Delta-opioid Receptor Antagonist Naltrindole Research Articles

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.

Effects of the acute administration of delta-opioid receptor ligands on the excitability of rat hippocampal glutamate and brainstem monoamine neurons in vivo.

It was previously reported that the delta opioid receptor (DOR) agonist SNC80 and antagonist naltrindole modulate the excitability of hippocampal glutamate neurons in primary cultures. The present study aimed to investigate the acute effects of these ligands on the firing activity of hippocampal cornu ammonis 1/3 (CA1/3) glutamate, dorsal raphe nucleus (DRN) serotonin (5-HT), locus coeruleus (LC) noradrenaline, and ventral tegmental area (VTA) dopamine neurons in in vivo conditions. Adult Wistar male rats were used. SNC80 and naltrindole were administered intravenously. Neuronal firing activity was assessed using extracellular single-unit electrophysiology. SNC80, administered first at 1-3 mg/kg, dose-dependently inhibited CA1/3 glutamate, DRN 5-HT, and VTA dopamine neurons. Naltrindole, administered at 1-3 mg/kg after SNC80, did not have any additional effect. Naltrindole, administered first at 1-3 mg/kg, stimulated DRN 5-HT neurons in a dose-dependent manner; this stimulation was dose-dependently reversed by 1-3 mg/kg of SNC80. SNC80 and naltrindole inhibited LC noradrenaline neurons when only they were co-administered at 3 mg/kg, and only when SNC80 was administered first. In conclusion, DOR ligands alter the firing activity of hippocampal glutamate and brainstem monoamine neurons in in vivo conditions. The psychoactive effects of DOR ligands, reported in previous studies, might be explained, at least in part, by their ability to modulate the firing activity of hippocampal glutamate and brainstem monoamine neurons.

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.

Supraspinal melatonin MT2 receptor agonism alleviates pain via a neural circuit that recruits mu opioid receptors.

Melatonin, through its G protein-coupled receptor (GPCR) (MTNR1B gene)MT2 , is implicated in analgesia, but the relationship between MT2 receptors and the opioid system remains elusive. In a model of rodent neuropathic pain (spared nerve injured [SNI]), the selective melatonin MT2 agonist UCM924 reversed the allodynia (a pain response to a non-noxious stimulus), and this effect was nullified by the pharmacological blockade or genetic inactivation of the mu opioid receptor (MOR), but not the delta opioid receptor (DOR). Indeed, SNI MOR, but not DOR knockout mice, did not respond to the antiallodynic effects of the UCM924. Similarly, the nonselective opioid antagonist naloxone and the selective MOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the effects of UCM924 in SNI rats, but not the DOR antagonist naltrindole (NTI). Electrophysiological recordings in the rostral-ventromedial medulla (RVM) revealed that the typical reduction of the firing activity of pronociceptive ON-cells, and the enhancement of the firing of the antinociceptive OFF-cells, induced by the microinjection of the MT2 agonist UCM924 into the ventrolateral periaqueductal gray (vlPAG) were blocked by MOR, but not DOR, antagonism. Immunohistochemistry studies showed that MT2 receptors are expressed in both excitatory (CaMKIIα+ ) and inhibitory (GAD65+ ) neuronal cell bodies in the vlPAG (~2.16% total), but not RVM. Only 0.20% of vlPAG neurons coexpressed MOR and MT2 receptors. Finally, UCM924 treatment induced an increase in the enkephalin precursor gene (PENK) in the PAG of SNI mice. Collectively, the melatonin MT2 receptor agonism requires MORs to exert its antiallodynic effects, mostly through an interneuronal circuit involving MOR and MT2 receptors.

