Orexin antagonism and substance-P: Effects and interactions on polycystic ovary syndrome in the wistar rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder without definitive treatments. Orexin and Substance-P (SP) neuropeptides can affect the ovarian steroidogenesis. Moreover, there are limited studies about the role of these neuropeptides in PCOS. We aimed here to clarify the effects of orexins and SP in PCOS as well as any possible interactions between them. METHODS: For this purpose, the animals (n = five rats per group) received intraperitoneally a single dose of SB-334,867-A (orexin-1 receptor antagonist; OX1Ra), JNJ-10,397,049 (orexin-2 receptor antagonist; OX2Ra), and CP-96,345 (neurokinin-1 receptor antagonist; NK1Ra), alone or in combination with each other after two months of PCOS induction. The blocking of orexin and SP receptors was studied in terms of ovarian histology, hormonal changes, and gene expression of ovarian steroidogenic enzymes. RESULTS: The antagonists' treatment did not significantly affect the formation of ovarian cysts. In the PCOS groups, the co-administration of OX1Ra and OX2Ra as well as their simultaneous injections with NK1Ra significantly reversed testosterone levels and Cyp19a1 gene expression when compared to the PCOS control group. There were no significant interactions between the PCOS groups that received NK1Ra together with one or both OX1R- and OX2R-antagonists. CONCLUSION: The blocking of the orexin receptors modulates abnormal ovarian steroidogenesis in the PCOS model of rats. This suggests that the binding of orexin-A and -B to their receptors reduces Cyp19a1 gene expression while increasing testosterone levels.

Hypothalamic deep brain stimulation as a strategy to manage anxiety disorders.

Fear is essential for survival, but excessive anxiety behavior is debilitating. Anxiety disorders affecting millions of people are a global health problem, where new therapies and targets are much needed. Deep brain stimulation (DBS) is established as a therapy in several neurological disorders, but is underexplored in anxiety disorders. The lateral hypothalamus (LH) has been recently revealed as an origin of anxiogenic brain signals, suggesting a target for anxiety treatment. Here, we develop and validate a DBS strategy for modulating anxiety-like symptoms by targeting the LH. We identify a DBS waveform that rapidly inhibits anxiety-implicated LH neural activity and suppresses innate and learned anxiety behaviors in a variety of mouse models. Importantly, we show that the LH DBS displays high temporal and behavioral selectivity: Its affective impact is fast and reversible, with no evidence of side effects such as impaired movement, memory loss, or epileptic seizures. These data suggest that acute hypothalamic DBS could be a useful strategy for managing treatment-resistant anxiety disorders.

The role of orexin in Alzheimer disease: From sleep-wake disturbance to therapeutic target.

Accumulating evidence has shown that sleep disturbance is a common symptom in Alzheimer's disease (AD), which is regarded as a modifiable risk factor for AD. Orexin is a key modulator of the sleep-wake cycle and has been found to be dysregulated in AD patients. The increased orexin in cerebrospinal fluid (CSF) is associated with decreased sleep efficiency and REM sleep, as well as cognitive impairment in AD patients. The orexin system has profuse projections to brain regions that are implicated in arousal and cognition and has been found to participate in the progression of AD pathology. Conversely the orexin receptor antagonists are able to consolidate sleep and reduce AD pathology. Therefore, improved understanding of the mechanisms linking orexin system, sleep disturbance and AD could make orexin receptor antagonists a promising target for the prevention or treatment of AD.

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

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

Intermittent self-administration of fentanyl induces a multifaceted addiction state associated with persistent changes in the orexin system.

The orexin (hypocretin) system plays a critical role in motivated drug taking. Cocaine self-administration with the intermittent access (IntA) procedure produces a robust addiction-like state that is orexin-dependent. Here, we sought to determine the role of the orexin system in opioid addiction using IntA self-administration of fentanyl. Different groups of male rats were either given continuous access in 1-h period (short access [ShA]), 6-h period (long access [LgA]), or IntA (5 min of access separated by 25 min of no access for 6 h) to fentanyl for 14 days. IntA produced a greater escalation of fentanyl intake, increased motivation for fentanyl on a behavioral economics task, persistent drug seeking during abstinence, and stronger cue-induced reinstatement compared with rats given ShA or LgA. We found that addiction behaviors induced by IntA to fentanyl were reversed by the orexin-1 receptor antagonist SB-334867. IntA to fentanyl was also associated with a persistent increase in the number of orexin neurons. Together, these results indicate that the IntA model is a useful tool in the study of opioid addiction and that the orexin system is critical for the maintenance of addiction behaviors induced by IntA self-administration of fentanyl.

