The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only delta opioid receptors. Activation profiles of most of these ligands at delta opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production.

Opioid neuropeptides and their receptors are evolutionarily conserved neuromodulatory systems that profoundly influence behavior. In dorsal striatum, which expresses the endogenous opioid enkephalin, patches (or striosomes) are limbic-associated subcompartments enriched in mu opioid receptors. The functional implications of opioid signaling in dorsal striatum and the circuit elements in patches regulated by enkephalin are unclear. Here, we examined how patch output is modulated by enkephalin and identified the underlying circuit mechanisms. We found that patches are relatively devoid of parvalbumin-expressing interneurons and exist as self-contained inhibitory microcircuits. Enkephalin suppresses inhibition onto striatal projection neurons selectively in patches, thereby disinhibiting their firing in response to cortical input. The majority of this neuromodulation is mediated by delta, not mu-opioid, receptors, acting specifically on intra-striatal collateral axons of striatopallidal neurons. These results suggest that enkephalin gates limbic information flow in dorsal striatum, acting via a patch-specific function for delta opioid receptors.

The purpose of the present study was to explore the neuroprotective role of delta opioid receptors (DOR) in the rat cortex in hypoxic preconditioning.

Delta opioid receptor (DOR) agonists have been identified as a promising novel therapy for headache disorders. DORs are broadly expressed in several peripheral and central regions important for pain processing and mood regulation; and it is unclear which receptors regulate headache associated symptoms. In a model of chronic migraine-associated pain using the human migraine trigger, nitroglycerin, we observed increased expression of DOR in cortex, hippocampus, and striatum; suggesting a role for these forebrain regions in the regulation of migraine. To test this hypothesis, we used conditional knockout mice with DORs deleted from forebrain GABAergic neurons (Dlx-DOR), and investigated the outcome of this knockout on the effectiveness of the DOR agonist SNC80 in multiple headache models. In DOR loxP controls SNC80 blocked the development of acute and chronic cephalic allodynia in the chronic nitroglycerin model, an effect that was lost in Dlx-DOR mice. In addition, the anti-allodynic effects of SNC80 were lost in a model of opioid induced hyperalgesia/medication overuse headache in Dlx-DOR conditional knockouts. In a model reflecting negative affect associated with migraine, SNC80 was only effective in loxP controls and not Dlx-DOR mice. Similarly, SNC80 was ineffective in the cortical spreading depression model of migraine aura in conditional knockout mice. Taken together, these data indicate that forebrain DORs are necessary for the action of DOR agonists in relieving headache-related symptoms and suggest that forebrain regions may play an important role in migraine modulation.

This study investigated the role of opioid receptor (OR) subtypes as a mechanism by which endurance exercise promotes cardioprotection against myocardial ischemia-reperfusion (IR) injury. Wistar rats were randomly divided into one of seven experimental groups: 1) control; 2) exercise-trained; 3) exercise-trained plus a non-selective OR antagonist; 4) control sham; 5) exercise-trained plus a kappa OR antagonist; 6) exercise-trained plus a delta OR antagonist; and 7) exercise-trained plus a mu OR antagonist. The exercised animals underwent 4 consecutive days of treadmill training (60 min/day at ∼70% of maximal oxygen consumption). All groups except the sham group were exposed to an in vivo myocardial IR insult, and the myocardial infarct size (IS) was determined histologically. Myocardial capillary density, OR subtype expression, heat shock protein 72 (HSP72) expression, and antioxidant enzyme activity were measured in the hearts of both the exercised and control groups. Exercise training significantly reduced the myocardial IS by approximately 34%. Pharmacological blockade of the kappa or mu OR subtypes did not blunt exercise-induced cardioprotection against IR-mediated infarction, whereas treatment of animals with a non-selective OR antagonist or a delta OR antagonist abolished exercise-induced cardioprotection. Exercise training enhanced the activities of myocardial superoxide dismutase (SOD) and catalase but did not increase the left ventricular capillary density or the mRNA levels of HSP72, SOD, and catalase. In addition, exercise significantly reduced the protein expression of kappa and delta ORs in the heart by 44% and 37%, respectively. Together, these results indicate that ORs contribute to the cardioprotection conferred by endurance exercise, with the delta OR subtype playing a key role in this response.