Delta Opioid Receptor (DOR) Agonist Activity of Buprenorphine in Mice

Buprenorphine is an approved method of treatment for opioid use disorder (OUD), and it is under investigation for cocaine use disorder-- for which there are no FDA-approved, pharmacological treatments. Buprenorphine is considered a mixed efficacy opioid ligand, and its partial agonist activity at the mu opioid receptor (MOR) is thought to mediate the therapeutic effects for OUD. There is also evidence that it acts as a kappa opioid receptor (KOR) and delta opioid receptor (DOR) antagonist and a nociception/orphanin FQ receptor (NOPR) agonist. However, one recent report found buprenorphine to act as a partial DOR agonist in cells expressing human opioid receptors (Bidlack et al., 2018). The goal of the present study was to investigate the in vivo activity of buprenorphine at DOR in rodents. Male C57BL6/N mice were used to evaluate the effects of buprenorphine on pentylenetetrazol (PTZ)-induced convulsive activity and in the mouse forced swim test. Buprenorphine did not produce overt convulsions in mice but dose-dependently increased PTZ-induced convulsions with 32 mg/kg buprenorphine as the maximally effective dose, and these effects were attenuated by the DOR antagonist naltrindole. Also, buprenorphine (0.01-0.1 mg/kg) decreased immobility in the mouse forced swim test, which was blocked by pretreatment with naltrindole. Together, these data demonstrate that buprenorphine produces DOR-mediated antidepressant-like effects and potentiates PTZ-induced convulsions, suggesting that buprenorphine acts as a partial DOR agonist in mice. Interestingly, there is a large separation between doses that produce potential antidepressant-like effects and pro-convulsive effects. Future studies will investigate whether this large separation between DOR-mediated effects are due to the complex pharmacology of buprenorphine. Further, these data offer additional insight into the activity of buprenorphine, which could help elucidate the mechanism needed for successful treatment of opioid or cocaine use disorder. These studies were supported in part by R21 DA041565 and DA007315.

Delta-Opioid Receptors Play a Role in the Control of Selected Parameters Related to Stress and Brain Plasticity Under Non-stress and/or Stress Conditions.

There is some evidence that delta-opioid receptors may be involved in the brain processes related to neuroprotection. The aim of the present studies was to test the hypothesis that endogenous opioid peptides acting via delta-opioid receptors can protect against stress-induced changes in factors related to brain plasticity and stress hormone release. Forty male adult Wistar rats were used. Half of the animals were exposed to sustained partial restraint stress (hypokinesis) lasting48h. Rats were treated with vehicle (isotonic saline) or the delta-opioid receptor antagonist naltrindole (3mg/kg/ml, s.c.) six times a day. The stressfulness of the model was confirmed by increased plasma concentrations of corticosterone and prolactin, the increase in anxiety behavior in the open field test, as well as the reduction of BrdU incorporation into newly formed DNA in the hippocampus. Treatment with naltrindole potentiated the stress-induced rise in aldosterone concentrations. The blockade of delta-opioid receptors resulted in a decrease in hippocampal BDNF gene expression independently of control or stress conditions. Treatment with naltrindole enhanced plasma concentrations of copeptin, a stable precursor of vasopressin. In conclusion, these results suggest that endogenous opioid peptides might play an inhibitory role in aldosterone release under stress conditions and in the control of vasopressin release independently of stress exposure. Endogenous opioids might stimulate hippocampal gene expression of the important neurotrophic factor BDNF via delta-opioid receptors.

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.

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.

Characterization of Delta Opioid Receptor Activity of MMP2200, a Mixed Efficacy Mu and Delta Opioid Receptor Agonist

Pharmacological modulation of the delta opioid receptor (DOR) in conjunction with a mu‐opioid receptor (MOR) agonist produces antinociceptive effects with potentially a better side effect profile. MMP2200 (a.k.a.Lactomorphin) is a CNS permeable, glycosylated peptide and an agonist at both MORs and DORs. Previous reports characterized the MOR agonist effects of MMP2200; however, the DOR agonist effects of MMP2200in vivo have not been explored. DOR agonists, such as SNC80, have been shown to produce convulsions and discriminative stimulus effects in rodents. Therefore, based on the DOR agonist activity of MMP2200, we hypothesized that MMP2200 would produce DOR‐mediated convulsions in mice and SNC80‐like discriminative stimulus effects in rats. Convulsions were evaluated by continual observation for up to 60 min in an observation cage. For SNC80 discrimination experiments, rats were trained to discriminate an injection of 3.2 mg/kg SNC80 from saline under a fixed ratio 10 schedule of food reinforcement, and different doses of SNC80 or MMP2200 were substituted to evaluate percent generalization and response rates. MMP2200 produced brief, non‐lethal convulsions in C57BL/6N, but not ICR, mice that were blocked by the DOR antagonist naltrindole and by genetic deletion of DOR, indicating that MMP2200‐induced convulsions are DOR mediated. Following acute drug administration, mice were then given a second dose of the DOR agonist 24 h later. These mice were tolerant to convulsions induced by SNC80, but not to MMP2200‐induced convulsions. Tolerance to the convulsive effects of MMP2200 was apparent 8 h following the initial injection. SNC80 induced convulsions were enhanced in mice lacking arrestin 2 but were unaltered in mice lacking arrestin 3. However, MMP2200‐induced convulsions were attenuated in mice lacking either arrestin 2 or 3. In rats trained to discriminate SNC80, MMP2200 fully generalized to the discriminative stimulus effects of SNC80 and only slightly decreased rates of responding. Overall, these results demonstrate that the dual MOR/DOR agonist MMP2200 produces many behavioral effects that are similar to the prototypical DOR agonist SNC80. Interestingly, there are some distinct differences in these DOR agonist effects, which may be mediated by concurrent MOR agonist activity with MMP2200 or by activating differential intracellular signaling molecules downstream of DOR activation. Future studies will evaluate the effects of DOR agonists and signaling pathways mediating these effects.