Nonadditivity Analysis.

We introduce the statistics behind a novel type of SAR analysis named "nonadditivity analysis". On the basis of all pairs of matched pairs within a given data set, the approach analyzes whether the same transformations between related molecules have the same effect, i.e., whether they are additive. Assuming that the experimental uncertainty is normally distributed, the additivities can be analyzed with statistical rigor and sets of compounds can be found that show significant nonadditivity. Nonadditivity analysis can not only detect nonadditivity, potential SAR outliers, and sets of key compounds but also allow estimating an upper limit of the experimental uncertainty in the data set. We demonstrate how complex SAR features that inform medicinal chemistry can be found in large SAR data sets. Finally, we show how the upper limit of experimental uncertainty for a given biochemical assay can be estimated without the need for repeated measurements of the same protein-ligand system.

Pharmacological treatment for insomnia in patients with major depressive disorder.

INTRODUCTION: Insomnia in Major Depressive Disorder (MDD) is highly prevalent and associated with increased suffering and functional impairment. Effective, evidence-based treatments for insomnia in MDD are an unmet need in clinical practice. AREAS COVERED: Herein, the authors provide a review of the clinical correlates, putative neurobiological mechanisms and treatment options for the management of insomnia in individuals with MDD. EXPERT OPINION: Sleep disturbances in MDD should be recognized as at least one of the following: (1) a domain of depressive psychopathology; (2) a consequence of rhythm disruptions; (3) a manifestation of comorbidities of sleep disturbances; (4) a manifestation of the influence of sex hormones in the brain in MDD; (5) a general medical comorbidity; and (6) a side effect of antidepressant medications. Assessment of insomnia in clinical practices is routinely performed with the use of non-structured interviews. Other methods such as standardized questionnaires and sleep diaries, along with complementary methods such as actigraphy and polysomnography are more scarcely applied. Smartphones and personal devices offer a promising strategy with the use of passive, long lasting, and ecologically valid assessments despite the lack of studies specifically targeting insomnia in individuals with MDD. New therapeutic approaches are essential, including novel targets such as orexins/hypocretins and the endocannabinoid system.

Neurophysiological and Behavioral Effects of Anti-Orexinergic Treatments in a Mouse Model of Huntington's Disease.

Huntington's disease (HD) is associated with sleep and circadian disturbances in addition to hallmark motor and cognitive impairments. Electrophysiological studies on HD mouse models have revealed an aberrant oscillatory activity at the beta frequency, during sleep, that is associated with HD pathology. Moreover, HD animal models display an abnormal sleep-wake cycle and sleep fragmentation. In this study, we investigated a potential involvement of the orexinergic system dysfunctioning in sleep-wake and circadian disturbances and abnormal network (i.e., beta) activity in the R6/1 mouse model. We found that the age at which orexin activity starts to deviate from normal activity pattern coincides with that of sleep disturbances as well as the beta activity. We also found that acute administration of Suvorexant, an orexin 1 and orexin 2 receptor antagonist, was sufficient to decrease the beta power significantly and to improve sleep in R6/1 mice. In addition, a 5-day treatment paradigm alleviated cognitive deficits and induced a gain of body weight in female HD mice. These results suggest that restoring normal activity of the orexinergic system could be an efficient therapeutic solution for sleep and behavioral disturbances in HD.

Increased Number and Activity of a Lateral Subpopulation of Hypothalamic Orexin/Hypocretin Neurons Underlies the Expression of an Addicted State in Rats.

BACKGROUND: The orexin (hypocretin) system is important for reward-driven motivation but has not been implicated in the expression of a multiphenotype addicted state. METHODS: Rats were assessed for economic demand for cocaine before and after 14 days of short access, long access, or intermittent access (IntA) to cocaine. Rats were also assessed for a number of other DSM-5-relevant addiction criteria following differential access conditions. Orexin system function was assessed by quantification of numbers and activity of orexin cells, pharmacological blockade of the orexin-1 receptor, and subregion-specific knockdown of orexin cell populations. RESULTS: IntA produced a cluster of addiction-like behaviors that closely recapitulate key diagnostic criteria for addiction to a greater extent than long access or short access. IntA was accompanied by an increase in number and activity of orexin-expressing neurons within the lateral hypothalamic subregion. This increase in orexin cell number and activity persisted during protracted withdrawal from cocaine for at least 150 days and was accompanied by enhanced incubation of craving in the same rats. Selective knockdown of lateral hypothalamic orexin neurons reduced motivation for cocaine, and orexin-1 receptor signaling played a larger role in drug seeking after IntA. CONCLUSIONS: We provide the first evidence that lateral hypothalamic orexin system function extends beyond general reward seeking to play a critical role in expression of a multiphenotype addiction-like state. Thus, the orexin system is a potential novel target for pharmacotherapies designed to treat cocaine addiction. In addition, these data point to the IntA model as a preferred approach to modeling addiction-like behavior in rats.

