Blockade of orexin receptors in the ventral tegmental area reduced the extinction period of the lateral hypothalamic-induced conditioned place preference in rats.

The orexinergic connection between the lateral hypothalamus (LH) and the ventral tegmental area (VTA) is involved in modulating the reward circuit. The conditioned place preference (CPP) can be induced by microinjection of carbachol, a cholinergic agonist, into the LH. The current research was conducted to understand whether intra-VTA orexin receptors (OXRs) could influence the duration of the extinction period or maintenance of the intra-LH carbachol-induced CPP. To this end, the rats unilaterally received intra-LH carbachol (250 nM) within a 3-day conditioning period. Animals that have already passed the conditioning test were unilaterally administered by intra-VTA microinjection of SB334867, an OX1R antagonist, or TCS OX2 29, an OX2R antagonist during the extinction phase of the LH stimulation-induced CPP. For the first time, our data indicated that daily intra-VTA administration of either SB334867 (30 nM) or TCS OX2 29 (10 and 30 nM) during the extinction period decreased the maintenance of intra-LH carbachol-induced CPP. In conclusion, OXRs in the VTA play crucial roles in the maintenance of reward processes.

Orexin-1 Receptor Signaling in Ventral Pallidum Regulates Motivation for the Opioid Remifentanil.

Signaling at the orexin-1 receptor (OxR1) is important for motivated drug taking. Using a within-session behavioral economics (BE) procedure, we previously found that pharmacologic blockade of the OxR1 decreased motivation (increased demand elasticity) for the potent and short-acting opioid remifentanil and reduced low-effort remifentanil consumption. However, the mechanism through which orexin regulates remifentanil demand is currently unknown. Previous work implicated OxR1 signaling within ventral pallidum (VP) as a potential target. VP is densely innervated by orexin fibers and is known to regulate opioid reward. Accordingly, this study sought to determine the role of VP OxR1 signaling in remifentanil demand and cue-induced reinstatement of remifentanil seeking in male rats. Intra-VP microinjections of the OxR1 antagonist SB-334867 (SB) decreased motivation (increased demand elasticity; α) for remifentanil without affecting remifentanil consumption at low effort. Baseline α values predicted the degree of cue-induced remifentanil seeking, and microinjection of SB into VP attenuated this behavior without affecting extinction responding. Baseline α values also predicted SB efficacy, such that SB was most effective in attenuating reinstatement behavior in highly motivated rats. Together, these findings support a selective role for VP OxR1 signaling in motivation for the opioid remifentanil. Our findings also highlight the utility of BE in predicting relapse propensity and efficacy of treatment with OxR1 antagonists.SIGNIFICANCE STATEMENT Abuse of opioids has risen rapidly and continues to be a major health crisis. Thus, there is an urgent need to better understand the neurobiological and behavioral mechanisms underlying opioid addiction. Here, we investigate the role of orexin-1 receptor signaling (OxR1) within ventral pallidum (VP) in remifentanil demand and cue-induced reinstatement of remifentanil seeking. Using a within-session behavioral economics procedure, we show that intra-VP microinjections of the OxR1 antagonist SB-334867 decreased motivation (increased demand elasticity) without affecting remifentanil consumption at low effort. We also found that SB microinjected intra-VP attenuated cue-induced reinstatement of remifentanil seeking. Together, our results support a role for VP OxR1 signaling in opioid reward.

Orexin A alleviates neuroinflammation via OXR2/CaMKKß/AMPK signaling pathway after ICH in mice.

