Myeloid cell expression of CD200R is modulated in active TB disease and regulates Mycobacterium tuberculosis infection in a biomimetic model.

A robust immune response is required for resistance to pulmonary tuberculosis (TB), the primary disease caused by Mycobacterium tuberculosis (Mtb). However, pharmaceutical inhibition of T cell immune checkpoint molecules can result in the rapid development of active disease in latently infected individuals, indicating the importance of T cell immune regulation. In this study, we investigated the potential role of CD200R during Mtb infection, a key immune checkpoint for myeloid cells. Expression of CD200R was consistently downregulated on CD14+ monocytes in the blood of subjects with active TB compared to healthy controls, suggesting potential modulation of this important anti-inflammatory pathway. In homogenized TB-diseased lung tissue, CD200R expression was highly variable on monocytes and CD11b+HLA-DR+ macrophages but tended to be lowest in the most diseased lung tissue sections. This observation was confirmed by fluorescent microscopy, which showed the expression of CD200R on CD68+ macrophages surrounding TB lung granuloma and found expression levels tended to be lower in macrophages closest to the granuloma core and inversely correlated with lesion size. Antibody blockade of CD200R in a biomimetic 3D granuloma-like tissue culture system led to significantly increased Mtb growth. In addition, Mtb infection in this system reduced gene expression of CD200R. These findings indicate that regulation of myeloid cells via CD200R is likely to play an important part in the immune response to TB and may represent a potential target for novel therapeutic intervention.

Characterization of a putative orexin receptor in Ciona intestinalis sheds light on the evolution of the orexin/hypocretin system in chordates.

Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.

The Role of the Orexin System in Craniocerebral Trauma-Induced Epilepsy in Mice.

BACKGROUND: Following traumatic brain injury (TBI), an imbalance arises in the central nervous system within the hippocampus region, resulting in the proliferation of mossy cell fibers, causing abnormal membrane discharge. Moreover, disruptions in cellular neurotransmitter secretion induce post-traumatic epilepsy. Extensive experimental and clinical data indicate that the orexin system plays a regulatory role in the hippocampal central nervous system, but the specific regulatory effects are unclear. Therefore, further experimental evaluation of its relevance is needed. OBJECTIVE: This study aims to investigate the effects of orexin receptor agonists (OXA) on the seizure threshold and intensity in controlled cortical impact (CCI) mice, and to understand the role of the orexin system in post-traumatic epilepsy (PTE). METHODS: Male C57BL/6 mice weighing 18-22 g were randomly divided into three groups: Sham, CCI, and CCI+OXA. The three groups of mice were sequentially constructed with models, implanted with electrodes, and established drug-delivery cannulas. After a 30-day recovery, the Sham and CCI groups were injected with physiological saline through the administration cannulas, while the CCI+OXA group was injected with OXA. Subsequently, all mice underwent electrical stimulation every 30 minutes for a total of 15 times. Epileptic susceptibility, duration, intensity, and cognitive changes were observed. Concurrently, the expression levels and changes of GABAergic neurons in the hippocampus of each group were examined by immunofluorescence. RESULTS: Injecting OXA into hippocampal CA1 reduces the threshold of post-traumatic seizures, prolongs the post-discharge duration, prolongs seizure duration, reduces cognitive ability, and exacerbates the loss of GABAergic neurons in the hippocampal region. CONCLUSIONS: Based on the results, we can find that injecting OXA antagonists into the CA1 region of the hippocampus can treat or prevent the occurrence and progression of post-traumatic epilepsy.

The role of orexin receptors within the CA1 area in the acquisition and expression of methamphetamine place preference.