The activation of delta-opioid receptors (DORs) in the caudate-putamen nucleus (CPN) produces regionally distinct changes in motor functions, many of which are also influenced by opioids active at micro-opioid receptors (MORs). These actions most likely occur in MOR-enriched patch compartments in the CPN. To determine the functional sites for DOR activation and potential interactions involving MOR in these regions, immunoperoxidase and immunogold-silver labeling methods were applied reversibly for the ultrastructural localization of DOR and MOR in single rat brain sections containing patches of the CPN. DOR immunoreactivity was commonly seen within the cytoplasm of spiny and aspiny neurons, many of which also expressed MOR. In dendrites and spines, DOR labeling was preferentially localized to membranes of the smooth endoplasmic reticulum and spine apparatus, whereas MOR showed a prominent plasmalemmal distribution. DOR- and/or MOR-labeled spines received asymmetric, excitatory synapses, some of which showed notable perforations, suggesting the involvement of these receptors in activity-dependent synaptic plasticity. DORs were more frequently detected than were MORs within axon terminals that formed either asymmetric synapses with spine heads or symmetric synapses with spine necks. Our results suggest that in striatal patches, DORs, often in cooperation with MORs, play a direct modulatory role in controlling the postsynaptic excitability of spines, whereas presynaptic neurotransmitter release onto spines is mainly influenced by DOR activation. In comparison with MOR, the prevalent association of DOR with cytoplasmic organelles that are involved in intracellular trafficking of cell surface proteins suggests major differences in availability of these receptors to extracellular opioids.

The hippocampal formation (HF) is an important site at which stress circuits and endogenous opioid systems intersect, likely playing a critical role in the interaction between stress and drug addiction. Prior study findings suggest that the stress-related neuropeptide corticotropin releasing factor (CRF) and the delta opioid receptor (DOR) may localize to similar neuronal populations within HF lamina. Here, hippocampal sections of male and cycling female adult Sprague-Dawley rats were processed for immunolabeling using antisera directed against the DOR and CRF peptide, as well as interneuron subtype markers somatostatin or parvalbumin, and analyzed by fluorescence and electron microscopy. Both DOR- and CRF-labeling was observed in interneurons in the CA1, CA3, and dentate hilus. Males and normal cycling females displayed a similar number of CRF immunoreactive neurons co-labeled with DOR and a similar average number of CRF-labeled neurons in the dentate hilus and stratum oriens of CA1 and CA3. In addition, 70% of DOR/CRF dual-labeled neurons in the hilar region co-labeled with somatostatin, suggesting a role for these interneurons in regulating perforant path input to dentate granule cells. Ultrastructural analysis of CRF-labeled axon terminals within the hilar region revealed that proestrus females have a similar number of CRF-labeled axon terminals that contain DORs compared to males but an increased number of CRF-labeled axon terminals without DORs. Taken together, these findings suggest that while DORs are anatomically positioned to modulate CRF immunoreactive interneuron activity and CRF peptide release, their ability to exert such regulatory activity may be compromised in females when estrogen levels are high.

Peripherally-restricted opiate compounds attenuate hyperalgesia in experimental models of inflammatory pain, but have little discernable effect on nociceptive behavior in normal animals. This suggests that activation of opioid receptors on peripheral sensory axons contributes to decreased afferent activity after injury. Previously, we reported that direct application of morphine to cutaneous receptive fields decreased mechanical and heat-evoked responses in a population of C-fiber nociceptors in inflamed skin. Consistent with reported behavioral studies, direct application of morphine had no effect on fiber activity in control skin. The aim of the present study was to determine whether mechanical responsiveness of nociceptors innervating inflamed skin was attenuated by direct activation of delta opioid receptors (DORs) on peripheral terminals. An ex vivo preparation of rat plantar skin and tibial nerve was used to examine effects of a selective DOR agonist, deltorphin II, on responsiveness of single fibers innervating inflamed skin. Electrical recordings were made eighteen hours after injection of complete Freund's adjuvant into the hindpaw. Deltorphin II produced an inhibition of the mechanical responsiveness of single fibers innervating inflamed skin; an effect blocked by the DOR-selective antagonist, naltrindole. The population of units responsive to deltorphin II was identified as consisting of C fiber mechanical nociceptors.