The opioid receptor positive allosteric modulator BMS‐986187 enhances some, but not all, delta‐opioid receptor mediated behaviors

Activation of the delta opioid receptor (DOR) by orthosteric agonists produces antinociception, antihyperalgesia, antidepressant‐like effects, and convulsions in animal models. Positive allosteric modulators (PAMs) bind to a receptor site distinct from the orthosteric binding site and can augment the activity of the endogenous system. Therefore, a DOR PAM could be an effective strategy for eliciting potential therapeutic effects without convulsions. To evaluate this hypothesis, we investigated the ability of the opioid receptor PAM BMS‐986187 to elicit DOR‐mediated behaviors in mice alone or in combination with the DOR orthosteric agonist SNC80 and evaluated the potential contribution of multiple opioid receptor subtypes to the observed behavioral effects. Antidepressant‐like effects were evaluated in the mouse forced swim test and convulsions were evaluated by continuous observation. The contribution of DORs and mu‐opioid receptors (MORs) was evaluated through pharmacological and genetic manipulation of the receptor subtypes. BMS‐986187 alone produced antidepressant‐like effects without convulsions. These antidepressant‐like effects were blocked by the DOR antagonist naltrindole and absent in DOR knockout mice, indicating a DOR‐mediated effect. The antidepressant‐like effects of BMS‐986187 were enhanced in MOR knockout mice and following betaFNA pretreatment, suggesting that activation of MORs functionally inhibited DOR‐mediated antidepressant‐like effects. BMS‐986187 increased the potency of the orthosteric DOR agonist SNC80 to decrease immobility in the forced swim test but not to elicit convulsions. In addition, SNC80, but not BMS‐986187, enhanced the convulsive effects of pentylenetetrazol. Overall, these findings suggest that BMS‐986187 has activity at DORs and MORs in vivo and enhances some, but not all, behavioral effects of DOR agonists.Support or Funding InformationThis work was funded in part by a Research Starter Grant from the PhRMA Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of the discriminative stimulus effects of the delta‐opioid receptor agonist, SNC80, in rats

Delta opioid receptor (DOR) agonists produce antihyperalgesia and antidepressant‐like effects, but they also induce convulsions limiting their clinical development. Recently, some work has shown that not all DOR agonists produce convulsions in rodents. Therefore, the current study aimed to further evaluate the pharmacological properties of these DOR ligands using a drug discrimination paradigm in male Sprague‐Dawley rats. The drug discrimination assay is useful for identifying compounds with similar interoceptive properties and evaluating the pharmacological activity of ligands in vivo. For this study, rats were trained to discriminate 3.2 mg/kg SNC80 from saline on a fixed ratio (FR) 10 schedule of reinforcement in a food‐reinforced, drug discrimination paradigm. Substitution patterns with purported DOR agonists AZD2327, DPI287, KNT127, and MMP2200 were evaluated as well as profiles of antagonism following pretreatment with naltrindole. Agonists that are structurally similar to SNC80, AZD2327 and DPI287, generalized to SNC80 while structurally unrelated agonists, KNT127 and MMP2200, did not. Morphine, ketamine, desipramine, cocaine, and nicotine failed to generalize to SNC80 up to doses that suppressed responding; however, amphetamine produced partial generalization to the discriminative stimulus effects of SNC80. The DOR antagonist naltrindole produced parallel, rightward shifts in the SNC80 dose effect curve. Overall, these data suggest that interoceptive effects differ among DOR agonists, which may contribute to their distinct profiles of behavioral activity and potentially diverse mechanisms of action. Future studies will further probe the pharmacological and/or signaling mechanisms that differentiate the behavioral effects of DOR agonists.Support or Funding InformationThis work is supported in part by R01 DA042092 (EMJ) and the IRPs of NIDA and NIAAA (KCR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The initial fall in arterial pressure evoked by endotoxin is mediated by the ventrolateral periaqueductal gray.