Treatment of Methamphetamine (METH) use disorder has become a crucial public health issue. The orexin system manipulation has provided promising evidence to attenuate addictive-like behaviors. This study explored the role of the orexin 1 receptor and orexin 2 receptor (OX1R and OX2R) in the CA1 area of the hippocampal formation in the acquisition and expression of METH-induced place preference. Animals were subjected to bilateral administration of different dosages (1, 3, 10, and 30 nmol/0.5 µl DMSO per side) of a selective OX1R antagonist, SB334867, or selective OX2R antagonist, TCS OX2 29 into the CA1 area throughout the conditioning phase or once on the post-conditioning phase in separate control and experimental groups. Behavioral data revealed that both OX1R (10 nmol; P < 0.01 and 30 nmol; P < 0.001) and OX2R (10 nmol; P < 0.05 and 30 nmol; P < 0.001) antagonism during the conditioning phase could block the formation of METH place preference dose-dependently. In addition, intra-CA1 microinjection of SB334867 on the post-conditioning phase attenuated the expression of METH place preference in a dose-dependent manner (3 nmol; P < 0.05, 10 nmol; P < 0.01 and 30 nmol; P < 0.001) whereas intra-CA1 administration of TCS OX2 29 only at the highest dosage (30 nmol) declined the expression of METH place preference (P < 0.01). It was also indicated that the suppressive effects of orexin receptor blockade on the METH-seeking behavior in the CA1 area were anatomically specific to this area. These findings support the possibility of targeting the orexin system to develop novel and successful pharmacological options for the treatment of METH dependence.

Synthesis and Characterization of a New Carbon-11 Labeled Positron Emission Tomography Radiotracer for Orexin 2 Receptors Neuroimaging.

Purpose: Orexin receptors (OXRs) play a crucial role in modulating various physiological and neuropsychiatric functions within the central nervous system (CNS). Despite their significance, the precise role of OXRs in the brain remains elusive. Positron emission tomography (PET) imaging is instrumental in unraveling CNS functions, and the development of specific PET tracers for OXRs is a current research focus. Methods: The study investigated MDK-5220, an OX2R-selective agonist with promising binding properties (EC50 on OX2R: 0.023 µM, Ki on hOX2R: 0.14 µM). Synthesized and characterized as an OX2R PET probe, [11C]MDK-5220 was evaluated for its potential as a tracer. Biodistribution studies in mice were conducted to assess OX2R binding selectivity, with particular attention to its interaction with P-glycoprotein (P-gp) on the blood-brain barrier. Results: [11C]MDK-5220 exhibited promising attributes as an OX2R PET probe, demonstrating robust OX2R binding selectivity in biodistribution studies. However, an observed interaction with P-gp impacted its brain uptake. Despite this limitation, [11C]MDK-5220 presents itself as a potential candidate for further development. Discussion: The study provides insights into the functionality of the OX system and the potential of [11C]MDK-5220 as an OX2R PET probe. The observed interaction with P-gp highlights a consideration for future modifications to enhance brain uptake. The findings pave the way for innovative tracer development and propel ongoing research on OX systems, contributing to a deeper understanding of their role in the CNS. Conclusion: [11C]MDK-5220 emerges as a promising OX2R PET probe, despite challenges related to P-gp interaction. This study lays the foundation for further exploration and development of PET probes targeting OXRs, opening avenues for advancing our understanding of OX system functionality within the brain.

Whole Brain Mapping of Orexin Receptor mRNA Expression Visualized by Branched In Situ Hybridization Chain Reaction.

