sb-334867-a and Pain

Stress activates multiple neural pathways and neurotransmitters that often suppress pain perception, the phenomenon called stress-induced analgesia (SIA). Orexin neurons from the lateral hypothalamus project to entire brain structures such as the hippocampus. The present study examined this hypothesis that orexinergic receptors in the CA1 region of the hippocampus may play a modulatory role in the development of SIA in formalin test as an animal model of persistent inflammatory pain. One hundred-two adult male Wistar rats were administered with intra-CA1 orexin-1 receptor (OX1r) antagonist, SB334867, at the doses of 3, 10, 30, and 100 nmol or TCS OX2 29 as orexin-2 receptor (OX2r) antagonist at the doses of 1, 3, 10, and 30 nmol. Five min later, rats were exposed to forced swim stress (FSS) for a 6-min period. Then, pain-related behaviors induced by formalin injection were measured at the 5-min blocks during a 60-min period of formalin test. The current study indicated that solely stress exposure elicits antinociception in the early and late phases of the formalin test. The FSS-induced analgesia was prevented by intra-CA1 administration of SB334867 or TCS OX2 29 during either phase of the formalin test. Moreover, the contribution of the OX2r in the mediation of analgesic effect of stress was more prominent than that of the OX1r during both phases of the formalin test. It is suggested that OX1r and OX2r in the CA1 region of the hippocampus are involved in stress-induced analgesia in the animal model of persistent inflammatory pain.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzoxazoles; CA1 Region, Hippocampal; Cyclophosphamide; Disease Models, Animal; Doxorubicin; Etoposide; Inflammation; Isoquinolines; Male; Microinjections; Naphthyridines; Orexin Receptor Antagonists; Orexin Receptors; Pain; Pain Measurement; Prednisone; Pyridines; Rats, Wistar; Stress, Psychological; Urea; Vincristine

Orexins are produced in the restricted regions of the lateral hypothalamus (LH). However, orexinergic receptors and projections are localized in wide regions like nucleus accumbens, ventral tegmental area, periaqueductal gray area and spinal cord which are involved in the pain modulation. This study was carried out to investigate the effects of intrathecal administration of orexin-1 receptor antagonist (SB-334867) in the spinal antinociception induced by intra-LH administration of carbachol (cholinergic receptor agonist) in both early and late phases of pain related behaviors in formalin test. In this study, pain-related behaviors (pain scores) were evaluated using the formalin test during 5-min block intervals for a 60-min period in seventy male Wistar rats were given SB-334867 (3, 10, 30 and 100 μM/10 μl) or vehicle (DMSO 12%; 10 μl) intrathecally following intra-LH administration of carbachol (250 nM/rat). Our data showed that intra-LH injection of carbachol attenuated the formalin-induced biphasic pain responses, and intrathecal administration of SB-334867 dose-dependently decreased LH stimulation-induced antinociceptive responses during both phases. Moreover, administration of different doses of SB-334867 during the early phase were more effective than those during the late phase. The antinociceptive role of orexinergic system in the formalin test through a neural pathway from the LH to the spinal cord provides evidence that orexins can be useful in therapeutic targets for pain relief.

Topics: Analgesics; Animals; Benzoxazoles; Hypothalamic Area, Lateral; Injections, Spinal; Male; Naphthyridines; Nociception; Orexin Receptor Antagonists; Orexin Receptors; Pain; Pain Measurement; Rats, Wistar; Spinal Cord; Urea