This study tested the hypothesis that the initial fall in arterial pressure evoked by lipopolysaccharide (LPS) is mediated by the ventrolateral column of the midbrain periaqueductal gray region (vlPAG). To test this hypothesis, the local anaesthetic lidocaine (2%; 0.1μL, 0.2μL or 1.0μL), the delta opioid receptor antagonist naltrindole (2nmol) or saline was microinjected into the vlPAG of isoflurane-anaesthetized rats bilaterally and LPS (1mg/kg) or saline was administered intravenously 2min later. Both lidocaine and naltrindole inhibited LPS-evoked hypotension significantly but did not affect arterial pressure in saline-treated control animals. Neither lidocaine nor naltrindole altered heart rate significantly in either LPS-treated or control animals. Microinjection of lidocaine or naltrindole into the dorsolateral PAG was ineffective. These data indicate that the vlPAG plays an important role in the initiation of endotoxic hypotension and further show that delta opioid receptors in the vlPAG participate in the response.

The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors.

Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially.

Ferulic Acid Alleviates Neuropathic Pain: Involvement of Descending Monoaminergic System and Opioid Receptors

Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress‐related depression and respond to similar treatment. FA is a major active component of angelica sinensis and has been reported to exert antidepressant‐like effects; however, it remains unknown whether resveratrol ameliorate CCI‐induced neuropathic pain. Chronic treatment with FA (10, 20, 40 and 80 mg/kg) twice daily for 3 weeks ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal monoamine levels. Pharmacological depletion of spinal noradrenaline (NA) or serotonin (5‐HT) by 6‐hydroxydopamine (6‐OHDA) or p‐chlorophenylalanine (PCPA) completely blocked FA's effects on mechanical allodynia or thermal hyperalgesia, respectively. The anti‐allodynic action of FA on mechanical stimuli was prevented by co‐treatment with beta2‐adrenoceptor antagonist ICI 118,551, or by co‐treatment with delta‐opioid receptor antagonist naltrindole. Moreover, the anti‐hyperalgesia on thermal stimuli induced by FA was blocked by co‐treatment with 5‐HT1A receptor antagonist WAY‐100635, or with the irreversible mu‐opioid receptor antagonist beta‐funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via modulation of descending monoaminergic system that coupled with spinal b2‐ and 5‐HT1A receptors and the downstream opioid receptors.

Inhibition of DOR prevents remifentanil induced postoperative hyperalgesia through regulating the trafficking and function of spinal NMDA receptors in vivo and in vitro.

Several studies have demonstrated that intraoperative remifentanil infusions have been associated with opioid-induced hyperalgesia (OIH). Activation of delta opioid receptor (DOR) and augmentation of N-methyl-d-aspartate (NMDA) receptor expression and function may play an important role in the development of OIH. The aim of this study was to investigate whether DOR inhibition could prevent remifentanil-induced hyperalgesia via regulating spinal NMDA receptor expression and function in vivo and in vitro. A rat model of remifentanil-induced postoperative hyperalgesia was performed with the DOR agonist deltorphin-deltorphin II or the DOR antagonist naltrindole injected intrathecally 10 min before remifentanil infusion. Mechanical and thermal hyperalgesia were measured at -24h, 2, 6, 24 and 48 h after remifentanil infusion. Western blot was applied to detect the membrane and total expression of DOR and NMDA receptor subunits (NR1, NR2A and NR2B) in spinal cord L4-L6 segments. In addition, whole-cell patch-clamp recording was used to investigate the effect of DOR inhibition on NMDA receptor-induced current in spinal cord slices in vitro. We found that membrane trafficking of DOR, NR1 and NR2B subunits in the spinal cord increased after remifentanil administration and surgery. The DOR antagonist naltrindole could attenuate mechanical and thermal hyperalgesia without affecting baseline nociceptive threshold, reduce membrane expression of DOR and decrease the membrane and total expressions of NR1 and NR2B subunits. Furthermore, the amplitude and the frequency of NMDA receptor-induced current were significantly increased by remifentanil incubation in neurons of the dorsal horn, which was reversed by the application of naltrindole. The above results indicate that inhibition of DOR could significantly inhibit remifentanil-induced hyperalgesia via modulating the total protein level, membrane trafficking and function of NMDA receptors in the dorsal horn of spinal cord, suggesting that naltrindole could be a potential anti-hyperalgesic agent for treating OIH.