Over the past few years, significant progress has been made in cancer therapy. Indeed, the lifespan of cancer patients has significantly increased. Although patients live longer, cancer-related pain remains a daily problem affecting their quality of life, especially when metastases reach the bone. In patients coping with cancer-induced bone pain, morphine and NSAIDs, often used in combination with other medications, are the most commonly used drugs to alleviate pain. However, these drugs have dose-limiting side effects. Morphine and other routinely used opioids are mu opioid receptor (MOPR) agonists. The MOPR is responsible for most opioid-related adverse effects. In the present study, we revealed potent analgesic effects of an intrathecally-administered selective delta opioid receptor (DOPR) agonist, deltorphin II, in a recently developed rat bone cancer model. Indeed, we found that deltorphin II dose-dependently reversed mechanical allodynia 14 days post-surgery in this cancer pain model, which is based on the implantation of mammary MRMT-1 cells in the femur. This effect was DOPR-mediated as it was completely blocked by naltrindole, a selective DOPR antagonist. Using the complete Freund's adjuvant model of inflammatory pain, we further demonstrated that deltorphin II was equipotent at alleviating inflammatory and cancer pain (i.e. similar ED50 values). Altogether, the present results show, for the first time, that activation of spinal DOPRs causes significant analgesia at doses sufficient to reduce inflammatory pain in a rat bone cancer pain model. Our results further suggest that DOPR represents a potential target for the development of novel analgesic therapies to be used in the treatment of cancer-related pain.

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca(2+) release from IP(3)-sensitive stores and Ca(2+) entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y(1) receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca(2+)-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.

The noradrenergic nucleus locus coeruleus is a key component of the stress circuitry of the brain. During stress, the neuropeptide corticotropin-releasing factor (CRF) is secreted onto LC, increasing LC output and norepinephrine concentration in the brain, which is thought to promote anxiety-like behavior. LC is also innervated by several structures that synthesize and release the endogenous opioid peptide enkephalin onto LC upon stressor termination. While the role of CRF neurotransmission within LC in mediating anxiety-like behavior and the behavioral response to stress has been well characterized, the role of enkephalinergic signaling at LC-expressed δ-opioid receptors has been comparatively understudied. We have previously shown that acute stressor exposure increases LC activity and anxiety-like behavior for at least one week. Here, we extend these findings by showing that these effects may be mediated at least in part through stress-induced downregulation of DORs within LC. Furthermore, overexpression of DORs in LC blocks the effects of stress on both LC firing properties and anxiety-like behavior. In addition, intra-LC infusions of enkephalin blocked stress-induced freezing behavior and promoted conditioned place preference. These findings indicate that enkephalinergic neurotransmission at DORs within LC is an important component of the behavioral response to stress and may drive reward-related behavior as well.

