Delta-opioid Receptor Activation Research Articles

Maternal treatment with a selective delta-opioid receptor agonist during gestation has a sex-specific pro-cognitive action in offspring: mechanisms involved.

Background: There is growing evidence that the treatment of several mental disorders can potentially benefit from activation of delta-opioid receptors. In the future, delta-agonists with a safe pharmacological profile can be used for the treatment of mood disorders in pregnant women. However, the data on prenatal exposure to delta-opioid agonists are missing. The present study is aimed to test the hypothesis that the activation of delta-opioid receptors during gravidity has positive effects on the behaviour accompanied by changes in glutamate and monoamine neurotransmission. Methods: Gestating Wistar rats were chronically treated with a selective delta-agonist SNC80 or vehicle. Adult male and female offspring underwent novel object recognition (for the assessment of cognition) and open field (for the assessment of anxiety and habituation) tests, followed by in vivo electrophysiological examination of the activity of hippocampal glutamate and midbrain serotonin (5-HT) and dopamine neurons. Results: We found that the maternal treatment with SNC80 did not affect the offspring's anxiety, habituation, and 5-HT neuronal firing activity. Female offspring of SNC80-treated dams exhibited improved novelty recognition associated with decreased firing rate and burst activity of glutamate and dopamine neurons. Conclusion: Maternal treatment with delta-opioid agonists during gestation may have a pro-cognitive effect on offspring without any negative effects on anxiety and habituation. The putative pro-cognitive effect might be mediated via mechanism(s) involving the firing activity of hippocampal glutamate and mesolimbic dopamine neurons.

The Opioid Receptor Influences Circadian Rhythms in Human Keratinocytes through the β-Arrestin Pathway.

The recent emphasis on circadian rhythmicity in critical skin cell functions related to homeostasis, regeneration and aging has shed light on the importance of the PER2 circadian clock gene as a vital antitumor gene. Furthermore, delta-opioid receptors (DOPrs) have been identified as playing a crucial role in skin differentiation, proliferation and migration, which are not only essential for wound healing but also contribute to cancer development. In this study, we propose a significant association between cutaneous opioid receptor (OPr) activity and circadian rhythmicity. To investigate this link, we conducted a 48 h circadian rhythm experiment, during which RNA samples were collected every 5 h. We discovered that the activation of DOPr by its endogenous agonist Met-Enkephalin in N/TERT-1 keratinocytes, synchronized by dexamethasone, resulted in a statistically significant 5.6 h delay in the expression of the core clock gene PER2. Confocal microscopy further confirmed the simultaneous nuclear localization of the DOPr-β-arrestin-1 complex. Additionally, DOPr activation not only enhanced but also induced a phase shift in the rhythmic binding of β-arrestin-1 to the PER2 promoter. Furthermore, we observed that β-arrestin-1 regulates the transcription of its target genes, including PER2, by facilitating histone-4 acetylation. Through the ChIP assay, we determined that Met-Enkephalin enhances β-arrestin-1 binding to acetylated H4 in the PER2 promoter. In summary, our findings suggest that DOPr activation leads to a phase shift in PER2 expression via β-arrestin-1-facilitated chromatin remodeling. Consequently, these results indicate that DOPr, much like its role in wound healing, may also play a part in cancer development by influencing PER2.

The opioid peptide met-enkephalin modulates thalamo-cortical excitation inhibition balance in a medial thalamus-anterior cingulate cortex circuit

Activation of opioid receptors in the anterior cingulate cortex (ACC) mediates aspects of analgesia induced by both exogenous and endogenous opioids. We have previously shown that opioid signaling disrupts both afferent excitatory and indirect inhibitory synaptic transmission from the medial thalamus (MThal) to the ACC, but the effects of non-selective opioid peptides such as [Met]5-enkephalin (ME) within this circuit remain poorly understood. The goal of the current study was to understand how ME modulates thalamic-driven excitatory and inhibitory synaptic transmission onto layer V pyramidal neurons in the ACC. We used pharmacology, brain slice electrophysiology and optogenetic stimulation to study opioid-mediated modulation of optically evoked glutamatergic and GABAergic transmission. The results revealed that bath perfused ME inhibited both AMPA-mediated excitatory and GABA-mediated inhibitory synaptic transmission in the ACC. However, inhibitory transmission was more potently inhibited than excitatory transmission by ME. This preferential reduction in GABAA-mediated synaptic transmission was primarily due to the activation of delta opioid receptors by ME and resulted in a net disinhibition of MThal-ACC excitatory pathway. These results suggest that moderate concentrations of ME can lead to a net increase in excitatory drive of ACC circuitry and that analgesia may be associated with disinhibition rather than inhibition of ACC subcircuits.

Nitro-benzylideneoxymorphone, a bifunctional mu and delta opioid receptor ligand with high mu opioid receptor efficacy.