Orexins, which are produced within neurons of the lateral hypothalamic area, play a pivotal role in the regulation of various behaviors, including sleep/wakefulness, reward behavior, and energy metabolism, via orexin receptor type 1 (OX1R) and type 2 (OX2R). Despite the advanced understanding of orexinergic regulation of behavior at the circuit level, the precise distribution of orexin receptors in the brain remains unknown. Here, we develop a new branched in situ hybridization chain reaction (bHCR) technique to visualize multiple target mRNAs in a semiquantitative manner, combined with immunohistochemistry, which provided comprehensive distribution of orexin receptor mRNA and neuron subtypes expressing orexin receptors in mouse brains. Only a limited number of cells expressing both Ox1r and Ox2r were observed in specific brain regions, such as the dorsal raphe nucleus and ventromedial hypothalamic nucleus. In many brain regions, Ox1r-expressing cells and Ox2r-expressing cells belong to different cell types, such as glutamatergic and GABAergic neurons. Moreover, our findings demonstrated considerable heterogeneity in Ox1r- or Ox2r-expressing populations of serotonergic, dopaminergic, noradrenergic, cholinergic, and histaminergic neurons. The majority of orexin neurons did not express orexin receptors. This study provides valuable insights into the mechanism underlying the physiological and behavioral regulation mediated by the orexin system, as well as the development of therapeutic agents targeting orexin receptors.

Orexin receptors in paraventricular nucleus influence sexual behavior via regulating the sympathetic outflow in males.

BACKGROUND: Orexins are hypothalamic neuropeptides associated with various neurophysiological activities such as sleep, arousal, and reward. However, there are few studies investigating the relationships between orexin receptors in the paraventricular nucleus and sexual behaviors. OBJECTIVES: To explore the roles of orexin receptors in the paraventricular nucleus on sexual behaviors and uncover its potential mechanisms in males. MATERIALS AND METHODS: Orexin A, orexin 1 receptor antagonist SB334867, and orexin 2 receptor antagonist TCS-OX2-29 were microinjected into the paraventricular nucleus to investigate the effects of orexin receptors on copulatory behavior testing of C57BL/6 mice. To explore if ejaculation could activate orexin 1 receptor-expressing neurons in the paraventricular nucleus, fluorescence immunohistochemical double staining was utilized. The levels of serum norepinephrine were measured and the lumbar sympathetic nerve activity was recorded to reflect the sympathetic nervous system activity. Moreover, the bulbospongiosus muscle-electromyogram was recorded and analyzed. To test whether perifornical/lateral hypothalamic area orexinergic neurons directly projected to the paraventricular nucleus, virus retrograde tracing technology was utilized. RESULTS: Orexin A significantly enhanced sexual performance by shortening the intromission and ejaculation latencies, and increasing the mount and intromission frequencies, while the opposite outcomes appeared with SB334867. However, TCS-OX2-29 had no significant effects on sexual behaviors. Moreover, orexin A increased lumbar sympathetic nerve activity and the levels of serum norepinephrine, while SB334867 decreased lumbar sympathetic nerve activity and norepinephrine, which caused a significant decrease in sympathetic nervous system outflow. Meanwhile, a robust increase in the bulbospongiosus muscle-electromyogram activity was identified after microinjecting orexin A. Furthermore, cFos immunopositive cells were increased and double stained with orexin 1 receptor-expressing neurons in the mating group. Additionally, the retrograde tracing results demonstrated that orexinergic neurons in the perifornical/lateral hypothalamic area directly projected to the paraventricular nucleus. CONCLUSIONS: Orexin 1 receptor in the paraventricular nucleus could influence the ejaculatory reflex via mediating the sympathetic nervous system activity, which might be of great importance in the treatment of premature ejaculation in the future.

Imaging flow cytometry of tumoroids: A new method for studying GPCR expression.

