Potentiation of μ-opioid receptor-mediated signaling by ketamine

Ketamine, a clinically relevant drug, has been shown to enhance opioid-induced analgesia and prevent hyperalgesia. However, the molecular mechanisms involved are not clearly understood. As previous studies found that activation of opioid receptors leads to the phosphorylation of mitogen-activated protein kinases, we investigated whether ketamine could modulate μ-opioid receptor (μOR)-mediated ERK1/2 phosphorylation. We find that acute treatment with ketamine enhances (～2- to 3-fold) the levels of opioid-induced ERK1/2 phosphorylation in recombinant as well as cells endogenously expressing μOR. Interestingly, we find that in the absence of ketamine ERK1/2 signaling is desensitized 10 min after opioid exposure whereas in its presence significant levels (～3-fold over basal) are detected. In addition, ketamine increases the rate of resensitization of opioid-mediated ERK1/2 signaling (15 min in its presence vs. 30 min in its absence). These results suggest that ketamine increases the effectiveness of opiate-induced signaling by affecting multiple mechanisms. In addition, these effects are observed in heterologous cells expressing μOR suggesting a non-NMDA receptor-mediated action of ketamine. Together this could, in part, account for the observed effects of ketamine on the enhancement of the analgesic effects of opiates as well as in the duration of opiate-induced analgesia.     

Repeated exposure to cocaine alters medial prefrontal cortex dopamine D₂-like receptor modulation of glutamate and dopamine neurotransmission within the mesocorticolimbic system

Repeated exposure to cocaine progressively increases drug-induced locomotor activity, which is termed behavioral sensitization. Previous studies have demonstrated that sensitization to cocaine is associated with a decrease in dopamine D? receptor function in the medial prefrontal cortex. The present report tested the hypothesis that reduced medial prefrontal cortex D? receptor function as a result of repeated cocaine exposure results in augmented excitatory transmission to the nucleus accumbens and ventral tegmental area, possibly as a partial result of enhanced inhibition of local dopamine release. Dual probe microdialysis experiments were conducted in male Sprague-Dawley rats 1, 7 or 30 days following the last of four daily injections of saline (1.0 mL/kg) or cocaine (15 mg/kg). Infusion of quinpirole (0.01, 1.0 and 100 μM), a D?-like receptor agonist, into the medial prefrontal cortex produced a dose-dependent decrease in cortical, nucleus accumbens and ventral tegmental area extracellular glutamate levels in control but not sensitized animals. Quinpirole also reduced basal dopamine levels in the medial prefrontal cortex in sensitized animals following 1 day of withdrawal from cocaine. Following 30 days of withdrawal, quinpirole also reduced dopamine levels in sensitized animals relative to saline controls, but not relative to baseline levels. These findings indicate that the expression of sensitization to cocaine is associated with altered modulation of mesocorticolimbic glutamatergic transmission at the level of the medial prefrontal cortex.     

Inhibition of forebrain μ-opioid receptor signaling by low concentrations of rimonabant does not require cannabinoid receptors and directly involves μ-opioid receptors

Increasing number of publications shows that cannabinoid receptor 1 (CB(1)) specific compounds might act in a CB(1) independent manner, including rimonabant, a potent CB(1) receptor antagonist. Opioids, cannabinoids and their receptors are well known for their overlapping pharmacological properties. We have previously reported a prominent decrease in μ-opioid receptor (MOR) activity when animals were acutely treated with the putative endocannabinoid noladin ether (NE). In this study, we clarified whether the decreased MOR activation caused by NE could be reversed by rimonabant in CB(1) receptor deficient mice. In functional [(35)S]GTPγS binding assays, we have elucidated that 0.1mg/kg of intraperitoneal (i.p.) rimonabant treatment prior to that of NE treatment caused further attenuation on the maximal stimulation of Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO), which is a highly specific MOR agonist. Similar inhibitory effects were observed when rimonabant was injected i.p. alone and when it was directly applied to forebrain membranes. These findings are cannabinoid receptor independent as rimonabant caused inhibition in both CB(1) single knockout and CB(1)/CB(2) double knockout mice. In radioligand competition binding assays we highlighted that rimonabant fails to displace effectively [(3)H]DAMGO from MOR in low concentrations and is highly unspecific on the receptor at high concentrations in CB(1) knockout forebrain and in their wild-type controls. Surprisingly, docking computational studies showed a favorable binding position of rimonabant to the inactive conformational state of MOR, indicating that rimonabant might behave as an antagonist at MOR. These findings were confirmed by radioligand competition binding assays in Chinese hamster ovary cells stably transfected with MOR, where a higher affinity binding site was measured in the displacement of the tritiated opioid receptor antagonist naloxone. However, based on our in vivo data we suggest that other, yet unidentified mechanisms are additionally involved in the observed effects.     