BACKGROUND: Orexins are two neuropeptides (orexin A, OXA; orexin B, OXB) secreted mainly from the lateral hypothalamus, which exert a wide range of physiological effects by activating two types of receptors (orexin receptor 1, OXR1; orexin receptor 2, OXR2). OXA has equal affinity for OXR1 and OXR2, whereas OXB binds preferentially to OXR2. OXA rapidly crosses the blood-brain barrier by simple diffusion. Many studies have reported OXA's protective effect on neurological diseases via regulating inflammatory response which is also a fundamental pathological process in intracerebral hemorrhage (ICH). However, neuroprotective mechanisms of OXA have not been explored in ICH. METHODS: ICH models were established using stereotactic injection of autologous arterial blood into the right basal ganglia of male CD-1 mice. Exogenous OXA was administered intranasally; CaMKKß inhibitor (STO-609), OXR1 antagonist (SB-334867), and OXR2 antagonist (JNJ-10397049) were administered intraperitoneally. Neurobehavioral tests, hematoma volume, and brain water content were evaluated after ICH. Western blot and ELISA were utilized to evaluate downstream mechanisms. RESULTS: OXA, OXR1, and OXR2 were expressed moderately in microglia and astrocytes and abundantly in neurons. Expression of OXA decreased whereas OXR1 and OXR2 increased after ICH. OXA treatment significantly improved not only short-term but also long-term neurofunctional outcomes and reduced brain edema in ipsilateral hemisphere. OXA administration upregulated p-CaMKKß, p-AMPK, and anti-inflammatory cytokines while downregulated p-NFκB and pro-inflammatory cytokines after ICH; this effect was reversed by STO-609 or JNJ-10397049 but not SB-334867. CONCLUSIONS: OXA improved neurofunctional outcomes and mitigated brain edema after ICH, possibly through alleviating neuroinflammation via OXR2/CaMKKß/AMPK pathway.

The orexin receptors in the ventral tegmental area are involved in the development of sensitization to expression of morphine-induced preference in rats.

Recent studies have shown that orexin neurons in the lateral hypothalamus send a compelling project to the ventral tegmental area (VTA). Besides, orexin-1 (OX1) and orexin-2 (OX2) in the VTA are necessary for the development of morphine-induced place preference. Also, sensitivity to morphine can reinforce the rewarding effects of morphine. The current study aims to determine the role of VTAs orexin receptors in morphine sensitization in rats. In 84 adult male albino Wistar rats, two separate cannulae bilaterally implanted into the VTA. They received intra-VTA infusions of SB334867 (0.1, 1 and 10 nM) and TCS OX2 29 (1, 7 and 20 nM) as OX1 and OX2 receptor antagonists, respectively, 10 min before subcutaneous administration of morphine (5 mg/kg) during 3-day sensitization period. After a 5-day drug-free period, the conditioned place preference (CPP) paradigm induced by subthreshold doses of morphine (0.5 mg/kg), and CPP scores were measured by EthoVision software. The results revealed that the blockade of both OX1 and OX2 receptors within the VTA reduced the expression of morphine-induced CPP in the sensitized rats. It is plausible that VTAs orexin receptors are involved in the development/acquisition of sensitization to morphine-induced CPP in the rats.

Spinal Orexin-2 Receptors are Involved in Modulation of the Lateral Hypothalamic Stimulation-Induced Analgesia.

Role of the orexinergic system in pain modulation is well studied and involvement of the spinal orexin-1 receptors is well documented. In this study, we examined role of the spinal orexin-2 receptors in modulation of inflammatory pain in rat. Fifty-one adult male Wistar rats were implanted unilaterally with a guide cannula into the LH and intrathecal tubing in the lumbar space between L4 and L5. Chemical stimulation of LH by carbachol (250 nM/0.5 µL saline) induced remarkable analgesia during the two phases of formalin test and Intrathecal administration of different doses of TCS OX2 29 (10, 30 and 100 µM/ 0.5 µL DMSO) prior to LH stimulation, dose-dependently antagonized the antinociceptive effect of the LH-stimulation during the two phases of formalin test. The effect size of the TCS OX2 29 was η2 = 0.47 and η2 = 0. 87 for the early and late phases of the test, respectively. Also, intrathecal administration of TCS OX2 29 alone (without stimulation of the LH) had no significant effect on formalin induced pain-related behaviors. Our results showed that spinal orexin-2 receptors are involved in modulation of the LH-stimulation induced analgesia in a persistent inflammatory pain model. These findings may suggest spinal orexin-2 receptors in particular and the orexin system in general as a useful therapeutic target for treatment of chronic pains.

Synthesis and biological evaluation of F-18 labeled tetrahydroisoquinoline derivatives targeting orexin 1 receptor.