Orexinergic neurons in the lateral hypothalamus (LH) play an important role in pain modulation. In addition, ventral tegmental area (VTA) is known as a part of descending pain modulatory circuitry. Little is known about the interaction between the LH and neural substrates involving in modulation of formalin-induced nociception. Accordingly, we aimed to examine the pain modulatory role of VTA orexin receptors in the formalin test.. Seventy-eight male Wistar rats were unilaterally implanted with two cannulae above the LH and VTA. Intra-VTA administration of SB-334867 (orexin-1 receptor antagonist) or TCS OX2 29 (orexin-2 receptor antagonist) was performed 5 min before intra-LH microinjection of carbachol (a cholinergic receptor agonist). The procedure was followed by subcutaneous injection of formalin after 5-min interval time.. Carbachol attenuated formalin-induced biphasic pain responses and SB-334867 or TCS OX2 29 administration dose-dependently antagonized the LH-induced analgesia during both phases. Blockade of orexin-1 and -2 receptors had more profound effects on the reduction of antinociception during the late phase compared to the early phase. Also, contribution of orexin-1 receptors in mediation of LH-induced analgesia was greater than orexin-2 receptors during the late phase.. Formalin test, a model of persistent inflammatory pain, mimics the conditions encountered in clinical situations. Pain modulatory role of orexinergic system in the formalin test through a neural pathway from the LH to the VTA provides the evidence that orexins can be useful therapeutic targets for chronic pain treatment. WHAT DOES THIS STUDY ADD?: There is a pathway from the lateral hypothalamus (LH) to the ventral tegmental area (VTA) which modulates biphasic formalin-induced pain. Blockade of VTA orexin receptors dose-dependently reduces LH-induced analgesia during both phases. Anti-analgesic effect of orexin receptor antagonists is more considerable during the late phase. Contribution of orexin-1 receptors to mediation of LH-induced analgesia is more than orexin-2 receptors during the late phase.

Topics: Animals; Benzoxazoles; Carbachol; Hypothalamic Area, Lateral; Isoquinolines; Male; Naphthyridines; Nociception; Orexin Receptor Antagonists; Orexin Receptors; Pain; Pain Measurement; Pyridines; Rats; Rats, Wistar; Urea; Ventral Tegmental Area

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

Topics: Animals; Benzoxazoles; Carbachol; Disease Models, Animal; Hypothalamic Area, Lateral; Male; Microinjections; Naphthyridines; Orexin Receptors; Orexins; Pain; Pain Measurement; Periaqueductal Gray; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Stimulation, Chemical; Urea

Locus coeruleus (LC) nucleus is involved in noradrenergic descending pain modulation. LC receives dense orexinergic projections from the lateral hypothalamus. Orexin-A and -B are hypothalamic peptides which modulate a variety of brain functions via orexin type-1 (OX1) and orexin type-2 (OX2) receptors. Previous studies have shown that activation of OX1 receptors induces endocannabinoid synthesis and alters synaptic neurotransmission by retrograde signaling via affecting cannabinoid type-1 (CB1) receptors. In the present study the interaction of orexin-A and endocannabinoids was examined at the LC level in a rat model of inflammatory pain. Pain was induced by formalin (2%) injection into the hind paw. Intra-LC microinjection of orexin-A decreased the nociception score during both phases of formalin test. Furthermore, intra-LC microinjection of either SB-334867 (OX1 receptor antagonist) or AM251 (CB1 receptor antagonist) increased flinches and also the nociception score during phase 1, 2 and the inter-phase of formalin test. The analgesic effect of orexin-A was diminished by prior intra-LC microinjection of either SB-334867 or AM251. This data show that, activation of OX1 receptors in the LC can induce analgesia and also the blockade of OX1 or CB1 receptors is associated with hyperalgesia during formalin test. Our findings also suggest that CB1 receptors may modulate the analgesic effect of orexin-A. These results outline a new mechanism by which orexin-A modulates the nociceptive processing in the LC nucleus.

Topics: Analgesics; Analysis of Variance; Animals; Benzoxazoles; Carrier Proteins; Dose-Response Relationship, Drug; Formaldehyde; Locus Coeruleus; Male; Microinjections; Naphthyridines; Orexins; Pain; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Wistar; Urea