Fluorescence confocal microscopy is commonly used to analyze the regulation membrane proteins expression such as G protein-coupled receptors (GPCRs). With this approach, the internal movement of GPCRs within the cell can be observed with a high degree of resolution. However, these microscopy techniques led to complex and time-consuming analysis and did not allow a large population of events to be sampled. A recent approach termed imaging flow cytometry (IFC), which combines flow cytometry and fluorescence microscopy, had two main advantages to study the regulation of GPCRs expression such as orexins receptors (OXRs): the ability (1) to analyze large numbers of cells and; (2) to visualize cell integrity and fluorescent markers localization. Here, we compare these two technologies using the orexin A (OxA) ligand coupled to rhodamine (OxA-rho) to investigate anti-tumoral OX1R expression in human digestive cancers. IFC has been adapted for cancer epithelial adherent cells and also to 3D cell culture tumoroids which partially mimic tumoral structures. In the absence of specific antibody, expression of OX1R is examined in the presence of OxA-rho. 2D-culture of colon cancer cells HT-29 exhibits a maximum level of OX1R internalization induced by OxA with 19% ± 3% colocalizing to early endosomes. In 3D-culture of HT-29 cells, internalization of OX1R/OxA-rho reached its maximum at 60 min, with 30.7% ± 6.4% of OX1R colocalizing with early endosomes. This is the first application of IFC to the analysis of the expression of a native GPCR, OX1R, in both 2D and 3D cultures of adherent cancer cells.

The integrative role of orexin-1 and orexin-2 receptors within the hippocampal dentate gyrus in the modulation of the stress-induced antinociception in the formalin pain test in the rat.

The stressful experiences, by triggering a cascade of hormonal and neural changes, can produce antinociception commonly referred to as stress-induced antinociception (SIA). Orexin neuropeptides have an essential role in stress responses and pain modulation. The dentate gyrus receives orexinergic projections and has been shown to be involved in pain processing. The current study investigated the possible role of orexin-1 and orexin-2 receptors (OX1r and OX2r, respectively) within the dentate gyrus in SIA in a rat model of formalin-induced pain behavior in one hind paw. Male Wistar rats weighing 230-250 g underwent stereotaxic surgery and a cannula was implanted in their brains, above the dentate gyrus region. Either SB334867 or TCS OX2 29 (OX1r and OX2r antagonists, respectively) was microinjected into the dentate gyrus region at a range of doses at 1, 3, 10, and 30 nmol (control group received DMSO 12% as vehicle), 5 min before the forced swim stress (FSS) exposure. The formalin test was performed to assess pain-related behaviors. The results indicated that FSS exposure relieves pain-related behavior in the early and late phases of the formalin test. Blockade of intra-dentate gyrus OX1 or OX2 receptors reduced the antinociceptive responses induced by FSS in the formalin test, with more impact during the late phase. Our findings support the potential role of intra-dentate gyrus orexin receptors as target sites of orexin neurons in painful and stressful situations. Therefore, understanding the exact mechanisms of SIA and the role of the orexinergic system in this phenomenon can lead to identifying the strategies to guide future research and offer a new approach to discovering new pain therapeutic agents.

Intra-accumbal orexinergic system contributes to the stress-induced antinociceptive behaviors in the animal model of acute pain in rats.

Stress and pain are interleaved at numerous levels - influencing each other. Stress can increase the nociception threshold in animals, long-known as stress-induced analgesia (SIA). Orexin is known as a neuropeptide that modulates pain. The effect of stress on the mesolimbic system in the modulation of pain is known. The role of the intra-accumbal orexin receptors in the modulation of acute pain by forced swim stress (FSS) is unclear. In this study, 117 adult male albino Wistar rats (270-300 g) were used. The animals were unilaterally implanted with cannulae above the NAc. The antagonist of the orexin-1 receptor (OX1r), SB334867, and antagonist of the orexin-2 receptor (OX2r), TCS OX2 29, were microinjected into the NAc in different doses (1, 3, 10, and 30 nmol/0.5 µl DMSO) before exposure to FSS for a 6-min period. The tail-flick test was carried out as an assay nociception of acute pain, and the nociceptive threshold [tail-flick latency (TFL)] was measured for 60-minute. The findings demonstrated that exposure to acute stress could remarkably increase the TFLs and antinociceptive responses. Moreover, intra-accumbal microinjection of SB334867 or TCS OX2 29 blocked the antinociceptive effect of stress in the tail-flick test. The contribution of orexin receptors was almost equally modulating SIA. The present study's findings suggest that OX1r and OX2r within the NAc modulate stress-induced antinociceptive responses. The intra-accumbal microinjection of orexin receptors antagonists declares inducing antinociceptive responses by FSS in acute pain. Proposedly, intra-accumbla orexinergic receptors have a role in the development of SIA.