The control of pain perception is a challenge in clinical dentistry, most prominent during tooth pulp inflammation. The tooth pulp is a well-defined target, and is densely supplied by a sensory trigeminal innervation. Opioids are signaling molecules that are suggested to participate in pain perception. Here we analysed the presence of delta opioid receptor (DOR) in trigeminal neurons innervating the tooth pulp of rat molars. Immunohistochemical and ultrastructural analysis revealed that DOR was identified in peripheral nerves in the molar dental pulp, both in the root and the coronal pulpal parts, with branching in the highly innervated subodontoblast layer. DOR was localised in about one third of all the trigeminal dental neurons, identified by means of retrograde neuronal transport of fluorogold (FG) from the dental pulp. Of the DOR-labeled neurons, nearly all were small and medium-sized (147.5-1,810.2 microm(2), mean 749.1 +/- 327.3 microm(2)). Confocal microscopy confirmed that DOR-immunoreactivity was distributed as granules in the neuronal cytoplasm. Approximately 70% of the DOR-immunoreactive neurons were also immunopositive for vanilloid receptor 1 (TRPV1). Ultrastructural analysis demonstrated DOR-immunoreactivity in the unmyelinated and in some of the myelinated nerve fibers in the dental pulp. These results indicate that DOR may influence the function in a subset of small and medium-sized trigeminal sensory neurons supporting the tooth, which are mainly known for their ability to mediate nociceptive stimuli. Agonists, acting on DOR, may thus have an influence on a subpopulation of nociceptive neurons supporting the rat tooth.

Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed by homologous recombination and these animals show complete loss of analgesic responses to morphine as well as loss of place-preference activity and physical dependence on this opioid. We report here quantitative autoradiographic mapping of opioid receptor subtypes in the brains of wild-type, heterozygous and homozygous mutant mice to demonstrate the deletion of the MOR gene, to investigate the possible existence of any mu-receptor subtypes derived from a different gene and to determine any modification in the expression of other opioid receptors. Mu-, delta-, kappa1- and total kappa-receptors, in adjacent coronal sections in fore- and midbrain and in sagittal sections, were labelled with [3H]DAMGO (D-Ala2-MePhe4-Gly-ol5 enkephalin), [3H]DELTI (D-Ala2 deltorphinI), [3H]CI-977 and [3H]bremazocine (in the presence of DAMGO and DPDPE) respectively. In heterozygous mice, deficient in one copy of the MOR gene, mu-receptors were detectable throughout the brain at about 50% compared to wild-type. In brains from mu-knockout mice there were no detectable mu-receptors in any brain regions and no evidence for mu-receptors derived from another gene. Delta-, kappa1- and total kappa-receptor binding was present in all brain regions in mutant mice where binding was detected in wild-type animals. There were no major quantitative differences in kappa- or delta-binding in mutant mice although there were some small regional decreases. The results indicate only subtle changes in delta- and kappa-receptors throughout the brains of animals deficient in mu-receptors.

The existence of opioid receptors in mammalian myometrial tissue is now widely accepted. Previously enkephalin degrading enzymes have been shown to be elevated in pregnant rat uterus and a met-enkephalin analogue has been shown to alter spontaneous contractility of rat myometrium. Here we have undertaken studies to determine the effects of met-enkephalin on in vitro human myometrial contractility and investigate the expression of opioid receptors in pregnant myometrium. Myometrial biopsies were taken from women undergoing elective caesarean delivery at term. Organ bath experiments were used to investigate the effect of the met-enkephalin analogue [d-Ala 2, d-met 5] enkephalin (DAMEA) on spontaneous contractility. A confocal immunofluorescent technique and real time PCR were used to determine the expression of protein and mRNA, respectively for two opioid receptor subtypes, mu and delta. DAMEA had a concentration dependent inhibitory effect on contractile activity (1 × 10(-7)M-1 × 10(-4)M; 54% reduction in contractile activity, P<0.001 at 1 × 10(-4)M concentration). Mu and delta opioid receptor protein sub-types and their respective mRNA were identified in all tissues sampled. This is the first report of opioid receptor expression and of an opioid mediated uterorelaxant action in term human non-labouring myometrium in vitro.