Facilitation of μ-opioid receptor activity by preventing δ-opioid receptor-mediated codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR?(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane is?a potential strategy to improve opioid analgesic therapies.     

Selective interactions of spinophilin with the C-terminal domains of the δ- and μ-opioid receptors and G proteins differentially modulate opioid receptor signaling

Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners (Leontiadis et al., 2009, Cell Signal. 21, 1218-1228; Georganta et al., 2010, Neuropharmacology, 59(3), 139-148). In the present study we demonstrate that spinophilin a dendritic-spine enriched scaffold protein associates with δ- and μ-opioid receptors (δ-ΟR, μ-OR) constitutively in HEK293 an interaction that is altered upon agonist administration and enhanced upon forskolin treatment for both μ-OR and δ-ΟR. Spinophilin association with the opioid receptors is mediated via the third intracellular loop and a conserved region of the C-terminal tails. The portion of spinophilin responsible for interaction with the δ-OR and μ-OR is narrowed to a region encompassing amino acids 151-444. Spinophilin, RGS4, Gα and Gβγ subunits of G proteins form a multi-protein complex using specific regions of spinophilin and a conserved amino acid stretch of the C-terminal tails of both δ-μ-ORs. Expression of spinophilin in HEK293 cells potentiated DPDPE-mediated adenylyl-cyclase inhibition of δ-OR leaving unaffected the levels of cAMP accumulation mediated by the μ-OR. Moreover, measurements of extracellular signal regulated kinase (ERK1,2) phosphorylation indicated that the presence of spinophilin attenuated agonist-driven ERK1,2 phosphorylation mediated upon activation of the δ-OR but not the μ-OR. Collectively, these findings suggest that spinophilin associates with both δ- and μ-ΟR and G protein subunits in HEK293 cells participating in a multimeric signaling complex that displays a differential regulatory role in opioid receptor signaling.     

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via G_s/olf or G_i/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gα_q activation in rat brain membranes was investigated. Agonist-induced Gα_q activation was assessed by increase in guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to Gα_q determined by [³⁵S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gα_q functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D₁-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D₂-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D₁-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT_2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT_2A receptor, but not by dopamine D₁-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D₁-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT_2A receptor has been mentioned.     

Stabilization of μ-opioid receptor facilitates its cellular translocation and signaling

The G protein-coupled μ-opioid receptor (μ-OR) mediates the majority of analgesia effects for morphine and other pain relievers. Despite extensive studies of its structure and activation mechanisms, the inherently low maturation efficiency of μ-OR represents a major hurdle to understanding its function. Here we computationally designed μ-OR mutants with altered stability to probe the relationship between cell-surface targeting, signal transduction, and agonist efficacy. The stabilizing mutation T315Y enhanced μ-OR trafficking to the plasma membrane and significantly promoted the morphine-mediated inhibition of downstream signaling. In contrast, the destabilizing mutation R165Y led to intracellular retention of μ-OR and reduced the response to morphine stimulation. These findings suggest that μ-OR stability is an important factor in regulating receptor signaling and provide a viable avenue to improve the efficacy of analgesics.     

Induction of conditioned place preference and dopamine release by salsolinol in posterior VTA of rats: involvement of μ-opioid receptors

Salsolinol (Sal), locally administered into the posterior VTA (pVTA) of rats, produces psychomotor responses and reinforcing effects, probably, through the activation of μ-opioid receptors (MORs). The neurochemical correlates of these phenomena are, however, practically unknown. In this paper, we explore the neurochemical events and the mechanisms involved in these behaviors. To do that, we test the ability of Sal, directly microinjected into the pVTA, to induce conditioned place preference (CPP) and to increase dopamine levels in the nucleus accumbens shell. Bilateral injections of 30 pmol of Sal induced a strong CPP (rats spent around 70% of the total test time), a result that could be explained by the fact that Sal microinjected into the pVTA increased DA levels in the ipsilateral accumbens up to 141% of baseline. The local pretreatment with β-FNA, an antagonist of MORs, prevented this increase, supporting our hypothesis on the involvement of MORs in the Sal-derived effects.     