Orexin receptors in the hippocampal dentate gyrus modulated the restraint stress-induced analgesia in the animal model of chronic pain.

Previous studies have shown that stressful stimuli induced an adaptive response of reduced nociception, known as stress-induced analgesia (SIA). Since orexin neuropeptides are involved in pain modulation, and orexin neurons, primarily located in the lateral hypothalamus (LH), project to various hippocampal regions, such as the dentate gyrus (DG), the current study aimed to examine the role of orexin receptors within the DG region in the restraint SIA in the animal model of chronic pain. One hundred-thirty adult male Wistar rats (230-250 g) were unilaterally implanted with a cannula above the DG region. Animals were given SB334867 or TCS OX2 29 (1, 3, 10, and 30 nmol, 0.5 µl/rat) into the DG region as orexin-1 receptor (OX1r) and orexin-2 receptor (OX2r) antagonists, respectively, five min before exposure to a 3-hour restraint stress (RS) period. Animals were then undergone the formalin test to assess pain-related behaviors as the animal model of chronic pain. The results showed that RS produces an analgesic response during the early and late phases of the formalin test. However, intra-DG microinjection of OX1r and OX2r antagonists attenuated the restraint SIA. OX2r antagonist was more potent than OX1r antagonist in the early phase of the formalin test, while OX1r antagonist was little more effective in the late phase. Predominantly, it could be concluded that the orexinergic system in the DG region might act as a potential endogenous pain control system and a novel target for treating stress-related disorders.

Contribution of the intra-hippocampal orexin system in the regulation of restraint stress response to pain-related behaviors in the formalin test.

Stress-induced antinociception (SIA) is due to the activation of several neural pathways and neurotransmitters that often suppress pain perception. Studies have shown that the orexin neuropeptide system is essential in pain modulation. Therefore, this study aimed to investigate the role of orexinergic receptors in the hippocampal CA1 region in modulating SIA response during the formalin test as an animal model of inflammatory pain. The orexin-1 receptor (OX1r) antagonist, SB334867, at 1, 3, 10, and 30 nmol or TCS OX2 29 as an orexin-2 receptor (OX2r) antagonist at the same doses were microinjected into the CA1 region in rats. Five minutes later, rats were exposed to restraint stress (RS) for 3 h, and pain-related behaviors were monitored in 5-min blocks for the 60-min test period in the formalin test. Results showed that applying RS for 3 h reduced pain responses in the early and late phases of the formalin test. The main findings showed that intra-CA1 injection of orexin receptor antagonists reduced the antinociception caused by stress in both phases of the formalin test. In addition, the contribution of OX2r in mediating the antinociceptive effect of stress was more prominent than that of OX1r in the early phase of the formalin test. However, in the late phase, both receptors worked similarly. Accordingly, the orexin system and its two receptors in the CA1 region of the hippocampus regulate SIA response to this animal model of pain in formalin test.

Orexin 2 receptor antagonism sex-dependently improves sleep/wakefulness and cognitive performance in tau transgenic mice.