Introduction: There is a major societal need for analgesics with less tolerance, dependence, and abuse liability. Preclinical rodent studies suggest that bifunctional ligands with both mu (MOPr) and delta (DOPr) opioid peptide receptor activity may produce analgesia with reduced tolerance and other side effects. This study explores the structure-activity relationships (SAR) of our previously reported MOPr/DOPr lead, benzylideneoxymorphone (BOM) with C7-methylene-substituted analogs. Methods: Analogs were synthesized and tested in vitro for opioid receptor binding and efficacy. One compound, nitro-BOM (NBOM, 12) was evaluated for antinociceptive effects in the warm water tail withdrawal assay in C57BL/6 mice. Acute and chronic antinociception was determined, as was toxicologic effects on chronic administration. Molecular modeling experiments were performed using the Site Identification by Ligand Competitive Saturation (SILCS) method. Results: NBOM was found to be a potent MOPr agonist/DOPr partial agonist that produces high-efficacy antinociception. Antinociceptive tolerance was observed, as was weight loss; this toxicity was only observed with NBOM and not with BOM. Modeling supports the hypothesis that the increased MOPr efficacy of NBOM is due to the substituted benzylidene ring occupying a nonpolar region within the MOPr agonist state. Discussion: Though antinociceptive tolerance and non-specific toxicity was observed on repeated administration, NBOM provides an important new tool for understanding MOPr/DOPr pharmacology.

Efficacy of dual enkephalinase inhibition in a preclinical migraine model is mediated by activation of peripheral delta opioid receptors.

The aim of this study was to evaluate whether elevating levels of enkephalin by inhibiting their degradation can attenuate stress-induced migraine-like behaviors in mice. Previous studies in animals have suggested the delta opioid receptor (DOR) as a novel migraine target. The primary endogenous ligands for DOR are enkephalins and their levels can be increased by pharmacological inhibition of enkephalinases; however, it is not clear whether enkephalinase inhibition can be efficacious in preclinical migraine models through activation of DOR or whether other opioid receptors might be involved. Further, it is not clear whether opioid receptors in the central nervous system are necessary for these effects. This study used a model of repetitive restraint stress in mice that induces periorbital hypersensitivity and priming to the nitric oxide donor sodium nitroprusside (SNP; 0.1 mg/kg). Von Frey filaments were used to measure periorbital mechanical thresholds and grimace scores were evaluated by observing mouse facial features. Animals were treated with the dual enkephalinase inhibitor (DENKI) PL37. On day two post-stress, PL37 given to mice via either intravenous injection (10 mg/kg) or oral gavage (20 mg/kg) significantly attenuated stress-induced periorbital hypersensitivity and facial grimace responses. Additionally, both intravenous (10 mg/kg) and oral gavage (20 mg/kg) of PL37 prior to SNP (0.1 mg/kg) administration on day 14 post-stress significantly reduced SNP-induced facial hypersensitivity. Injection of the DOR antagonist naltrindole (0.1 mg/kg) but not the mu-opioid receptor antagonist CTAP (1 mg/kg) prior to PL37 treatment blocked the effects. Finally, pretreatment of mice with the peripherally restricted opioid receptor antagonist naloxone methiodide (5 mg/kg) blocked the effects of PL37. These data demonstrate that inhibiting enkephalinases, and thus protecting enkephalins from degradation, attenuates stress-induced migraine-like behavior via activation of peripheral DOR. Peripheral targeting of endogenous opioid signaling may be an effective therapeutic strategy for migraine.

Activation of Delta-Opioid Receptor Protects ARPE19 Cells against Oxygen-Glucose Deprivation/Reoxygenation-Induced Necroptosis and Apoptosis by Inhibiting the Release of TNF-α.