Central Orexin A Affects Reproductive Axis by Modulation of Hypothalamic Kisspeptin/Neurokinin B/Dynorphin Secreting Neurons in the Male Wistar Rats.

It is an established fact that orexin plays an important role in regulating the reproductive axis and the secretions of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH). However, its precise cellular and molecular mechanisms are not fully recognized. Accordingly, the aim of the present study is to find out whether the central injection of orexin A (OXA) and its antagonists, SB-334867 (as orexin receptor antagonist 1; OX1RA) and JNJ-10397049 (as orexin receptor antagonist 2; OX2RA), either alone or in combination, can leave any impact on the reproductive axis (either hormonal or behavioral) in the male Wistar rats. Furthermore, in order to see whether OXA signals can be relayed through the pathway of kisspeptin/neurokinin B/dynorphin (known as KNDy neurons, a neural network which works upstream of GnRH neurons) or not, the relative gene expression of these neuropeptides were measured. Overall, the data from radioimmunoassay revealed that OXA significantly decreases the mean serum level of LH and testosterone and, in a similar vein, its antagonists neutralize this impact. Moreover, data from real-time quantitative PCR indicated that OXA has significantly reduced the hypothalamic expression of Gnrh. In this line, the gene expressions of Kisspeptin and Neurokinin b decreased. However, OXA antagonists neutralize this impact. Also, the expression of Dynorphin gene was upregulated by the following application of the OXA. The results of this study are related to the impact of orexin on the reproductive axis. It is recommended that KNDy neurons as the interneural pathway relay the information of orexin to the GnRH neurons.

Hypocretin/orexin antagonists decrease cocaine self-administration by female rhesus monkeys.

BACKGROUND: The hypocretin/orexin system is involved in regulating arousal, and much recent work demonstrates that decreasing hypocretin receptor-1 (HCRTr1) activity using antagonists decreases appetitive behavior, including stimulant drug self-administration and reinstatement. METHODS: The present study determined the effects of hypocretin-1 and HCRTr1 antagonists on responding reinforced by intravenous (i.v.) cocaine self-administration (0.0125 - 0.05 mg/kg/infusion) in 5 female rhesus monkeys. Responding was examined using 3 schedules of reinforcement: 1) a Fixed interval 1 min, Fixed ratio 10 Chain schedule [FI 1-min (FR10:S)], 2) a Progressive Ratio (PR) schedule, and 3) a cocaine vs. candy. RESULTS: Choice schedule: the HCRTr1 antagonist SB-334867 (8-24 mg/kg, i.m.) decreased cocaine taking under the Chain schedule and PR schedule in all 5 monkeys and in 4 of the 5 monkeys under the Choice schedule. d- Amphetamine (0.06 - 0.25 mg/kg, i.m.), tested as a control manipulation, decreased cocaine taking in all 5 monkeys under the Chain schedule. The peptide hypocretin-1 (0.072 mg/kg, i.v.) increased cocaine taking in the monkeys with low rates of cocaine taking under the Chain (3/4) and Choice (4/5) schedules. Reinstatement of extinguished cocaine responding following response-independent delivery of a large dose of cocaine (0.3 mg/kg) was attenuated in 3 of the 5 monkeys by the HCRTr1 antagonist SB-334867. CONCLUSIONS: These data expand upon work accomplished in predominantly male rodents suggesting that the hypocretin system modulates the response to appetitive stimuli. A better understanding of this system offers promise as a novel approach in medication development for appetitive disorders.

Reduced Orexin System Function Contributes to Resilience to Repeated Social Stress.

Exposure to stress increases the risk of developing affective disorders such as depression and post-traumatic stress disorder (PTSD). However, these disorders occur in only a subset of individuals, those that are more vulnerable to the effects of stress, whereas others remain resilient. The coping style adopted to deal with the stressor, either passive or active coping, is related to vulnerability or resilience, respectively. Important neural substrates that mediate responses to a stressor are the orexins. These neuropeptides are altered in the cerebrospinal fluid of patients with stress-related illnesses such as depression and PTSD. The present experiments used a rodent social defeat model that generates actively coping rats and passively coping rats, which we have previously shown exhibit resilient and vulnerable profiles, respectively, to examine if orexins play a role in these stress-induced phenotypes. In situ radiolabeling and qPCR revealed that actively coping rats expressed significantly lower prepro-orexin mRNA compared with passively coping rats. This led to the hypothesis that lower levels of orexins contribute to resilience to repeated social stress. To test this hypothesis, rats first underwent 5 d of social defeat to establish active and passive coping phenotypes. Then, orexin neurons were inhibited before each social defeat for three additional days using designer receptors exclusively activated by designer drugs (DREADDs). Inhibition of orexins increased social interaction behavior and decreased depressive-like behavior in the vulnerable population of rats. Indeed, these data suggest that lowering orexins promoted resilience to social defeat and may be an important target for treatment of stress-related disorders.