Orexin 1 receptor (OX1R) is thought to be involved in various body functions, including arousal maintenance and emotional control, but the full details of its function remain unknown. OX1R imaging with positron emission tomography (PET) would be useful in elucidating the orexin system including OX1R, but no PET probes targeting OX1R have been reported. We, therefore, designed and synthesized tetrahydroisoquinoline (THIQ) derivatives as novel PET probes targeting OX1R, and evaluated their utility. In an in vitro competitive binding assay, THIQ-1 and THIQ-2 showed significantly higher binding to OX1R (IC50 = 30 and 31 nM, respectively) than OX2R (IC50 = 160 and 332 nM, respectively). These features were also observed in a cell binding assay using [18F]THIQ-1 and [18F]THIQ-2, demonstrating their OX1R-specific binding property in vitro. In a biodistribution study using normal mice, the brain uptake of [18F]THIQ-1 was higher than that of [18F]THIQ-2, but further improvement is required for in vivo imaging with PET. Taken together, [18F]THIQ-1 and [18F]THIQ-2 have the potential to become useful imaging probes for PET targeting the OX1R, but require additional structural changes to improve their brain uptake.

Modulation by orexin A of spontaneous excitatory and inhibitory transmission in adult rat spinal substantia gelatinosa neurons.

Hypothalamic neuropeptides, orexins A and B, differently inhibit nociceptive behavior. This difference is possibly due to a distinction between orexins A and B in modulating synaptic transmission in spinal substantia gelatinosa (SG) neurons that play a pivotal role in regulating nociceptive transmission. Although we previously reported a modulatory action of orexin B on synaptic transmission in adult rat SG neurons, it has not been fully examined how the transmission is affected by orexin A. The present study examined the effects of orexin A on spontaneous excitatory and inhibitory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. Like orexin B, orexin A produced an inward current at -70 mV and/or increased the frequency of spontaneous excitatory postsynaptic current without changing its amplitude. Half-maximal effective concentration values for their effects were 0.0045 and 0.030 µM, respectively; the former value was four-fold smaller than that of orexin B while the latter value was comparable to that of orexin B. Orexin A enhanced not only glycinergic but also GABAergic transmission, although only glycinergic transmission was facilitated by orexin B in the majority of neurons tested. Orexin A activities were inhibited by an orexin-1 receptor antagonist (SB334867) but not an orexin-2 receptor antagonist (JNJ10397049), as different from orexin B whose activation was depressed by JNJ10397049 but not SB334867. These results indicate that orexin A has a different action from orexin B in SG neurons in efficacy for inward current production and in GABAergic transmission enhancement, possibly owing to orexin-1 but not orexin-2 receptor activation. This difference could contribute to at least a part of the distinction between orexins A and B in antinociceptive effects.

Orexin A Suppresses Oxidized LDL Induced Endothelial Cell Inflammation via MAPK p38 and NF-κB Signaling Pathway.

Orexin A is a multifaceted peptide produced in hypothalamus. We examined the effect of orexin A on vascular endothelial cells. Our study showed that orexin A had a profound inhibitory effect against endothelial inflammation by oxidized low-density lipoprotein (ox-LDL) in endothelial cells. Orexin A partially suppressed ox-LDL-induced monocytes THP-1 cells attachment to endothelial cells by limiting expression of vascular molecules including VCAM-1, ICAM-1, and E-selectin. Mechanistically, orexin A ameliorated endothelial dysfunction by reducing MAP kinase p38 and NF-κB activation via its receptor-OX1R. Orexin A suppressed phosphorylation of MAP kinase p38 and the NF-κB cascade kinases IKKα and IκBα, and prevented the shuttle of p65 protein into nuclear. Additionally, we reported that OX1R was expressed in HUVECs. Silence of OX1R completely abolished the inhibitory function of orexin in attachment of THP-1 cells. Collectively, our data suggest that orexin A ameliorated endothelial dysfunction under inflammatory stimuli. © 2018 IUBMB Life, 70(10):961-968, 2018.

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.

Role of orexin-2 and CB1 receptors within the periaqueductal gray matter in lateral hypothalamic-induced antinociception in rats.