Retinal ischemia-reperfusion injury (RIRI) is the basis of the pathology that leads to many retinal diseases and induces necroptosis and apoptosis. Tumor necrosis factor-α (TNF-α) is critically involved in necroptosis and apoptosis. Delta-opioid receptor (DOR) activation inhibits TNF-α release in our previous studies, it might prevent necroptosis and apoptosis by inhibiting the release of TNF-α. However, the role of TNF-α and DOR in necroptosis and apoptosis of retinal pigment epithelial (RPE) cells remains largely unknown. Here, we explored the mechanisms of TNF-α and DOR in necroptosis and apoptosis using an oxygen-glucose deprivation/reoxygenation (OGD/R) model of adult retinal pigment epithelial cell line-19 (ARPE19) cells. ARPE19 cells were exposed to OGD/R conditions to mimic RIRI in vitro. Cell viability was quantified using the Cell Counting Kit-8 (CCK-8) assay. Morphological changes were observed by inverted microscopy. TNF-α protein levels in cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). The DOR agonist TAN-67 and antagonist naltrindole (NTI) were used to pretreat cells for 1 or 2 hours before OGD24/R36 administration. Calcein acetoxymethylester/propidium iodide (Calcein-AM/PI) and Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to detect necroptotic and apoptotic ARPE19 cells, respectively. The protein expression of DOR, p-RIP1 (RIP1), p-RIP3 (RIP3), p-MLKL (MLKL), and cleaved Caspase3 (Caspase3) was measured by western blotting. OGD severely damaged ARPE19 cells. Prolonged reoxygenation significantly increased TNF-α level and decreased DOR expression in ARPE19 cells. Pretreatment with the DOR agonist TAN-67 (10 µM) significantly improved ARPE19 cell viability after OGD24/R36 by reducing the number of necroptotic and apoptotic cells. Furthermore, DOR activation significantly inhibited TNF-α release and suppressed the expression of proteins related to necroptosis and apoptosis, including p-RIP1, p-RIP3, p-MLKL, and cleaved Caspase3, after OGD24/R36. This effect was reversed by the DOR antagonist NTI. These results strongly suggest that DOR activation inhibits necroptosis and apoptosis by decreasing TNF-α release, leading to the prevention of OGD/R-induced injury in ARPE19 cells. This study provides an innovative idea for clinical treatment strategies for retinal damage and vision loss due to RIRI.

The Effects of Activation and Blockade of Delta Opioid Receptors on the Acquisition of Responding for an Opioid‐Associated Stimulus

Drug‐associated stimuli take on conditioned reinforcing properties that promote drug‐seeking and relapse. Previously, we have demonstrated that daily systemic injections of SNC80 (delta opioid receptor agonist) resulted in dose‐dependent increases in responding for the opioid‐associated stimulus. In the current experiment, we assessed the extent to which activating and/or blocking delta opioid receptors via a systemic injection of SNC80 or naltrindole, respectively, modified the conditioned reinforcing properties of an opioid‐associated stimulus. We hypothesized that a pre‐session injection of SNC80 would increase the conditioned reinforcing properties of the remifentanil‐associated stimulus; however, an injection of naltrindole would have no effect, and an injection of naltrindole would block the ability of SNC80 to enhance the conditioned reinforcing properties of the stimulus. First, we exposed rats to 20 i.v. infusions of remifentanil (3.2 ug/kg/infusion) and 20 stimulus presentations that were delivered response‐independently each day for 5 days. For the experimental group (Paired Conditioning), the remifentanil infusions and stimulus presentations were delivered concurrently. The control group (Random control) received the same number of infusions and stimulus presentations, but they were not explicitly paired. For the next 7 sessions (acquisition), we tested the extent to which the stimulus functioned as a conditioned reinforcer by allowing rats to freely respond via a nosepoke for presentations of the remifentanil‐associated stimulus only. On the fourth acquisition session, rats received one of the following combinations of injections: (a) vehicle + vehicle, (b) vehicle + naltrindole (3.2 mg/kg), (c) vehicle + SNC80 (3.2 mg/kg), or (d) SNC80 (3.2 mg/kg) + naltrindole (3.2 mg/kg). We found that rats in the Paired Pavlovian Conditioning group responded for the remifentanil‐associated stimulus significantly more than controls (p= 0.003, hp2= 0.15). Responding for remifentanil‐paired stimuli depended on treatment condition (p < 0.001, hp2= 0.35). Post‐hoc tests revealed that SNC80 administration increased responding for the remifentanil‐associated stimulus relative to all other treatment conditions (p’s < 0.001). These findings demonstrated that a remifentanil‐associated stimulus takes on conditioned reinforcing properties and that the conditioned reinforcing properties of the stimulus was augmented by delta opioid receptor activation and these effects were blocked via naltrindole. These findings support previous work that delta opioid receptors play a role in mediating conditioned reinforcing properties of drug‐associated stimuli.

Naltrindole Pretreatment Exhibits Robust Cardioprotection in an Acute In Vivo Model of Ischemia/Reperfusion