Delta-opioid receptor blockade in the ventral pallidum increases perceived palatability and consumption of saccharin solution in rats

The ventral pallidum (VP) is involved in ingestive behaviour. It receives dense GABAergic projections from the nucleus accumbens. GABAergic terminals in the VP co-express enkephalin, an endogenous ligand of delta-opioid receptors. The role of the delta-opioid receptors in the VP in the context of ingestive behaviour remains unclear, in contrast to the well-understood involvement of the mu-opioid receptors. We used the single-bottle test to examine the effects of VP microinjections of the delta-opioid receptor antagonist naltrindole on consumption of a saccharin solution. Naltrindole injections significantly increased the intake of saccharin, but not water, during a 2-h test session. We also investigated perceived palatability of saccharin using a taste reactivity test. The drug treatments increased ingestive responses to intraorally infused saccharin. Further experimentation explored the role of VP delta-opioid receptors in behavioural responses to saccharin that were previously paired with malaise upon the retrieval of conditioned taste aversion (CTA). Naltrindole-injected rats exhibited longer latency for the first occurrence of aversive responses than vehicle-injected control rats. However, there was no between-group difference in total aversive responses. These results suggest that naltrindole injections into the VP induce an enhancement of perceived palatability of a normally preferred saccharin solution, and thereby facilitate consumption of the solution. On the other hand, delayed aversive responses to the conditioned aversive saccharin suggest that the delta-opioid receptors in the VP mediate the initiation of aversive taste reactivity responses to the conditioned stimulus upon CTA retrieval.

Tolerance develops to the antiallodynic effects of the peripherally acting opioid loperamide hydrochloride in nerve-injured rats

Peripherally acting opioids are potentially attractive drugs for the clinical management of certain chronic pain states due to the lack of centrally mediated adverse effects. However, it remains unclear whether tolerance develops to peripheral opioid analgesic effects under neuropathic pain conditions. We subjected rats to L5 spinal nerve ligation (SNL) and examined the analgesic effects of repetitive systemic and local administration of loperamide hydrochloride, a peripherally acting opioid agonist. We found that the inhibition of mechanical hypersensitivity, an important manifestation of neuropathic pain, by systemic loperamide (1.5mg/kg subcutaneously) decreased after repetitive drug treatment (tolerance-inducing dose: 0.75 to 6.0mg/kg subcutaneously). Similarly, repeated intraplantar injection of loperamide (150μg/50μL intraplantarly) and D-Ala2-MePhe4-Glyol5 enkephalin (300μg/50μL), a highly selective mu-opioid receptor (MOR) agonist, also resulted in decreased inhibition of mechanical hypersensitivity. Pretreatment with naltrexone hydrochloride (5mg/kg intraperitoneally) and MK-801 (0.2mg/kg intraperitoneally) attenuated systemic loperamide tolerance. Western blot analysis showed that repetitive systemic administration of morphine (3mg/kg subcutaneously), but not loperamide (3mg/kg subcutaneously) or saline, significantly increased MOR phosphorylation in the spinal cord of SNL rats. In cultured rat dorsal root ganglion neurons, loperamide dose-dependently inhibited KCl-induced increases in [Ca2+]i. However, this drug effect significantly decreased in cells pretreated with loperamide (3μM, 72hours). Intriguingly, in loperamide-tolerant cells, the delta-opioid receptor antagonist naltrindole restored loperamide’s inhibition of KCl-elicited [Ca2+]i increase. Our findings indicate that animals with neuropathic pain may develop acute tolerance to the antiallodynic effects of peripherally acting opioids after repetitive systemic and local drug administration.