Opioid receptors (OR) are widely expressed in the central nervous system (CNS). Opioid antinociception might be initiated by activation of OR outside the CNS, indicating targeting of peripheral OR could be useful in the treatment of chronic pain. This study was designed to detect OR in skin tissues of healthy volunteers at both mRNA and protein levels. Skin samples from 10 healthy individuals were investigated. Total isolated RNAs were reverse transcribed, amplified and quantified by real-time PCR. Tissue and skin fibroblast OR protein was detected by immunohistochemistry, Western blot, and immunofluorescence. All skin tissue samples expressed delta- (DOR) and kappa-OR (KOR) mRNAs. Using immunohistochemistry, DOR and KOR were localized in skin fibroblast-like and mononuclear cells. Skin fibroblasts in culture expressed DOR and KOR mRNA. Using immunofluorescence, both DOR and KOR proteins were expressed predominantly on the cell membrane with minor staining in the cytoplasm. We suggest that enhanced expression of DOR and KOR in skin justifies the exploration of selective novel delta and kappa agonists for local pain treatment.

Inflammatory visceral pain is endogenously controlled by enkephalins locally released by mucosal CD4+ T lymphocytes in mice. The present study aimed at identifying opioid receptor(s) expressed on nociceptive sensory nerves involved in this peripheral opioid-mediated analgesia.

The diversity of receptor signaling is increased by receptor heteromerization leading to dynamic regulation of receptor function. While a number of studies have demonstrated that family A G-protein-coupled receptors are capable of forming heteromers in vitro, the role of these heteromers in normal physiology and disease has been poorly explored. In this study, direct interactions between CB(1) cannabinoid and delta opioid receptors in the brain were examined. Additionally, regulation of heteromer levels and signaling in a rodent model of neuropathic pain was explored. First we examined changes in the expression, function and interaction of these receptors in the cerebral cortex of rats with a peripheral nerve lesion that resulted in neuropathic pain. We found that, following the peripheral nerve lesion, the expression of both cannabinoid type 1 receptor (CB(1)R) and the delta opioid receptor (DOR) are increased in select brain regions. Concomitantly, an increase in CB(1)R activity and decrease in DOR activity was observed. We hypothesize that this decrease in DOR activity could be due to heteromeric interactions between these two receptors. Using a CB(1)R-DOR heteromer-specific antibody, we found increased levels of CB(1)R-DOR heteromer protein in the cortex of neuropathic animals. We subsequently examined the functionality of these heteromers by testing whether low, non-signaling doses of CB(1)R ligands influenced DOR signaling in the cortex. We found that, in cortical membranes from animals that experienced neuropathic pain, non-signaling doses of CB(1)R ligands significantly enhanced DOR activity. Moreover, this activity is selectively blocked by a heteromer-specific antibody. Together, these results demonstrate an important role for CB(1)R-DOR heteromers in altered cortical function of DOR during neuropathic pain. Moreover, they suggest the possibility that a novel heteromer-directed therapeutic strategy for enhancing DOR activity, could potentially be employed to reduce anxiety associated with chronic pain.

Opioids are powerful analgesics but the clinical utility of these compounds is reduced by aversive outcomes, including the development of affective and substance use disorders. Opioid systems do not function in isolation so understanding how these interact with other neuropharmacological systems could lead to novel therapeutics that minimize withdrawal, tolerance, and emotional dysregulation. The cannabinoid system is an obvious candidate as anatomical, pharmacological, and behavioral studies point to opioid-cannabinoid interactions in the mediation of these processes. The aim of our study is to uncover the role of specific cannabinoid and opioid receptors in addiction-related behaviors, specifically nociception, withdrawal, anxiety, and depression. To do so, we tested the effects of a selective CB1 agonist, arachidonyl-2-chloroethylamide (ACEA), on mouse behavior in tail immersion, naloxone-precipitated withdrawal, light-dark, and splash tests. We examined cannabinoid-opioid interactions in these tests by comparing responses of wildtype (WT) mice to mutant lines lacking either Mu or Delta opioid receptors. ACEA, both acute or repeated injections, had no effect on nociceptive thresholds in WT or Mu knockout (KO) mice suggesting that analgesic properties of CB1 agonists may be restricted to chronic pain conditions. The opioid antagonist, naloxone, induced similar levels of withdrawal in all three genotypes following ACEA treatment, confirming an opioidergic contribution to cannabinoid withdrawal. Anxiety-like responses in the light-dark test were similar across WT and KO lines; neither acute nor repeated ACEA injections modified this behavior. Similarly, administration of the Delta opioid receptor antagonist, naltrindole, alone or in combination with ACEA, did not alter responses of WT mice in the light-dark test. Thus, there may be a dissociation in the effect of pharmacological blockade vs. genetic deletion of Delta opioid receptors on anxiety-like behavior in mice. Finally, our study revealed a biphasic effect of ACEA on depressive-like behavior in the splash test, with a prodepressive state induced by acute exposure, followed by a shift to an anti-depressive state with repeated injections. The initial pro-depressive effect of ACEA was absent in Mu KO mice. In sum, our findings confirm interactions between opioid and cannabinoid systems in withdrawal and reveal reduced depressive-like symptoms with repeated CB1 receptor activation.