BACKGROUND AND PURPOSE: Tau pathology contributes to a bidirectional relationship between sleep disruption and neurodegenerative disease. Tau transgenic rTg4510 mice model tauopathy symptoms, including sleep/wake disturbances, which manifest as marked hyperarousal. This phenotype can be prevented by early transgene suppression; however, whether hyperarousal can be rescued after onset is unknown. EXPERIMENTAL APPROACH: Three 8-week experiments were conducted with wild-type and rTg4510 mice after age of onset of hyperarousal (4.5 months): (1) Tau transgene suppression with doxycycline (200 ppm); (2) inactive phase rapid eye movement (REM) sleep enhancement with the dual orexin receptor antagonist suvorexant (50 mg·kg-1 ·day-1 ); or (3) Active phase non-NREM (NREM) and REM sleep enhancement using the selective orexin 2 (OX2 ) receptor antagonist MK-1064 (40 mg·kg-1 ·day-1 ). Sleep was assessed using polysomnography, cognition using the Barnes maze, and tau pathology using immunoblotting and/or immunohistochemistry. KEY RESULTS: Tau transgene suppression improved tauopathy and hippocampal-dependent spatial memory, but did not modify hyperarousal. Pharmacological rescue of REM sleep deficits did not improve spatial memory or tau pathology. In contrast, normalising hyperarousal by increasing both NREM and REM sleep via OX2 receptor antagonism restored spatial memory, independently of tauopathy, but only in male rTg4510 mice. OX2 receptor antagonism induced only short-lived hypnotic responses in female rTg4510 mice and did not improve spatial memory, indicating a tau- and sex-dependent disruption of OX2 receptor signalling. CONCLUSIONS AND IMPLICATIONS: Pharmacologically reducing hyperarousal corrects tau-induced sleep/wake and cognitive deficits. Tauopathy causes sex-dependent disruptions of OX2 receptor signalling/function, which may have implications for choice of hypnotic therapeutics in tauopathies.

Orexin-A attenuated motion sickness through modulating neural activity in hypothalamus nuclei.

BACKGROUND AND PURPOSE: We evaluated the hypothesis that central orexin application could counteract motion sickness responses through regulating neural activity in target brain areas. EXPERIMENTAL APPROACH: Thec effects of intracerebroventricular (i.c.v.) injection of orexin-A and SB-334867 (OX1 antagonist) on motion sickness-induced anorexia, nausea-like behaviour (conditioned gaping), hypoactivity and hypothermia were investigated in rats subjected to Ferris wheel-like rotation. Orexin-A responsive brain areas were identified using Fos immunolabelling and were verified via motion sickness responses after intranucleus injection of orexin-A, SB-334867 and TCS-OX2-29 (OX2 antagonist). The efficacy of intranasal application of orexin-A versus scopolamine on motion sickness symptoms in cats was also investigated. KEY RESULTS: Orexin-A (i.c.v.) dose-dependently attenuated motion sickness-related behavioural responses and hypothermia. Fos expression was inhibited in the ventral part of the dorsomedial hypothalamus (DMV) and the paraventricular nucleus (PVN), but was enhanced in the ventral part of the premammillary nucleus ventral part (PMV) by orexin-A (20 µg) in rotated animals. Motion sickness responses were differentially inhibited by orexin-A injection into the DMV (anorexia and hypoactivity), the PVN (conditioned gaping) and the PMV (hypothermia). SB-334867 and TCS-OX2-29 (i.c.v. and intranucleus injection) inhibited behavioural and thermal effects of orexin-A. Orexin-A (60 µg·kg-1) and scopolamine inhibited rotation-induced emesis and non-retching/vomiting symptoms, while orexin-A also attenuated anorexia with mild salivation in motion sickness cats. CONCLUSION AND IMPLICATIONS: Orexin-A might relieve motion sickness through acting on OX1 and OX2 receptors in various hypothalamus nuclei. Intranasal orexin-A could be a potential strategy against motion sickness.

Orexins and Prostate Cancer: State of the Art and Potential Experimental and Therapeutic Perspectives.