Curcumin exerts antinociceptive effects in a mouse model of neuropathic pain: Descending monoamine system and opioid receptors are differentially involved

Curcumin, a phenolic compound present in Curcuma longa, has been reported to exert antinociceptive effects in some animal models, but the mechanisms remain to be elucidated. This work aimed to investigate the antinociceptive action of curcumin on neuropathic pain and the underlying mechanism(s). Chronic constriction injury (CCI), a canonical animal model of neuropathic pain, was produced by loosely ligating the sciatic nerve in mice and von Frey hair or hot plate test was used to assess mechanical allodynia or thermal hyperalgesia (to heat), respectively. Chronic, but not acute, curcumin treatment (5, 15 or 45 mg/kg, p.o., twice per day for three weeks) alleviated mechanical allodynia and thermal hyperalgesia in CCI mice, accompanied by increasing spinal monoamine (or metabolite) contents. Chemical ablation of descending noradrenaline (NA) by 6-hydroxydopamine (6-OHDA), or depletion of descending serotonin by p-chlorophenylalanine (PCPA), abolished curcumin’s antinociceptive effect on mechanical allodynia or thermal hyperalgesia, respectively. The anti-allodynic action of curcumin on mechanical stimuli was totally blocked by chronic co-treatment with the β 2-adrenoceptor antagonist ICI 118,551, or by acute co-treatment with the delta-opioid receptor antagonist naltrindole. Meanwhile, co-treatment with the 5-HT 1A receptor antagonist WAY-100635 chronically, or with the irreversible mu-opioid receptor antangonist β-funaltrexamine acutely, completely abrogated the anti-hyperalgesic action of curcumin on thermal stimuli. Collectively, these findings indicate that the descending monoamine system (coupled with spinal β 2-adrenoceptor and 5-HT 1A receptor) is critical for the modality-specific antinociceptive effect of curcumin in neuropathic pain. Delta- and mu-opioid receptors are likely rendered as downstream targets, accordingly. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.

Nucleus accumbens facilitates nociception

We have previously demonstrated an opioid link in nucleus accumbens (NAc) that mediates antinociception produced by a novel ascending pain modulation pathway. For example, noxious stimulation induces heterosegmental antinociception that is mediated by both mu- and delta-opioid receptors in NAc. However, spinal intrathecal administration of the mu-receptor agonist [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO) also induces heterosegmental antinociception. The aim of the present study in the rat was to identify the intra-NAc opioid receptors that mediate the antinociceptive effects of spinally administered DAMGO and also to determine the effect of NAc efferent activity on nociception. Intra-NAc administration of either the mu-opioid receptor antagonist Cys 2,Tyr 3, Orn 5,Pen 7amide (CTOP) or the delta-opioid receptor antagonist naltrindole blocked the antinociceptive effect of spinally administered DAMGO on the jaw-opening reflex (JOR). Injection of quaternary lidocaine (QX-314) attenuated the JOR, suggesting that the output of NAc is pronociceptive. In support of this, intra-NAc injection of the excitatory amino acid agonist kainate enhanced the JOR. Thus, it is possible to modulate activity in NAc to bidirectionally attenuate or enhance nociception, suggesting a potential role for NAc in setting nociceptive sensitivity.

Delta opioid peptide [ d-Ala2, d-Leu5] enkephalin (DADLE) triggers postconditioning against transient forebrain ischemia

Preconditioning with selective delta opioid peptide [ d-Ala2, d-Leu5] enkephalin (DADLE) provides ischemic tolerance following transient forebrain ischemia in rats. However, whether DADLE postconditioning retains its neuroprotective efficacy and the underlying molecular mechanism in ischemic brain is largely unknown. We investigated DADLE postconditioning protection of hippocampal CA1 neurons against transient forebrain ischemia. 6 days after being implanted with cannula at the right lateral ventricle, rats underwent 10 min of forebrain ischemia by four vessel occlusion. Hippocampal CA1 neuronal survival and degeneration were measured in the hippocampi of rats at 3 days after ischemia. The behavioral and cognitive improvements of DADLE treatment in rats were also evaluated on days 5–9 using open-field and Morris water maze tests. The results showed that DADLE at doses of 0.25 and 2.5 nmol, but not 25 nmol, could significantly protect CA1 neurons against ischemia/reperfusion injury. Co-administration with the delta-opioid receptor antagonist naltrindole or pretreatment with the Akt antagonist LY294002 completely abolished the DADLE postconditioning effect. Furthermore, DADLE postconditioning exhibited cognitive benefits in rats with transient forebrain ischemia. The study thus suggested a therapeutic opportunity of postconditioning neuroprotection by DADLE and also provided important information in understanding the mechanism of DADLE action in the ischemic brain.