Myocardial infarctions (MI), remain prevalent in the U.S. with over one million individuals experiencing this cardiac event every year. Delta opioid receptor activation is known to mimic pharmacologic preconditioning to reduce infarct size post‐MI. Surprisingly, results from our laboratory have shown that naltrindole (NTI), a selective delta opioid receptor antagonist, elicits unexpected cardioprotective effects ex vivo when given prior to 30 min global ischemia (I) followed by 45 min reperfusion (R). Whether NTI’s cardioprotective effects can be translated in vivo with intact neurohormonal compensatory mechanisms, has not yet been elucidated. In this study, we aim to determine the cardioprotective effects of NTI in an in vivo model that more closely mimics acute MI clinically.Female Sprague‐Dawley rats (~250g) were assigned into three cohorts: non‐treated control (n=15), high dose (HD, 4.0‐8.0 mg/kg, n=5) NTI, and low dose (LD, 0.2‐1.0 mg/kg, n=18) treatment groups. All three cohorts were anesthetized with ketamine‐xylazine (90 mg/kg, 9mg/kg). Control rats received normal saline (0.9% NaCl), while treatment groups received either HD or LD NTI. NTI was infused through the jugular vein (0.4mL/min) over 5 mins before inducing regional ischemia (pretreatment). The proximal left coronary artery was occluded for 30 min followed by 3 hr reperfusion. At the end of the experiment, blue dye was administered to determine the area at risk (AR). Planimetered photographs of heart slices were then stained with 1% triphenyltetrazolium chloride to determine area of necrosis (AN) and infarct size (AN/AR). Cardiac function was measured via a pressure transducer placed into the left ventricle (LV) in a small cohort of the LD NTI (0.4‐1.0 mg/kg, n=3) and HD NTI groups (n=5). Data were analyzed via ANOVA Student Newman Keuls post‐hoc analysis (infarct size) and unpaired t‐test (cardiac function), respectively, with p<0.05 considered statistically significant.HD NTI exhibited a robust ~60% reduction in infarct size (21±7 %; n=4, p<0.01) compared to control (57±2 %; n=15) and LD NTI (55±4 %; n=18) hearts. HD NTI (n=5) also exerted a significant depression of the maximal and minimal rate of LV pressure change on dP/dt max (6167±489 mmHg/s vs. 3714±645 mmHg/s) and dP/dt min (‐7015±942 mmHg/s vs. ‐4034±776 mmHg/s) respectively and heart rate (261±20 BPM vs. 208±3 BPM) at the end of pretreatment compared to LD NTI (0.4‐1.0 mg/kg, n=3 all p<0.05). This preconditioning effect was correlated with the robust decrease in infarct size compared to LD NTI and control groups. These results were similar to our ex‐vivo studies, although a 20x dose was needed in vivo to presumably overcome compensatory neurohormonal responses (i.e. 4mg/kg vs 0.2mg/kg). Results suggest that NTI pretreatment can protect against I/R injury. Future studies will examine the effects of drug infusion at different ischemic time points for optimal cardioprotection.

SRI 22136, A Novel Delta Opioid Receptor (DOR) Antagonist for the Treatment of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia, which affects 47 million people worldwide. There are over five million AD patients over the age of 65 in the U.S. which is predicted to increase to 16 million by 2050. There are currently no approved disease‐modifying therapies for AD and attempts to prevent or slow the progression of the formation of beta‐amyloid plaques by targeting both the beta‐secretase 1 (BACE1) and gamma‐secretase enzymes have not yet achieved clinical success.Studies suggest that indirect modulation of the function of these enzymes via G‐protein coupled receptors (GPCRs) may provide a novel strategy to reduce A‐beta peptide production with potentially less side effects. Among GPCRs that influence amyloidogenesis, the delta opioid receptor (DOR), in particular, has been shown to play an important role in the trafficking and function of BACE1 and gamma‐secretase and in the production of A‐beta peptide. DOR activation increases BACE1 and gamma‐secretase activity in vitro and in a mouse model of AD, and antagonism of DOR specifically blocks the amyloidogenic pathway and efficaciously prevents AD progression in mice. These effects were demonstrated using a known DOR antagonist, naltrindole. However, the potential of DOR antagonists as therapeutic agents for AD has yet to be explored.The aim of this project is to create novel DOR antagonists via medicinal chemistry and identify the most promising lead compound based on binding, selectivity, and functional profile in vitro. Further, select a small set of the most promising compounds and evaluate the compound’s ability to mitigate AD‐like pathology in vivo using APP/PS double‐transgenic mice. We report here the identification of several selective DOR antagonists with low nanomolar affinity/potency that have been shown to block BACE1 activity in vitro. Lead compound SRI‐22136, showed systemic stability and blood brain barrier penetration using the in vivo tail flick assay in CD‐1 mice. SRI‐22136 also showed efficacy in Novel Object Recognition (NOR) behavioral studies and in brain histological analysis studies we observed greater reduction in the AD pathological markers at a dose of 1mg/kg twice daily for 90 days continuous treatment in vivo using the APP/PS AD model double‐transgenic mice. Also, in our brain histological analysis the compound exhibited greater efficacy in reduction of the AD pathology markers such as Aβ signal, and secondary inflammation and activated glia detected by GFAP (astrocytes) and CD11b (microglia) in APP/PS AD model double‐transgenic mice. We further report our progress in testing this compound’s effect on biochemical marker evaluation using the cortex and hippocampus regions of the APP/PS AD model double‐transgenic mice.

Delta Opioid Receptor in Astrocytes Contributes to Neuropathic Cold Pain and Analgesic Tolerance in Female Mice.