Orexin plays an important role in pain modulation. Orexin-1 and orexin-2 receptors (Ox1r and Ox2r) are found at high density in the ventrolateral periaqueductal gray matter (vlPAG). Our previous study showed that chemical stimulation of the lateral hypothalamus with carbachol induces antinociception in the tail-flick test, a model of acute pain, and Ox1r-mediated antinociception in the vlPAG is modulated by the activity of vlPAG CB1 receptors. In the current study, TCS OX2 29, an Ox2r antagonist (5, 15, 50, 150, and 500 nmol/l), was microinjected into the vlPAG 5 min before the administration of carbachol (125 nmol/l). TCS OX2 29 dose dependently reduced carbachol-induced antinociception. In a second set of experiments, animals were treated with carbachol 5 min after intra-vlPAG administration of 15 nmol/l TCS OX2 29 and 1 nmol/l AM251 (a selective CB1 receptor antagonist), or 150 nmol/l TCS OX2 29 and 10 nmol/l AM251. The findings showed that the antinociceptive effect of orexin is partially mediated by activation of vlPAG Ox2 receptors. Furthermore, the administration of ineffective doses of Ox2 and CB1 receptor antagonists reduced the lateral hypothalamus-induced antinociception. It seems that Ox2 and CB1 receptors act through different pathways and Ox2r-mediated antinociception is not dependent on CB1 receptor activity.

Individual differences in orexin-I receptor modulation of motivation for the opioid remifentanil.

Orexin-1 receptors (Ox1Rs) have been implicated in the motivation for drugs of abuse. Here, we utilized a within-session behavioral-economics threshold procedure to screen for individual differences in economic demand for the ultra-short-acting opioid remifentanil and to test whether antagonism of Ox1Rs reduces remifentanil demand. The behavioral-economics procedure revealed robust individual differences in free consumption of remifentanil (Q0 parameter; hedonic set point). Rats with low baseline Q0 (low takers) displayed high demand elasticity (α parameter; reduced responding as drug price increased indicating low motivation for drug), whereas subjects with a higher Q0 (high takers) exhibit low demand elasticity (low α) by continuing to self-administer remifentanil despite increased cost (reflecting higher motivation for drug). In a punished responding paradigm utilizing footshock, subjects that were classified as high takers at baseline withstood twice as much shock as low takers to continue self-administering remifentanil. Interestingly, Ox1R antagonism with SB-334867 reduced Q0 and increased α in low takers but not in high takers. Similarly, the Ox1R antagonist attenuated cue-induced, but not drug-induced, reinstatement of remifentanil seeking in low takers but had no significant effect on reinstatement of drug seeking in high takers. Together, these data reveal a novel role of orexins in demand for remifentanil: Ox1Rs modulate demand in low takers but not in individuals that exhibit addictive-like behaviors (high takers). Finally, the behavioral assays in this study can serve as a novel laboratory model for studying individual differences in opioid use disorders.

Orexin signaling is necessary for hypoglycemia-induced prevention of conditioned place preference.

While the neural control of glucoregulatory responses to insulin-induced hypoglycemia is beginning to be elucidated, brain sites responsible for behavioral responses to hypoglycemia are relatively poorly understood. To help elucidate central control mechanisms associated with hypoglycemia unawareness, we first evaluated the effect of recurrent hypoglycemia on a simple behavioral measure, the robust feeding response to hypoglycemia, in rats. First, food intake was significantly, and similarly, increased above baseline saline-induced intake (1.1 ± 0.2 g; n = 8) in rats experiencing a first (4.4 ± 0.3; n = 8) or third daily episode of recurrent insulin-induced hypoglycemia (IIH, 3.7 ± 0.3 g; n = 9; P < 0.05). Because food intake was not impaired as a result of prior IIH, we next developed an alternative animal model of hypoglycemia-induced behavioral arousal using a conditioned place preference (CPP) model. We found that hypoglycemia severely blunted previously acquired CPP in rats and that recurrent hypoglycemia prevented this blunting. Pretreatment with a brain penetrant, selective orexin receptor-1 antagonist, SB-334867A, blocked hypoglycemia-induced blunting of CPP. Recurrently hypoglycemic rats also showed decreased preproorexin expression in the perifornical hypothalamus (50%) but not in the adjacent lateral hypothalamus. Pretreatment with sertraline, previously shown to prevent hypoglycemia-associated glucoregulatory failure, did not prevent blunting of hypoglycemia-induced CPP prevention by recurrent hypoglycemia. This work describes the first behavioral model of hypoglycemia unawareness and suggests a role for orexin neurons in mediating behavioral responses to hypoglycemia.