Differential response to morphine of the oligomeric state of μ-opioid in the presence of δ-opioid receptors

Prolonged morphine treatment induces extensive desensitization of the μ-opioid receptor (μOR) which is the G-protein-coupled receptor that primarily mediates the cellular response to morphine. To date, the molecular mechanism underlying this process is unknown. Here, we have used live cell fluorescence imaging to investigate whether prolonged morphine treatment affects the physical environment of μOR, or its coupling with G-proteins, in two neuronal cell lines. We find that chronic morphine treatment does not change the amount of enhanced yellow fluorescence protein (eYFP)-tagged μOR on the plasma membrane, and only slightly decreases its association with G-protein subunits. Additionally, morphine treatment does not have a detectable effect on the diffusion coefficient of eYFP-μOR. However, in the presence of another family member, the δ-opioid receptor (δOR), prolonged morphine exposure results in a significant increase in the diffusion rate of μOR. Number and brightness measurements suggest that μOR exists primarily as a dimer that will oligomerize with δOR into tetramers, and morphine promotes the dissociation of these tetramers. To provide a plausible structural context to these data, we used homology modeling techniques to generate putative configurations of μOR-δOR tetramers. Overall, our studies provide a possible rationale for morphine sensitivity.     

Dissociation of μ- and δ-opioid inhibition of glutamatergic synaptic transmission in superficial dorsal horn

Background

Although it has been widely accepted that the primary somatosensory (SI) cortex plays an important role in pain perception, it still remains unclear how the nociceptive mechanisms of synaptic transmission occur at the single neuron level. The aim of the present study was to examine whether noxious stimulation applied to the orofacial area evokes the synaptic response of SI neurons in urethane-anesthetized rats using an in vivo patch-clamp technique.

Results

In vivo whole-cell current-clamp recordings were performed in rat SI neurons (layers III-IV). Twenty-seven out of 63 neurons were identified in the mechanical receptive field of the orofacial area (36 neurons showed no receptive field) and they were classified as non-nociceptive (low-threshold mechanoreceptive; 6/27, 22%) and nociceptive neurons. Nociceptive neurons were further divided into wide-dynamic range neurons (3/27, 11%) and nociceptive-specific neurons (18/27, 67%). In the majority of these neurons, a proportion of the excitatory postsynaptic potentials (EPSPs) reached the threshold, and then generated random discharges of action potentials. Noxious mechanical stimuli applied to the receptive field elicited a discharge of action potentials on the barrage of EPSPs. In the case of noxious chemical stimulation applied as mustard oil to the orofacial area, the membrane potential shifted depolarization and the rate of spontaneous discharges gradually increased as did the noxious pinch-evoked discharge rates, which were usually associated with potentiated EPSP amplitudes.

Conclusions

The present study provides evidence that SI neurons in deep layers III-V respond to the temporal summation of EPSPs due to noxious mechanical and chemical stimulation applied to the orofacial area and that these neurons may contribute to the processing of nociceptive information, including hyperalgesia.     

The role of δ-opioid receptor subtypes in cocaine- and methamphetamine-induced place preferences

The effects of δ-receptor antagonists on cocaine- and methamphetamine-induced place preferences were examined in rats. Cocaine- and methamphetamine-induced place preferences were significantly attenuated by naltrindole (NTI: a non-selective δ-opioid receptor antagonist). Furthermore, naltriben (NTB: a selective δ₂-opioid receptor antagonist), but not 7-benzylidenenaltrexone (BNTX: a selective δ₁-opioid receptor antagonist), attenuated the cocaine- and methamphetamine-induce place preferences. These results suggest that δ-opioid receptors, particularly δ₂-opioid receptors, may be involved in the reinforcing effects of cocaine and methamphetamine.     

The mechanism of μ-opioid receptor (MOR)-TRPV1 crosstalk in TRPV1 activation involves morphine anti-nociception,tolerance and dependence

Initiated by the activation of various nociceptors, pain is a reaction to specific stimulus modalities. The μ-opioid receptor (MOR) agonists, including morphine, remain the most potent analgesics to treat patients with moderate to severe pain. However, the utility of MOR agonists is limited by the adverse effects associated with the use of these drugs, including analgesic tolerance and physical dependence. A strong connection has been suggested between the expression of the transient receptor potential vanilloid type 1 (TRPV1) ion channel and the development of inflammatory hyperalgesia. TRPV1 is important for thermal nociception induction, and is mainly expressed on sensory neurons. Recent reports suggest that opioid or TRPV1 receptor agonist exposure has contrasting consequences for anti-nociception, tolerance and dependence. Chronic morphine exposure modulates TRPV1 activation and induces the anti-nociception effects of morphine. The regulation of many downstream targets of TRPV1 plays a critical role in this process, including calcitonin gene-related peptide (CGRP) and substance P (SP). Additional factors also include capsaicin treatment blocking the anti-nociception effects of morphine in rats, as well as opioid modulation of TRPV1 responses through the cAMP-dependent PKA pathway and MAPK signaling pathways. Here, we review new insights concerning the mechanism underlying MOR-TRPV1 crosstalk and signaling pathways and discuss the potential mechanisms of morphine-induced anti-nociception, tolerance and dependence associated with the TRPV1 signaling pathway and highlight how understanding these mechanisms might help find therapeutic targets for the treatment of morphine induced antinociception, tolerance and dependence.     