Orexin A and B are hypothalamic peptides known to modulate arousal, feeding, and reward via OX1 and OX2 receptors. Orexins are also antinociceptive in the brain, but their mechanism(s) of action remain unclear. Here, we investigated the antinociceptive mechanism of orexin A in the rat ventrolateral periaqueductal gray (vlPAG), a midbrain region crucial for initiating descending pain inhibition. In vlPAG slices, orexin A (30-300 nm) depressed GABAergic evoked IPSCs. This effect was blocked by an OX1 [1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea (SB 334867)], but not OX2 [N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (compound 29)], antagonist. Orexin A increased the paired-pulse ratio of paired IPSCs and decreased the frequency, but not amplitude, of miniature IPSCs. Orexin A-induced IPSC depression was mimicked by (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a cannabinoid 1 (CB1) receptor agonist. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM 251), a CB1 antagonist, reversed depressant effects by both agonists. Orexin A-induced IPSC depression was prevented by 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and tetrahydrolipstatin, inhibitors of phospholipase C (PLC) and diacylglycerol lipase (DAGL), respectively, and enhanced by cyclohexyl[1,1'-biphenyl]-3-ylcarbamate (URB602), which inhibits enzymatic degradation of 2-arachidonoylglycerol (2-AG). Moderate DAGLα, but not DAGLβ, immunoreactivity was observed in the vlPAG. Orexin A produced an overall excitatory effect on evoked postsynaptic potentials and hence increased vlPAG neuronal activity. Intra-vlPAG microinjection of orexin A reduced hot-plate nociceptive responses in rats in a manner blocked by SB 334867 and AM 251. Therefore, orexin A may produce antinociception by activating postsynaptic OX1 receptors, stimulating synthesis of 2-AG, an endocannabinoid, through a Gq-protein-mediated PLC-DAGLα enzymatic cascade culminating in retrograde inhibition of GABA release (disinhibition) in the vlPAG.

Topics: Analysis of Variance; Animals; Animals, Newborn; Arachidonic Acids; Benzoxazines; Benzoxazoles; Biphenyl Compounds; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Disease Models, Animal; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Estrenes; gamma-Aminobutyric Acid; Glycerides; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Intracellular Signaling Peptides and Proteins; Lactones; Male; Morpholines; Naphthalenes; Naphthyridines; Neural Inhibition; Neural Pathways; Neuropeptides; Orexin Receptors; Orexins; Orlistat; Pain; Pain Measurement; Patch-Clamp Techniques; Periaqueductal Gray; Piperidines; Pyrazoles; Pyrrolidinones; Rats; Rats, Wistar; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Urea

Previous results have suggested that orexins causes a rise of intracellular free calcium ([Ca(2+)](i)) in cultured rat dorsal root ganglion (DRG) neurons, implicating a role in nociception, but the underlying mechanism is unknown. Hence, the aim of the present study was to investigate whether the orexins-mediated signaling involves the PKC pathways in these sensory neurons. Cultured DRG neurons were loaded with 1 micromol Fura-2 AM and [Ca(2+)](i) responses were quantified by the changes in 340/380 ratio using fluorescence imaging system. The orexin-1 receptor antagonist SB-334867-A (1 microM) inhibited the calcium responses to orexin-A and orexin-B (59.1+/-5.1 % vs. 200 nM orexin-A, n=8, and 67+/-3.8 % vs. 200 nM orexin-B, n=12, respectively). The PKC inhibitor chelerythrine (10 and 100 microM) significantly decreased the orexin-A (200 nM)-induced [Ca(2+)](i) increase (59.4+/-4.8 % P<0.01, n=10 and 4.9+/-1.6 %, P<0.01, n=9) versus response to orexin-A). It was also found that chelerythrine dose-dependently inhibited the [Ca(2+)](i) response to 200 nM orexin-B. In conclusion, our results suggest that orexins activate intracellular calcium signaling in cultured rat sensory neurons through PKC-dependent pathway, which may have important implications for nociceptive modulation and pain.

Topics: Animals; Benzoxazoles; Calcium Signaling; Cells, Cultured; Ganglia, Spinal; Intracellular Signaling Peptides and Proteins; Naphthyridines; Neuropeptides; Neurotransmitter Agents; Nociceptors; Orexins; Pain; Protein Kinase C; Rats; Rats, Wistar; Sensory Receptor Cells; Urea