Role of orexin-2 receptors in the nucleus accumbens in antinociception induced by carbachol stimulation of the lateral hypothalamus in formalin test.

Orexins, which are mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), play an important role in pain modulation. Previously, it has been established that the nucleus accumbens (NAc) is involved in the modulation of formalin-induced nociceptive responses, a model of tonic pain. In this study, the role of intra-accumbal orexin-2 receptors (OX2rs) in the mediation of formalin-induced pain was investigated. A volume of 0.5 µl of 10, 20, and 40 nmol/l solutions of TCS OX2 29, an OX2r antagonist, were unilaterally microinjected into the NAc 5 min before an intra-LH carbachol microinjection (0.5 µl of 250 nmol/l solution). After 5 min, animals received a subcutaneous injection of formalin 2.5% (50 µl) into the hind paw. Pain-related behaviors were assessed at 5 min intervals during a 60-min test period. The findings showed that TCS OX2 29 administration dose dependently blocked carbachol-induced antinociception during both phases of formalin-induced pain. The antianalgesic effect of TCS OX2 29 was greater during the late phase compared with the early phase. These observations suggest that the NAc, as a part of a descending pain-modulatory circuitry, partially mediates LH-induced analgesia in the formalin test through recruitment of OX2rs. This makes the orexinergic system a good potential therapeutic target in the control of persistent inflammatory pain.

Orexin-1 receptor signalling in the prelimbic cortex and ventral tegmental area regulates cue-induced reinstatement of ethanol-seeking in iP rats.

Orexins (hypocretins) are hypothalamic neuropeptides that innervate the entire neuraxis, including the prelimbic cortex and ventral tegmental area and have been implicated in ethanol-seeking behaviour. The present study aimed to use the orexin-1 (OX1 ) receptor antagonist SB-334867 to examine the role of prelimbic cortex and ventral tegmental area OX1 receptors in cue-induced reinstatement of ethanol-seeking. Ethanol-preferring rats (iP) rats were trained to self-administer ethanol (10 percent v/v, FR3) or sucrose (0.2-1 percent w/v, FR3) in the presence of reward-associated cues before being implanted with indwelling guide cannulae. Rats then underwent extinction training for 11 days. On test days, rats were given a microinjection of vehicle or SB-334867 (3 µg/side) and presented with reward-associated cues to precipitate reinstatement. Results show SB-334867 infused into the prelimbic cortex attenuated cue-induced reinstatement of ethanol-seeking, but not sucrose-seeking. OX1 antagonism in the ventral tegmental area also attenuated cue-induced reinstatement of ethanol-seeking. These findings suggest that OX1 receptors located in the prelimbic cortex and ventral tegmental area are part of a circuit driving cue-mediated ethanol-seeking behaviour.

Beyond Membrane Protein Structure: Drug Discovery, Dynamics and Difficulties.

Most of the previous content of this book has focused on obtaining the structures of membrane proteins. In this chapter we explore how those structures can be further used in two key ways. The first is their use in structure based drug design (SBDD) and the second is how they can be used to extend our understanding of their functional activity via the use of molecular dynamics. Both aspects now heavily rely on computations. This area is vast, and alas, too large to consider in depth in a single book chapter. Thus where appropriate we have referred the reader to recent reviews for deeper assessment of the field. We discuss progress via the use of examples from two main drug target areas; G-protein coupled receptors (GPCRs) and ion channels. We end with a discussion of some of the main challenges in the area.

Orexin A regulates plasma insulin and leptin levels in a time-dependent manner following a glucose load in mice.