Background: The delta opioid receptor (DOR) contributes to pain control, and a major challenge is the identification of DOR populations that control pain, analgesia, and tolerance. Astrocytes are known as important cells in the pathophysiology of chronic pain, and many studies report an increased prevalence of pain in women. However, the implication of astrocytic DOR in neuropathic pain and analgesia, as well as the influence of sex in this receptor activity, remains unknown.Experimental Approach: We developed a novel conditional knockout (cKO) mouse line wherein DOR is deleted in astrocytes (named GFAP-DOR-KO), and investigated neuropathic mechanical allodynia as well as analgesia and analgesic tolerance in mutant male and female mice. Neuropathic cold allodynia was also characterized in mice of both sexes lacking DOR either in astrocytes or constitutively.Results: Neuropathic mechanical allodynia was similar in GFAP-DOR-KO and floxed DOR control mice, and the DOR agonist SNC80 produced analgesia in mutant mice of both sexes. Interestingly, analgesic tolerance developed in cKO males and was abolished in cKO females. Cold neuropathic allodynia was reduced in mice with decreased DOR in astrocytes. By contrast, cold allodynia was exacerbated in full DOR KO females.Conclusions: These findings show that astrocytic DOR has a prominent role in promoting cold allodynia and analgesic tolerance in females, while overall DOR activity was protective. Altogether this suggests that endogenous- and exogenous-mediated DOR activity in astrocytes worsens neuropathic allodynia while DOR activity in other cells attenuates this form of pain. In conclusion, our results show a sex-specific implication of astrocytic DOR in neuropathic pain and analgesic tolerance. These findings open new avenues for developing tailored DOR-mediated analgesic strategies.

Hair Growth Promotion by δ-Opioid Receptor Activation.

Literature has revealed that the delta opioid receptor (DOR) exhibited diverse pharmacological effects on neuron and skin. In the present study, we have investigated whether the activation of DOR has hair-growth promotion effects. Compared with other opioid receptor, DOR was highly expressed in epidermal component of hair follicle in human and rodents. The expression of DOR was high in the anagen phase, but it was low in the catagen and telogen phases during mouse hair cycle. Topical application of UFP-512, a specific DOR agonist, significantly accelerated the induction of the anagen in C3H mice. Topical application of UFP-512 also increased the hair length in hair organ cultures and promoted the proliferation and the migration of outer root sheath (ORS) cells. Similarly, pharmacological inhibition of DOR by naltrindole significantly inhibited the anagen transition process and decreased hair length in hair organ cultures. Thus, we further examined whether Wnt/β-catenin pathway was related to the effects of DOR on hair growth. We found that Wnt/β-catenin pathway was activated by UFP-512 and siRNA for β-catenin attenuated the UFP-512 induced proliferation and migration of ORS cells. Collectively, result established that DOR was involved in hair cycle regulation, and that DOR agonists such as UFP-512 should be developed for novel hair-loss treatment.

Biphalin, a dimeric opioid peptide, reduces neonatal hypoxia-ischemia brain injury in mice by the activation of PI3K/Akt signaling pathway

Previous studies have demonstrated that the activation of delta opioid receptors is neuroprotective against neonatal hypoxia-ischemia (HI) brain injury. The aim of this study was to investigate the neuroprotective effects of biphalin, a dimeric opioid peptide, in a mouse model of neonatal HI and the underlying mechanisms. On postnatal day 10, mouse pups were subjected to unilateral carotid artery ligation followed by 1 h of hypoxia (10 % O2 in N2). For treatment, biphalin (5 mg/kg, 10 mg/kg, 20 mg/kg) was administered intraperitoneally immediately after HI. The opioid antagonist naloxone or phosphatidylinositol-3-kinase inhibitor Ly294002 was administered to determine the underlying mechanisms. Infarct volume, brain edema, phosphorylated Akt and apoptosis-related proteins levels were evaluated by using a combination of 2,3,5-triphenyltetrazolium chloride staining, brain water content and Western blotting at 24 h after HI. The long-term effects of biphalin were evaluated by brain atrophy measurement, Nissl staining and neurobehavioral tests at 3 weeks post-HI. Biphalin (10 mg/kg) significantly reduced the infarct volume and ameliorated brain edema. Biphalin also had long-term protective effects against the loss of ipsilateral brain tissue and resulted in improvements in neurobehavioral outcomes. However, naloxone or Ly294002 abrogated the neuroprotective effects of biphalin. Furthermore, biphalin treatment significantly preserved phosphorylated Akt expression, increased Bcl-2 levels, and decreased Bax and cleaved caspase 3 levels after HI. These effects were also reversed by naloxone and Ly294002 respectively. In conclusion, biphalin protects against HI brain injury in neonatal mice, which might be through activation of the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway.