The mechanism of μ-opioid receptor (MOR)-TRPV1 crosstalk in TRPV1 activation involves morphine anti-nociception,tolerance and dependence

Initiated by the activation of various nociceptors, pain is a reaction to specific stimulus modalities. The μ-opioid receptor (MOR) agonists, including morphine, remain the most potent analgesics to treat patients with moderate to severe pain. However, the utility of MOR agonists is limited by the adverse effects associated with the use of these drugs, including analgesic tolerance and physical dependence. A strong connection has been suggested between the expression of the transient receptor potential vanilloid type 1 (TRPV1) ion channel and the development of inflammatory hyperalgesia. TRPV1 is important for thermal nociception induction, and is mainly expressed on sensory neurons. Recent reports suggest that opioid or TRPV1 receptor agonist exposure has contrasting consequences for anti-nociception, tolerance and dependence. Chronic morphine exposure modulates TRPV1 activation and induces the anti-nociception effects of morphine. The regulation of many downstream targets of TRPV1 plays a critical role in this process, including calcitonin gene-related peptide (CGRP) and substance P (SP). Additional factors also include capsaicin treatment blocking the anti-nociception effects of morphine in rats, as well as opioid modulation of TRPV1 responses through the cAMP-dependent PKA pathway and MAPK signaling pathways. Here, we review new insights concerning the mechanism underlying MOR-TRPV1 crosstalk and signaling pathways and discuss the potential mechanisms of morphine-induced anti-nociception, tolerance and dependence associated with the TRPV1 signaling pathway and highlight how understanding these mechanisms might help find therapeutic targets for the treatment of morphine induced antinociception, tolerance and dependence.     

Chemical coding of neurons expressing δ- and κ-opioid receptor and type I vanilloid receptor immunoreactivities in the porcine ileum

Opioid drugs have profound antidiarrheal and constipating actions in the intestinal tract and are effective in mitigating abdominal pain. Mediators of intestinal inflammation and allergy produce increased mucosal secretion, altered bowel motility and pain due to their ability to evoke enteric secretomotor reflexes through primary afferent neurons. In this study, the distribution of delta- and kappa-opioid receptor (DOR and KOR, respectively) immunoreactivities in chemically identified neurons of the porcine ileum was compared with that of the capsaicin-sensitive type 1 vanilloid receptor (VR1). DOR and VR1 immunoreactivities were observed to be highly localized in choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-positive neurons and nerve fibers of the submucosal and myenteric plexuses and both receptors exhibited frequent colocalization. In the inner submucosal plexus, they also were colocalized in substance P (SP)-positive neurons. Neurons in the outer submucosal plexus expressed DOR immunoreactivity alone or in combination with VR1. KOR-immunoreactive neurons were found only in the myenteric plexus; these cells coexpressed immunoreactivity to ChAT, CGRP, vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS). In addition, some KOR-positive neurons coexpressed immunoreactivities to DOR and VR1. Based on their neurochemical coding, opioid and vanilloid receptor-immunoreactive neurons in the submucosal and myenteric plexuses may include primary afferents and constitute novel therapeutic targets for the palliation of painful intestinal inflammatory, hypersensitivity and dysmotility states.     

ßarrestin1-biased agonism at human δ-opioid receptor by peptidic and alkaloid ligands

We have previously reported on the differential regulation of the human δ-opioid receptor (hDOR) by alkaloid (etorphine) and peptidic (DPDPE and deltorphin I) ligands, in terms of both receptor desensitization and post-endocytic sorting. Since ßarrestins are well known to regulate G protein-coupled receptors (GPCRs) signaling and trafficking, we therefore investigated the role of ßarrestin1 (the only isoform expressed in our cellular model) in the context of the hDOR. We established clonal cell lines of SK-N-BE cells over-expressing ßarrestin1, its dominant negative mutant (ßarrestin1^319-418), and shRNA directed against endogenous ßarrestin1. Interestingly, both binding and confocal microscopy approaches demonstrated that ßarrestin1 is required for hDOR endocytosis only when activated by etorphine. Conversely, functional experiments revealed that ßarrestin1 is exclusively involved in hDOR desensitization promoted by the peptides. Taken together, these results provide substantial evidence for a ßarrestin1-biased agonism at hDOR, where ßarrestin1 is differentially involved during receptor desensitization and endocytosis depending on the ligand.     