The hypocretin/orexin system as a target for excessive motivation in alcohol use disorders.

The hypocretin/orexin (ORX) system has been repeatedly demonstrated to regulate motivation for drugs of abuse, including alcohol. In particular, ORX seems to be critically involved in highly motivated behaviors, as is observed in high-seeking individuals in a population, in the seeking of highly palatable substances, and in models of dependence. It seems logical that this system could be considered as a potential target for treatment for addiction, particularly alcohol addiction, as ORX pharmacological manipulations significantly reduce drinking. However, the ORX system also plays a role in a wide range of other behaviors, emotions, and physiological functions and is disrupted in a number of non-dependence-associated disorders. It is therefore important to consider how the ORX system might be optimally targeted for potential treatment for alcohol use disorders either in combination with or separate from its role in other functions or diseases. This review will focus on the role of ORX in alcohol-associated behaviors and whether and how this system could be targeted to treat alcohol use disorders while avoiding impacts on other ORX-relevant functions. A brief overview of the ORX system will be followed by a discussion of some of the factors that makes it particularly intriguing as a target for alcohol addiction treatment, a consideration of some potential challenges associated with targeting this system and, finally, some future directions to optimize new treatments.

Dynorphin Counteracts Orexin in the Paraventricular Nucleus of the Thalamus: Cellular and Behavioral Evidence.

The orexin (Orx) system plays a critical role in drug addiction and reward-related behaviors. The dynorphin (Dyn) system promotes depressive-like behavior and plays a key role in the aversive effects of stress. Orx and Dyn are co-released and have opposing functions in reward and motivation in the ventral tegmental area (VTA). Previous studies suggested that OrxA transmission in the posterior paraventricular nucleus of the thalamus (pPVT) participates in cocaine-seeking behavior. This study determined whether Orx and Dyn interact in the pPVT. Using the brain slice preparation for cellular recordings, superfusion of DynA onto pPVT neurons decreased the frequency of spontaneous and miniature excitatory postsynaptic currents (s/mEPSCs). OrxA increased the frequency of sEPSCs but had no effect on mEPSCs, suggesting a network-driven effect of OrxA. The amplitudes of s/mEPSCs were unaffected by the peptides, indicating a presynaptic action on glutamate release. Augmentation of OrxA-induced glutamate release was reversed by DynA. Utilizing a behavioral approach, separate groups of male Wistar rats were trained to self-administer cocaine or sweetened condensed milk (SCM). After extinction, rats received intra-pPVT administration of OrxA±DynA±the κ-opioid receptor antagonist nor-binaltorphimine (NorBNI) under extinction conditions. OrxA reinstated cocaine- and SCM-seeking behavior, with a greater effect in cocaine animals. DynA blocked OrxA-induced cocaine seeking but not SCM seeking. NorBNI did not induce or potentiate cocaine-seeking behavior induced by OrxA but reversed DynA effect. This indicates that the κ-opioid system in the pPVT counteracts OrxA-induced cocaine seeking, suggesting a novel therapeutic target to prevent cocaine relapse.

Role of orexin-A in the ventrolateral preoptic area on components of total energy expenditure.

BACKGROUND: Identifying whether components of total energy expenditure (EE) are affected by orexin receptor (OXR1 and OXR2) stimulation or antagonism with dual orexin receptor antagonists (DORAs) has relevance for obesity treatment. Orexin receptor stimulation reduces weight gain by increasing total EE and EE during spontaneous physical activity (SPA). OBJECTIVE: The purpose of this study was to determine if a DORA (TCS-1102) in the ventrolateral preoptic area (VLPO) reduced orexin-A-induced arousal, SPA, total EE and EE during sleep, rest, wake and SPA and whether the DORA alone reduced total EE and its components. We hypothesized that: (1) a DORA would reduce orexin-A induced increases in arousal, SPA, components of total EE, reductions in sleep and the EE during sleep and (2) the DORA alone would reduce baseline (non-stimulated) SPA and total EE. SUBJECTS/METHODS: Sleep, wakefulness, SPA and EE were determined after microinjection of the DORA (TCS-1102) and orexin-A in the VLPO of male Sprague-Dawley rats with a unilateral cannula targeted towards the VLPO. Individual components of total EE were determined based on time-stamped data. RESULTS: The DORA reduced orexin-A-induced increases in arousal, SPA, total EE and EE during SPA, wake, rest and sleep 1 h post injection (P<0.05). Orexin-A significantly reduced sleep and significantly increased EE during sleep 1 h post injection (P<0.05). Furthermore, the DORA alone significantly reduced total EE, EE during sleep (NREM and REM) and resting EE 2 h post injection (P<0.05). CONCLUSIONS: These data suggest that orexin-A reduces weight gain by stimulating total EE through increases in EE during SPA, rest and sleep. Residual effects of the DORA alone include decreases in total EE and EE during sleep and rest, which may promote weight gain.