The Effects of Delta Opioid Receptors Activation on the Acquisition of Responding for an Opioid‐associated Stimulus

Drug-associated stimuli take on conditioned reinforcing properties that promote drug-seeking and relapse. Previous research has shown that, following response-independent infusions of remifentanil (mu opioid receptor agonist) paired with a stimulus, rats learned to make a novel response for presentations of an opioid-associated stimulus to a greater extent than control rats. Further, research has demonstrated that stimulating delta opioid receptors in the nucleus accumbens results in dose-dependent increases in drug-seeking. In the current experiment, we assessed the extent to which activating delta opioid receptors via a systemic injection of SNC80 modified the conditioned reinforcing properties of an opioid-associated stimulus. We hypothesized that pre-session injections of SNC80 would increase the conditioned reinforcing properties of the remifentanil-associated stimulus. First, we exposed rats to 20 i.v. infusions of remifentanil (0, 1.0, or 3.2 mcg/kg/infusion) and 20 stimulus presentations that were delivered response-independently each day for 5 days. For the experimental group (Paired Conditioning), the remifentanil infusions and stimulus presentations were delivered concurrently. The control group (Random control) received the same number of infusions and stimulus presentations, but they were not explicitly paired. For the next 7 sessions (acquisition), we tested the extent to which the stimulus functioned as a conditioned reinforcer by allowing rats to freely respond via a nosepoke for presentations of the remifentanil-associated stimulus only. Prior to each acquisition session, rats received an injection of SNC80 (0, 1.0, or 3.2 mg/kg). We found that rats in the Paired Pavlovian Conditioning group responded for the remifentanil-associated stimulus significantly more than controls (p = 0.005, hp2 = 0.09). In addition, SNC80 increased responding for remifentanil-associated stimuli relative to vehicle treatment (p < 0.001, hp2 = 0.35). These findings demonstrated that a remifentanil-associated stimulus takes on conditioned reinforcing properties and that the conditioned reinforcing properties of the stimulus was augmented by delta opioid receptor activation. This may suggest that delta opioid receptors may play a role in mediating conditioned reinforcing properties.

Naltrindole Exhibits Robust Cardioprotection in Myocardial Ischemia Reperfusion Injury

The predominant opioid receptor subtypes in heart tissue are delta and kappa opioid receptors. Activation of delta and kappa opioid receptors during preconditioning have been shown to reduce infarct size in myocardial ischemia reperfusion (I/R) injury. This cardioprotective effect has been abolished with naloxone (NX), a broad-spectrum opioid receptor antagonist (e.g., delta, kappa, and mu). Previously, we demonstrated that a novel tripeptide, structurally similar to other delta or kappa opioid peptide receptor agonists, when given prior to global ischemia, significantly reduced infarct size and improved post-reperfused cardiac function using a Langendorf rat I/R heart ex vivo model. To identify the opioid receptor subtype responsible for tripeptide's cardioprotective effects, we used naltrindole (NTI), a selective delta opioid receptor antagonist. The cardioprotective effects of tripeptide were blocked by NX, but not NTI. To determine whether the cardioprotective effects of the combination was due to tripeptide or NTI, the effects of NTI and NX were evaluated individually in the same model. Male Sprague-Dawley rats (~300g) were subjected to global I(30min)/R(50min). NX (10 μM, n=6) or NTI (5 μM, n=8) or H2O vehicle (control n= 10) were given to hearts 5 min prior to ischemia and during the first 5 min of reperfusion in a Langendorf perfused model. Left ventricular (LV) cardiac function indices were measured using a pressure transducer. At the end of reperfusion (50min), hearts were frozen, sectioned (2 mm), and stained with 1% triphenyltetrazolium chloride (TTC). Infarcted heart tissue was compared to total tissue weight. Data were evaluated using ANOVA Student-Newman-Keuls post-hoc analysis. Control hearts (n=10) exhibited increased final LV end diastolic pressure (LVEDP) of 60±5 mmHg from baseline values (~8mmHg for all groups) and a mean infarct size of 36 ± 3% at 50 min post-reperfusion. NX showed no significant cardioprotective effect, since final LVEDP (64 ± 5%) and infarct size (35 ± 5%) was similar to control hearts. Surprisingly, the selective delta opioid antagonist, NTI, remarkably improved final LVEDP to 17 ± 3mmHg to near baseline values and reduced infarct size to 7 ± 2% compared to control and NX hearts (both p<0.01). The results indicate that NTI can prevent ischemic injury and restore post-reperfused heart function to pre-ischemic levels. In future studies, we will examine the intracellular mechanisms of NTI responsible for preventing myocardial ischemic injury.