μ- and δ-opioid receptor antagonists precipitate similar withdrawal phenomena in butorphanol and morphine dependence

The relative involvement of μ- and δ-opioid receptors in the mediation of butorphanol-, as compared to morphine-, dependence was examined with the use of highly selective antagonists at μ- and δ-opioid receptors. Extracellular fluid levels of glutamate (Glu) and aspartate (Asp) were measured within the pontine locus coeruleus following precipitation of withdrawal from dependence on either butorphanol or morphine in conscious Sprague-Dawley rats. Dependence was induced by intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/μl/h), morphine (26 nmol/μl/h) or saline vehicle (1 μl/h) for 3 days by means of an osmotic minipump. Microdialysis probes (2 mm tip) were inserted into the locus coeruleus 24 h before precipitation of withdrawal by i.c.v. injection of either the μ-opioid receptor antagonist,d-Pen-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; 4.8 nmol/5 μl or 48 nmol/5 μl), or the δ-opioid receptor antagonist, naltrindole (17-cyclopropylmethyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2′3′-indolmorphinan hydrochloride; 48 nmol/5 μl or 100 nmol/5 μl). Baseline levels of Glu ranged from 9.59±1.27 to 12.84 ±3.01 μM in the various treatment groups. Level of Asp were similar. Precipitation of withdrawal by CTOP elicited significant increases of Glu and Asp in both morphine- and butorphanol-dependent rats. Maximal increases in Glu of 425% and 258% above baseline levels were elicited in the first 15 min microdialysis sample following i.c.v. injection of CTOP in morphine- and butorphanol-dependent rats, respectively. Behavioral signs of withdrawal were greater in morphine than butorphanol-dependent groups. The i.c.v. treatment with naltrindole elicited increases in Glu and Asp that were similar, although less marked, than those precipitated by CTOP treatment. Administration of naltrindole produced equivalent signs of withdrawal in both morphine- and butorphanol-dependent rats. Withdrawal from dependence on both morphine and butorphanol is characterized by elevations in coerulear levels of excitatory amino acids. Responses elicited following the use of selective μ- and δ-opioid receptor antagonists to precipitate withdrawal suggest that the role played by these receptors in mediation of the signs and symptoms of withdrawal do not differ greatly between butorphanol- and morphine-dependent rats.     

Antagonistic effect of buprenorphine on the antitussive effect of morphine is mediated via the activation of μ1-opioid receptors

The effect of buprenorphine on the antitussive effect of morphine was examined in mice. Buprenorphine at doses of 0.1,0.3 and 1 mg/kg given i.p. alone have no effects on the % inhibition in the number of capsaicin-induced coughs. However, pretreatment with the same doses of buprenorphine for 2 hr significantly attenuated the antitussive effect of morphine (3 mg/Kg, I.p.). Naloxonazine, a selective μ₁-opioid receptor antagonist, had no effect on the antitussive effect of morphine, but blocks the antagonistic effect of buprenorphine on antitussive effect of morphine. These results suggest that buprenorphine antagonizes the antitussive effect of morphine via the activation of μ₁-opioid receptors.     

Differential mediation of cold water swim stress-induced antinociception by δ-opioid receptor subtypes in diabetic mice

The involvement of δ-opioid receptor subtypes in cold water swim stress (CWSS)-induced antinociception in diabetic mice was compared with that in non-diabetic mice. Three-minute swim stress produced significant antinociception in both diabetic and non-diabetic mice as determined by the tail-pinch test. However, the extent of CWSS-induced antinociception in diabetic mice was significantly greater than that in non-diabetic mice. Pretreatment with naltriben, a selective δ₂-opioid receptor antagonist, significantly attenuated CWSS-induced antinociception in both non-diabetic and diabetic mice. In contrast, although 7-benzylidenenaltrexone, a selective δ₁-opioid receptor antagonist, significantly attenuated CWSS-induced antinociception in diabetic mice, it had no effect in non-diabetic mice. These results suggest that CWSS-induced antinociceotion in non-diabetic mice is mediated by δ₂-opioid receptors, whereas CWSS-induced antinociception in diabetic mice is mediated by both δ₁- and δ₂-opioid receptors. Furthermore, the enhanced CWSS-induced antinociception in diabetic mice may be due to the activation of δ₁-opioid receptors.