AIMS/HYPOTHESIS: Orexin A (OXA) is a neuropeptide implicated in the regulation of arousal status and energy metabolism. Orexin receptors are expressed not only in the central nervous system but also in the pancreas and adipose tissue. However, little is known about the physiological function of orexins. This study investigated the role of exogenous OXA in blood glucose control after glucose load in mice. In addition, the effect of OXA on insulin secretion was also identified in mouse pancreatic beta cells. METHODS: Insulin secretion and intracellular Ca(2+) levels were measured in perifused mouse islets. To investigate the effects of exogenous OXA on blood glucose levels in vivo, intraperitoneal glucose tolerance tests were performed after a subcutaneous injection of OXA in normal and high-fat diet-induced diabetic mice. RESULTS: OXA significantly potentiated glucose-stimulated insulin secretion in vitro, which increased intracellular Ca(2+) levels, mainly through adenylate cyclase and ryanodine receptor activation. This Ca(2+)-dependent insulinotropic effect of OXA was blocked in Epac2 (Rapgef4)-deficient beta cells. After a glucose load in mice, exogenous OXA decreased blood glucose levels, compared with the control, by enhancing plasma insulin and decreasing plasma glucagon levels. Additionally, OXA caused a delayed increase in plasma leptin levels, resulting in lower plasma insulin levels when blood glucose levels fell to baseline. CONCLUSIONS/INTERPRETATION: These results suggest that OXA might be a critical regulator of insulin, glucagon and leptin secretion in response to glucose. Thus, exogenous OXA might have therapeutic potential in improving blood glucose control in patients with type 2 diabetes.

Anterior thalamic paraventricular nucleus is involved in intermittent access ethanol drinking: role of orexin receptor 2.

The paraventricular nucleus of the thalamus (PVT) has been shown to participate in hedonic feeding and is thought to influence drug seeking. This understudied nucleus contains anterior (aPVT) and posterior (pPVT) subregions, which receive dense projections from hypothalamic orexin/hypocretin (OX) but exhibit anatomical and functional differences. This study sought to characterize in Long-Evans rats the involvement of these PVT subregions and their OX receptor activity in consumption of the drug, ethanol. Compared with those maintained on water and chow only (water group), rats trained to drink pharmacologically relevant levels of ethanol (ethanol group) showed increased neuronal activation in the PVT, specifically the aPVT but not pPVT, as indicated by c-Fos immunoreactivity. Similar results were obtained in rats administered ethanol via oral gavage, indicating that this site-specific effect was due to ethanol exposure. In support of the involvement of OX, the ethanol group also showed increased mRNA levels of this neuropeptide in the hypothalamus and of OX 2 receptor (OX2R) but not OX 1 receptor (OX1R), again in the aPVT but not pPVT. Similarly, ethanol gavage increased double labeling of c-Fos with OX2R but not OX1R, specifically in the aPVT. Evidence directly supporting a role for aPVT OX2R in ethanol consumption was provided by results with local injections, showing ethanol intake to be enhanced by OX-A or OX-B in the aPVT but not pPVT and reduced by a local antagonist of OX2R but not OX1R. These results focus attention on the aPVT and specifically its OX2R in mediating a positive feedback relationship with ethanol intake.

Effects of methamphetamine on actigraphy-based sleep parameters in female rhesus monkeys: Orexin receptor mechanisms.

BACKGROUND: The orexin system has been implicated as a mechanism underlying insomnia and methamphetamine-induced sleep disruptions, with a potential role for OX2 receptors in the sleep-modulating effects of orexin. The aim of the present study was to investigate the extent to which orexin receptors mediate the effects of acute methamphetamine administration on actigraphy-based sleep in female rhesus monkeys. METHODS: Actigraphy-based sleep measures were obtained in female rhesus monkeys (n=5) under baseline and acute test conditions. First, morning (10h) i.m. injections of methamphetamine (0.03 - 0.56mg/kg) were administered to determine the effects of methamphetamine alone. Then, saline or methamphetamine (0.3mg/kg) were administered at 10h, and evening (17h30) oral treatments with vehicle, the non-selective orexin receptor antagonist suvorexant (1 - 10mg/kg, p.o.), or the OX2-selective orexin receptor antagonist MK-1064 (1 - 10mg/kg, p.o.) were given. The ability of suvorexant and MK-1064 (10mg/kg, p.o.) to improve actigraphy-based sleep was also assessed in a group of female monkeys quantitatively identified with "short-duration sleep" (n=4). RESULTS: Methamphetamine dose-dependently disrupted actigraphy-based sleep parameters. Treatment with either suvorexant or MK-1064 dose-dependently improved actigraphy-based sleep in monkeys treated with methamphetamine. Additionally, both suvorexant and MK-1064 promoted actigraphy-based sleep in a group of monkeys with baseline short actigraphy-based sleep. CONCLUSIONS: These findings suggest that orexin-mediated mechanisms play a role in the effects of methamphetamine on actigraphy-based sleep in female monkeys. Targeting the orexin system, in particular OX2 receptors, could be an effective option for treating sleep disruptions observed in individuals with methamphetamine use disorder.