Delta Opioid Receptor Activation with Delta Opioid Peptide [d-Ala2, d-Leu5] Enkephalin Contributes to Synaptic Improvement in Rat Hippocampus against Global Ischemia

Global cerebral ischemia induced by cardiac arrest usually leads to poor neurological outcomes. Numerous studies have focused on ways to prevent ischemic damage in the brain, however clinical therapies are still limited. Our previous studies revealed that delta opioid receptor (DOR) activation with [d-Ala2, d-Leu5] enkephalin (DADLE), a DOR agonist, not only significantly promotes neuronal survival on day 3, but also improves spatial memory deficits on days 5-9 after ischemia. However, the neurological mechanism underlying DADLE-induced cognitive recovery remains unclear. This study first examined the changes in neuronal survival in the CA1 region at the advanced time point (day 7) after ischemia/reperfusion (I/R) injury and found a significant amelioration of damaged CA1 neurons in the rats treated with DADLE (2.5 nmol) when administered at the onset of reperfusion. The structure and function of CA1 neurons on days 3 and 7 post-ischemia showed significant improvements in both the density of the injured dendritic spines and the basic transmission of the impaired CA3-CA1 synapses following DADLE treatment. The molecular changes involved in DADLE-mediated synaptic modulation on days 3 and 7 post-ischemia implied the time-related differential regulation of PKCα-MARCKS on the dendritic spine structure and of BDNF- ERK1/2-synapsin I on synaptic function, in response to ischemic/reperfusion injury as well as to DADLE treatment. Importantly, all the beneficial effects of DADLE on ischemia-induced cellular, synaptic, and molecular deficits were eliminated by the DOR inhibitor naltrindole (2.5 nmol). Taken together, this study suggested that DOR activation-induced protective signaling pathways of PKCα-MARCKS involved in the synaptic morphology and BDNF-ERK-synapsin I in synaptic transmission may be engaged in the cognitive recovery in rats suffering from advanced cerebral ischemia.

Switching of delta opioid receptor subtypes in central amygdala microcircuits is associated with anxiety states in pain

Anxiety is often comorbid with pain. Delta opioid receptors (DORs) are promising targets for the treatment of pain and mental disorders with little addictive potential. However, their roles in anxiety symptoms at different stages of pain are unclear. In the current study, mice with inflammatory pain at the fourth hour following complete Freund’s adjuvant (CFA) injection displayed significant anxiety-like behavior, which disappeared at the seventh day. Combining electrophysiology, optogenetics, and pharmacology, we found that activation of delta opioid receptor 1 (DOR1) in the central nucleus amygdala (CeA) inhibited both the anxiolytic excitatory input from the basolateral amygdala (BLA) and the anxiogenic excitatory input from the parabrachial nucleus (PBN). In contrast, activation of delta opioid receptor 2 (DOR2) did not affect CeA excitatory synaptic transmission in normal and 4-h CFA mice but inhibited the excitatory projection from the PBN rather than the BLA in 7-day CFA mice. Furthermore, the function of both DOR1 and DOR2 was downregulated to the point of not being detectable in the CeA of mice at the 21st day following CFA injection. Taken together, these results suggest that functional switching of DOR1 and DOR2 is associated with anxiety states at different stages of pain via modulating the activity of specific pathways (BLA-CeA and PBN-CeA).

δ-Opioid receptor activation ameliorates lipopolysaccharide-induced inflammation and apoptosis by inhibiting the MAPK/caspase-3 pathway in BV2 microglial cells.

Delta-opioid receptor (DOR) is widely distributed in the central nervous system, and its activation protects against ischaemic/hypoxic brain injury. However, the role of DOR in microglia in ischaemic stroke has not yet been fully investigated. We found that DOR was expressed in both human and mouse cerebral microglia, besides, it was upregulated in activated BV2 microglial cells by immunofluorescence staining and Western blot. DOR activation by the specific agonist TAN-67 significantly enhanced BV2 microglial cell viability and reduced apoptosis, as evidenced by decreased cleaved caspase-3 levels and TdT-mediated aUTP-X nick end labelling (TUNEL) staining after LPS stimulation. Furthermore, activation of DOR significantly inhibited inducible nitric oxide synthase (iNOS) production and dose-dependently inhibited the mRNA and protein expression levels of other pro-inflammatory cytokines, including IL-1β and IL-6, whereas it increased the expression of the anti-inflammatory cytokine IL-10 in LPS-stimulated BV2 microglial cells; these effects were correlated with diminished phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. Moreover, these effects could be reversed by the DOR antagonist naltrindole. DOR activation can activate microglia to switch to the beneficial phenotype and inhibit LPS-induced inflammation and apoptosis via the mitogen-activated protein kinase (MAPK)/caspase-3 pathway in BV2 microglial cells. This study provides new insight into neuroprotection against and treatment of ischaemic stroke.

Peripheral Deltorphin II Inhibits Nociceptors Following Nerve Injury.