Investigation of orexin-2 selective receptor antagonists: Structural modifications resulting in dual orexin receptor antagonists.

In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.

Timed Inhibition of Orexin System by Suvorexant Improved Sleep and Glucose Metabolism in Type 2 Diabetic db/db Mice.

Sleep disturbances are associated with type 2 diabetes; therefore, the amelioration of sleep may improve metabolic disorders. To investigate this possibility, we here examined the effects of suvorexant, an antiinsomnia drug targeting the orexin system, on sleep and glucose metabolism in type 2 diabetic mice. Diabetic db/db mice had a longer wakefulness time during the resting period, as compared with nondiabetic db/m+ control mice. The single or 7-day administration of suvorexant at lights-on (ie, the beginning of the resting phase) increased nonrapid eye movement sleep time during the resting phase and, as a consequence, reduced awake time. The daily resting-phase administration of suvorexant for 2-4 weeks improved impaired glucose tolerance in db/db mice without affecting body weight gain, food intake, systemic insulin sensitivity, or serum insulin, and glucagon levels. No changes were detected in the markers of lipid metabolism and inflammation, such as the hepatic triglyceride content and Tnf-α mRNA levels in liver and adipose tissues. The improving effect of suvorexant on glucose tolerance was associated with a reduction in the expression levels of hepatic gluconeogenic factors, including phosphoenolpyruvate carboxykinase and peroxisome proliferator-activated receptor-γ coactivator-1α in the liver in the resting phase. In contrast, the daily awake-phase administration of suvorexant had no beneficial effect on glucose metabolism. These results suggest that the suvorexant-induced increase of sleep time at the resting phase improved hepatic glucose metabolism in db/db mice. Our results provide insight into the development of novel pharmacological interventions for type 2 diabetes that target the orexin-operated sleep/wake regulatory system.

Hypoxia and hypercapnia inhibit hypothalamic orexin neurons in rats.

Evidence of impaired function of orexin neurons has been found in individuals with cardiorespiratory disorders, such as obstructive sleep apnea (OSA) and sudden infant death syndrome (SIDS), but the mechanisms responsible are unknown. Individuals with OSA and SIDS experience repetitive breathing cessations and/or rebreathing of expired air, resulting in hypoxia/hypercapnia (H/H). In this study, we examined the responses of fluorescently identified rat orexin neurons in the lateral hypothalamus to acute H/H to test if and how these neurons alter their activity and function during this challenge. Experiments were conducted in an in vitro slice preparation using voltage-clamp and current-clamp configurations. H/H (10 min) induced hyperpolarization, accompanied by rapid depression, and finally, cessation of firing activity in orexin neurons. Hypoxia alone had similar but less potent effects. H/H did not alter the frequency of inhibitory glycinergic postsynaptic currents. The frequency of GABAergic currents was diminished but only at 8-10 min of H/H. In contrast, the frequency of excitatory glutamatergic postsynaptic events was diminished as early as 2-4 min of H/H. In the presence of glutamatergic receptor blockers, the inhibitory effects of H/H on the firing activity and membrane potential of orexin neurons persisted but to a lesser extent. In conclusion, both direct alteration of postsynaptic membrane properties and diminished glutamatergic neurotransmission likely contribute to the inhibition of orexin neurons by H/H. These mechanisms could be responsible for the decreased function of orexin in individuals at risk for OSA and SIDS.

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.

BACKGROUND: 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. METHODS: One hundred thirty-two adult male albino Wistar rats weighing 180-250 g 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 50 nM), or AM251, as a selective CB1 receptor antagonist (1, 3, 10, 30 and 100 nM), was performed just 5 min before carbachol (125 nM) microinjection into the LH. RESULTS: 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. CONCLUSION: 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. SIGNIFICANCE: 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.