Clinical and preclinical data concur that sleep disruption causes hyperalgesia, but the brain mechanisms through which sleep and pain interact remain poorly understood. Evidence that pontine components of the ascending reticular activating system modulate sleep and nociception encouraged the present study testing the hypothesis that hypocretin-1 (orexin-A) and an adenosine receptor agonist administered into the pontine reticular nucleus, oral part (PnO) each alter thermal nociception. Adult male rats (n = 23) were implanted with microinjection guide tubes aimed for the PnO. The PnO was microinjected with saline (control), hypocretin-1, the adenosine A(1) receptor agonist N(6)-p-sulfophenyladenosine (SPA), the hypocretin receptor-1 antagonist N-(2-Methyl-6-benzoxazolyl)-N''-1,5-naphthyridin-4-yl-urea (SB-334867), and hypocretin-1 plus SB-334867. As an index of antinociceptive behavior, the latency (in seconds) to paw withdrawal away from a thermal stimulus was measured following each microinjection. Compared to control, antinociception was significantly increased by hypocretin-1 and by SPA. SB-334867 increased nociceptive responsiveness, and administration of hypocretin-1 plus SB-334867 blocked the antinociception caused by hypocretin-1. These results suggest for the first time that hypocretin receptors in rat PnO modulate nociception.. Widely distributed and overlapping neural networks regulate states of sleep and pain. Specifying the brain regions and neurotransmitters through which pain and sleep interact is an essential step for developing adjunctive therapies that diminish pain without disrupting states of sleep and wakefulness.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Aging; Animals; Benzoxazoles; Hot Temperature; Intracellular Signaling Peptides and Proteins; Male; Microinjections; Naphthyridines; Neuropeptides; Orexin Receptors; Orexins; Pain; Pain Measurement; Pons; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Reticular Formation; Time Factors; Urea

The posterior hypothalamus (PH) is known to reduce nociceptive pain, but the effect of PH stimulation on neuropathic pain is not known. Because neurons containing the neurotransmitter orexin-A are located in the PH in some strains of rat and intrathecal injection of orexin-A produces antinociception in a neuropathic pain model, we hypothesized that orexin-A from neurons in the PH modifies nociception in the spinal cord dorsal horn. To test this hypothesis, the cholinergic agonist carbachol or normal saline was microinjected into the PH of lightly anesthetized female Sprague-Dawley rats with chronic constriction injury (CCI) and foot withdrawal latencies (FWL) were measured. Carbachol-induced PH stimulation produced dose dependent antinociception as shown by significantly increased FWL compared to saline controls. To investigate the role of orexin-A in PH-induced antinociception, the orexin-1 receptor antagonist SB-334867 or dimethyl sulfoxide (DMSO) for control, was given intrathecally following carbachol-induced PH stimulation. SB-334867 decreased FWL compared to DMSO controls. These data are suggestive that stimulating the PH produces antinociception in a neuropathic pain model and that the antinociceptive effect is mediated in part by orexin-1 receptors in the spinal cord dorsal horn.

Topics: Analgesics; Animals; Benzoxazoles; Carbachol; Cholinergic Agonists; Dose-Response Relationship, Drug; Female; Hypothalamus; Naphthyridines; Neurons; Orexin Receptors; Pain; Pain Measurement; Posterior Horn Cells; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Sciatic Neuropathy; Spinal Cord; Urea

Orexins (orexin A and B) are initially known to be a hypothalamic peptide critical for feeding and normal wakefulness. In addition, emerging evidence from behavioral tests suggests that orexins are also involved in the regulation of nociceptive processing, suggesting a novel potential therapeutic approach for pain treatment. Both spinal and supraspinal mechanisms appear to contribute to the role of orexin in nociception. In the spinal cord, dorsal root ganglion (DRG) neurons are primary afferent neurons that transmit peripheral stimuli to the pain-processing areas. Morphological results show that both orexin A and orexin-1 receptor are distributed in DRG neurons. Moreover, by using whole-cell patch-clamp recordings and calcium imaging measurements we found that orexin A induced excitability and intracellular calcium concentration elevation in the isolated rat DRG neurons, which was mainly dependent on the activation of spinal orexin-1 receptor. Based on these findings, we propose a hypothesis that the direct effect of orexin A on DRG neurons would represent a possible mechanism for the orexinergic modulation of spinal nociceptive transmission.

Topics: Action Potentials; Animals; Benzoxazoles; Calcium Signaling; Ganglia, Spinal; Intracellular Signaling Peptides and Proteins; Male; Naphthyridines; Neurons; Neuropeptides; Orexin Receptors; Orexins; Pain; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Synaptic Transmission; Urea