Opioid-produced antinociception in mammals seems to be mediated in part by pathways originating in the periaqueductal gray (PAG) and the rostroventral medulla (RVM), and these pathways may include serotonergic neurons. In the present study, we examined the relationship of the cloned mu- and delta-receptors (MOR1 and DOR1, respectively) to PAG neurons projecting to the RVM, and RVM neurons projecting to the dorsal spinal cord. This was carried out by combining immunocytochemical staining for MOR1, DOR1, and serotonin with fluorescent retrograde tract-tracing. Of 133 retrogradely labeled cells in the RVM, 31% were immunoreactive for MOR1. Of the double-labeled cells, 41% also were immunoreactive for 5HT. Fifty-three percent of retrogradely labeled cells were apposed by DOR1-ir varicosities; 29% of the apposed cells were immunoreactive for 5HT. In the mesencephalon, cells retrogradely labeled from the RVM were usually surrounded by MOR1-ir structures; however, retrogradely labeled cells were never observed to be immunoreactive for MOR1. Similarly, retrogradely labeled cells in the caudal midbrain were seldom, if ever, labeled for DOR1; however, they frequently were apposed by DOR1-ir varicosities. Of 156 retrogradely labeled profiles from three rats, 52 (33%) were apposed by DOR1-ir varicosities. We conclude that both mu- and delta-opioid receptors could be involved in the antinociception mediated by the PAG-RVM-spinal cord circuit. In addition, opioids seem likely to have both direct and indirect effects on spinally projecting RVM cells in general, and on serotonergic RVM cells in particular.

Chronic inflammatory pain is present in many pathologies and diminishes the patient's quality of life. Moreover, most current treatments have a low efficacy and significant side effects. Recent studies demonstrate the analgesic properties of slow-releasing hydrogen sulfide (H2S) donors in animals with osteoarthritis or neuropathic pain, but their effects in inflammatory pain and related pathways are not completely understood. Several treatments potentiate the analgesic actions of δ-opioid receptor (DOR) agonists, but the role of H2S in modulating their effects and expression during inflammatory pain remains untested. In C57BL/6J male mice with inflammatory pain provoked by subplantar injection of complete Freund's adjuvant, we evaluated: (1) the antiallodynic and antihyperalgesic effects of different doses of two slow-releasing H2S donors, i.e., diallyl disulfide (DADS) and phenyl isothiocyanate (P-ITC) and their mechanism of action; (2) the pain-relieving effects of DOR agonists co-administered with H2S donors; (3) the effects of DADS and P-ITC on the oxidative stress and molecular changes caused by peripheral inflammation. Results demonstrate that both H2S donors inhibited allodynia and hyperalgesia in a dose-dependent manner, potentiated the analgesic effects and expression of DOR, activated the antioxidant system, and reduced the nociceptive and apoptotic pathways. The data further demonstrate the possible participation of potassium channels and the Nrf2 transcription factor signaling pathway in the pain-relieving activities of DADS and P-ITC. This study suggests that the systemic administration of DADS and P-ITC and local application of DOR agonists in combination with slow-releasing H2S donors are two new strategies for the treatment of inflammatory pain.