Prostate cancer (PCa) is the second most common cancer in humans. Peptides have recently been used as targeted therapeutics in cancers, due to their extensive multi-functional applications. Two hypothalamic peptides, orexins A (OXA) and B (OXB) and their specific receptors, orexin receptor 1 (OX1R) and 2 (OX2R), orchestrate several biological processes in the central nervous system and peripheral organs. However, in addition to their role in physiological responses, orexins are involved in numerous inflammatory and/or neoplastic pathologies. The presence and expression of orexins in different cancer models, including prostate cancer, and their role in inducing pro- or anti-apoptotic responses in tumor cell lines, suggest that the orexinergic system might have potential therapeutic action or function as a diagnostic marker in PCa. In addition to the traditional animal models for studying human PCa, the canine model might also serve as an additional tool, due to its clinical similarities with human prostate cancer.

Antinociceptive effect of intermittent fasting via the orexin pathway on formalin-induced acute pain in mice.

It has been suggested that stress responses induced by fasting have analgesic effects on nociception by elevating the levels of stress-related hormones, while there is limited understanding of pain control mechanisms. Here, we investigated whether acute or intermittent fasting alleviates formalin-induced pain in mice and whether spinal orexin A (OXA) plays a role in this process. 6, 12, or 24 h acute fasting (AF) and 12 or 24 h intermittent fasting (IF) decreased the second phase of pain after intraplantar formalin administration. There was no difference in walking time in the rota-rod test and distance traveld in the open field test in all groups. Plasma corticosterone level and immobility time in the forced swim test were increased after 12 h AF, but not after 12 h IF. 12 h AF and IF increased not only the activation of OXA neurons in the lateral hypothalamus but also the expression of OXA in the lateral hypothalamus and spinal cord. Blockade of spinal orexin 1 receptor with SB334867 restored formalin-induced pain and spinal c-Fos immunoreactivity that were decreased after 12 h IF. These results suggest that 12 h IF produces antinociceptive effects on formalin-induced pain not by corticosterone elevation but by OXA-mediated pathway.

Experimental sickness reduces hypocretin receptor 1 expression in the lateral hypothalamus and ventral tegmental area of female mice.

Recent studies have focused on how sickness behaviours, including lethargy, are coordinated in the brain in response to peripheral infections. Decreased hypocretin (orexin) signalling is associated with lethargy and previous research suggests that hypocretin signalling is downregulated during sickness. However, there are studies that find increases or no change in hypocretin signalling during sickness. It is further unknown whether hypocretin receptor expression changes during sickness. Using lipopolysaccharide (LPS) to induce sickness in female mice, we investigated how LPS-injection affects gene expression of hypocretin receptors and prepro-hypocretin as well as hypocretin-1 peptide concentrations in brain tissue. We found that hypocretin receptor 1 gene expression was downregulated during sickness in the lateral hypothalamus and ventral tegmental area, but not in the dorsal raphe nucleus or locus coeruleus. We found no changes in hypocretin receptor 2 expression. Using a gene expression calculation that accounts for primer efficiencies and multiple endogenous controls, we were unable to detect changes in prepro-hypocretin expression. Using radioimmunoassay, we found no change in hypocretin-1 peptide in rostral brain tissue. Our results indicate that hypocretin receptor expression can fluctuate during sickness, adding an additional level of complexity to understanding hypocretin signalling during sickness.

Age-dependent down-regulation of orexin receptors in trigeminal nucleus caudalis correlated with attenuation of orexinergic analgesia in rats.

Aging is related to a variety of physiological organ changes, including central and peripheral nervous systems. It has been reported that the orexin signaling has a potential analgesic effect in different models of pain, especially inflammatory pulpal pain. However, the age-induced alteration in dental pain perception and orexin analgesia has not yet been fully elucidated. Here, we tested that how aging may change the effect of orexin-A on nociceptive behaviors in a rat dental pulp pain model. The expression levels of orexin receptors and the nociceptive neuropeptides substance P (SP) and calcitonin-related gene peptide (CGRP) were also assessed in the trigeminal nucleus caudalis (TNC) of young and aged rats. Dental pulp pain was induced by intradental application of capsaicin (100 µg). The immunofluorescence technique was used to evaluate the expression levels. The results show less efficiency of orexin-A to ameliorate pain perception in aged rats as compared to young rats. In addition, a significant decrease in the number of orexin 1 and 2 receptors was observed in the TNC of aged as compared to young rats. Dental pain-induced SP and CGRP overexpression was also significantly inhibited by orexin-A injection into the TNC of young animals. In contrast, orexin-A could not produce such effects in the aged animals. In conclusion, the older age-related reduction of the antinociceptive effect of orexin may be due to the downregulation of its receptors and inability of orexin signaling to inhibit the expression of nociceptive neuropeptides such as SP and CGRP in aged rats.

