, thus establishing a behaviorally relevant connection between central ghrelin and VTA orexin. Further analysis revealed that food restriction increased the magnitude of dopamine spikes evoked by food independent of any pharmacological manipulations. The results support the regulation of food-evoked dopamine spikes by physiological state with endogenous fluctuations in ghrelin as a key contributor. Our data highlight a novel mechanism by which signals relating physiological state could influence food reinforcement and food-directed behavior.