Pivotal role of orexin signaling in the posterior paraventricular nucleus of the thalamus during the stress-induced reinstatement of oxycodone-seeking behavior.

BACKGROUND: The orexin (OX) system has received increasing interest as a potential target for treating substance use disorder. OX transmission in the posterior paraventricular nucleus of the thalamus (pPVT), an area activated by highly salient stimuli that are both reinforcing and aversive, mediates cue- and stress-induced reinstatement of reward-seeking behavior. Oral administration of suvorexant (SUV), a dual OX receptor (OXR) antagonist (DORA), selectively reduced conditioned reinstatement of oxycodone-seeking behavior and stress-induced reinstatement of alcohol-seeking behavior in dependent rats. AIMS: This study tested whether OXR blockade in the pPVT with SUV reduces oxycodone or sweetened condensed milk (SCM) seeking elicited by conditioned cues or stress. METHODS: Male Wistar rats were trained to self-administer oxycodone (0.15 mg/kg, i.v., 8 h/day) or SCM (0.1 ml, 2:1 dilution [v/v], 30 min/day). After extinction, we tested the ability of intra-pPVT SUV (15 µg/0.5 µl) to prevent reinstatement of oxycodone or SCM seeking elicited by conditioned cues or footshock stress. RESULTS: The rats acquired oxycodone and SCM self-administration, and oxycodone intake correlated with signs of physical opioid withdrawal, confirming dependence. Following extinction, the presentation of conditioned cues or footshock elicited reinstatement of oxycodone- and SCM-seeking behavior. Intra-pPVT SUV blocked stress-induced reinstatement of oxycodone seeking but not conditioned reinstatement of oxycodone or SCM seeking or stress-induced reinstatement of SCM seeking. CONCLUSIONS: The results indicate that OXR signaling in the pPVT is critical for stress-induced reinstatement of oxycodone seeking, further corroborating OXRs as treatment targets for opioid use disorder.

Neuropeptide W increases mean arterial pressure as a result of behavioral arousal.

Neuropeptide W (NPW), an endogenous ligand for G protein-coupled receptors NPBWR1 (GPR7) and NPBWR2 (GPR8), has been detected in neurons in limbic and reticular activating system areas known to be important in arousal, as well as hypothalamic nuclei known to be important in food and water intake and the neuroendocrine response to stress. In rat, central administration of NPW increased mean arterial pressure (MAP) and behaviors associated with locomotion and grooming. We hypothesized that the NPW-induced increase in MAP was secondary to those increases in physical activity. Since peptides that stimulate arousal have been shown to increase sympathetic activity (e.g., orexin), we tested the ability of the mixed α1- and α2-adrenergic antagonist, phentolamine, to block the NPW-23-induced rise in MAP. Phentolamine pretreatment abrogated the NPW-induced MAP increase. However, we noticed the animals no longer exhibited NPW-associated behavioral arousal when pretreated with phentolamine. Anesthesia also blocked the NPW-induced increase in MAP, although the animals still were able to respond with an increase in MAP to centrally administered ANG II. Additionally, pretreatment with an orexin type 1 receptor antagonist significantly reduced the behavioral action of NPW-23 and completely blocked the peptide's action to increase MAP, suggesting that orexin neurons are downstream targets of NPW. Our results suggest that NPW increased MAP secondary to increased behavioral arousal.