The role of orexin-1 receptors within the ventral tegmental area in the extinction and reinstatement of methamphetamine place preference.

Targeting the orexin system has recently been identified as one of the promising options for treating drug addiction. It may be more feasible and achievable if we investigate the accurate function of the orexin system in brain areas implicated in reward and addiction, such as the ventral tegmental area (VTA) by animal reward models. This study investigated the contribution of the orexin system, mainly the orexin-1 receptors (OX1R) in the VTA, in the extinction and reinstatement of methamphetamine (METH) related memories in the conditioned place preference (CPP) model. Animals after the acquisition of METH place preference were subjected to two separate sets of extinction and reinstatement experiments to receive various concentrations of selective OX1R antagonist, SB334867 into the bilateral VTA before extinction sessions (1, 3, and 10 nmol/0.3 µl DMSO per side) or only on the reinstatement phase (3, 10, and 30 nmol/0.3 µl DMSO per side), respectively. Intra-VTA infusion of SB334867 throughout the extinction phase could remarkably facilitate the extinction process and decrease the maintenance of reinforcing effects of METH at the highest dosage (10 nmol; p < 0.0001). Data also indicated a single microinfusion of SB334867 into the VTA before reinstatement of the METH-seeking behavior could considerably prevent the relapse of previously formed reward-context memories (10 nmol; p < 0.01 and 30 nmol; p < 0.001). The present study provided evidence supporting the potential therapeutic effects of the orexin system modulation, specifically in the VTA, on different stages of METH-induced place preference.

Anandamide improves food intake and orexinergic neuronal activity in the chronic sleep deprivation induction model in rats by modulating the expression of the CB1 receptor in the lateral hypothalamus.

Sleep deprivation alters orexinergic neuronal activity in the lateral hypothalamus (LH), which is the main regulator of sleep-wake, arousal, appetite, and energy regulation processes. Cannabinoid receptor (CBR) expression in this area is involved in modulating the function of orexin neurons. In this study, we investigated the effects of endocannabinoid anandamide (AEA) administration on improving food intake and appetite by modulating the activity of orexin neurons and CB1R expression after chronic sleep deprivation. Adult male Wistar rats (200-250 g) were randomly divided into three groups: control + vehicle (Control), chronic sleep deprivation + vehicle (SD), and chronic sleep deprivation +20 mg/kg AEA (SD + A). For SD induction, the rats were kept in a sleep deprivation device for 18 h (7 a.m. to 1 a.m.) daily for 21 days. Weight gain, food intake, the electrical power of orexin neurons, CB1R mRNA expression in hypothalamus, CB1R protein expression in the LH, TNF-α, IL-6, IL-4 levels and antioxidant activity in hypothalamus were measured after SD induction. Our results showed that AEA administration significantly improved food intake (p < 0.01), Electrical activity of orexin neurons (p < 0.05), CB1R expression in the hypothalamus (p < 0.05), and IL-4 levels (p < 0.05). AEA also reduced mRNA expression of OX1R and OX2R (p < 0.01 and p < 0.05 respectively), also IL-6 and TNF-α (p < 0.01) and MDA level (p < 0.05) in hypothalamic tissue. As a consequence, AEA modulates orexinergic system function and improves food intake by regulating the expression of the CB1 receptor in the LH in sleep deprived rats.