Clinical and preclinical studies have revealed that local administration of opioid agonists into peripheral tissue attenuates inflammatory pain. However, few studies have examined whether peripherally restricted opioids are effective in reducing mechanical allodynia and hyperalgesia that usually follows nerve injury. The aim of the present study was to determine whether the mechanical responsiveness of C-fiber mechanical nociceptors innervating skin under neuropathic pain conditions is depressed by direct activation of delta opioid receptors (DORs) on their peripheral terminals. A murine model of peripheral neuropathic pain was induced with a spared nerve (tibial) injury, in which mice survived 7 or 28 days after surgery before electrophysiological testing began. Control groups comprised naïve and sham-operated animals. An ex vivo preparation of mouse plantar skin with attached tibial nerve was used to examine electrophysiologically the effects of the selective DOR agonist, deltorphin II, on the response properties of individual cutaneous C-fiber nociceptors. In contrast to naïve and sham-operated animals, deltorphin II induced an inhibition of the mechanical responsiveness of C-fiber mechanical nociceptors innervating skin under neuropathic conditions. The effects of deltorphin II were concentration-dependent and prevented by pretreatment with naltrindole indicating DOR-mediated inhibitory effects of deltorphin II. Our results provide the first direct evidence for expression of functional DORs on mechanical nociceptors innervating skin in an animal model of neuropathic pain.

Delta opioid receptor activation modulates affective pain and modality-specific pain hypersensitivity associated with chronic neuropathic pain.

Delta opioid receptor (DOR) agonists alleviate nociceptive behaviors in various chronic pain models, including neuropathic pain, while having minimal effect on sensory thresholds in the absence of injury. The mechanisms underlying nerve injury-induced enhancement of DOR function are unclear. We used a peripheral nerve injury (PNI) model of neuropathic pain to assess changes in the function and localization of DORs in mice and rats. Intrathecal administration of DOR agonists reversed mechanical allodynia and thermal hyperalgesia. The dose-dependent thermal antinociceptive effects of DOR agonists were shifted to the left in PNI rats. Administration of DOR agonists produced a conditioned place preference in PNI, but not in sham, animals, whereas the DOR antagonist naltrindole produced a place aversion in PNI, but not in sham, mice, suggesting the engagement of endogenous DOR activity in suppressing pain associated with the injury. GTPγS autoradiography revealed an increase in DOR function in the dorsal spinal cord, ipsilateral to PNI. Immunogold electron microscopy and in vivo fluorescent agonist assays were used to assess changes in the ultrastructural localization of DORs in the spinal dorsal horn. In shams, DORs were primarily localized within intracellular compartments. PNI significantly increased the cell surface expression of DORs within lamina IV-V dendritic profiles. Using neonatal capsaicin treatment, we identified that DOR agonist-induced thermal antinociception was mediated via receptors expressed on primary afferent sensory neurons but did not alter mechanical thresholds. These data reveal that the regulation of DORs following PNI and suggest the importance of endogenous activation of DORs in regulating chronic pain states.

Delta opioid peptide [d-Ala2, d-Leu5] enkephalin confers neuroprotection by activating delta opioid receptor-AMPK-autophagy axis against global ischemia

BackgroundIschemic stroke poses a severe risk to human health worldwide, and currently, clinical therapies for the disease are limited. Delta opioid receptor (DOR)-mediated neuroprotective effects against ischemia have attracted increasing attention in recent years. Our previous studies revealed that DOR activation by [d-Ala2, d-Leu5] enkephalin (DADLE), a selective DOR agonist, can promote hippocampal neuronal survival on day 3 after ischemia. However, the specific molecular and cellular mechanisms underlying the DOR-induced improvements in ischemic neuronal survival remain unclear.ResultsWe first detected the cytoprotective effects of DADLE in an oxygen–glucose deprivation/reperfusion (OGD/R) model and observed increased viability of OGD/R SH-SY5Y neuronal cells. We also evaluated changes in the DOR level following ischemia/reperfusion (I/R) injury and DADLE treatment and found that DADLE increased DOR levels after ischemia in vivo and vitro. The effects of DOR activation on postischemic autophagy were then investigated, and the results of the animal experiment showed that DOR activation by DADLE enhanced autophagy after ischemia, as indicated by elevated LC3 II/I levels and reduced P62 levels. Furthermore, the DOR-mediated protective effects on ischemic CA1 neurons were abolished by the autophagy inhibitor 3-methyladenine (3-MA). Moreover, the results of the cell experiments revealed that DOR activation not only augmented autophagy after OGD/R injury but also alleviated autophagic flux dysfunction. The molecular pathway underlying DOR-mediated autophagy under ischemic conditions was subsequently studied, and the in vivo and vitro data showed that DOR activation elevated autophagy postischemia by triggering the AMPK/mTOR/ULK1 signaling pathway, while the addition of the AMPK inhibitor compound C eliminated the protective effects of DOR against I/R injury.ConclusionDADLE-evoked DOR activation enhanced neuronal autophagy through activating the AMPK/mTOR/ULK1 signaling pathway to improve neuronal survival and exert neuroprotective effects against ischemia.