Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia

Analgesic effects of delta opioid receptor (DOR) –selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) –knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266–273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II–induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR’s effects was also independent of β-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.     

Regulation of mu opioid receptor internalization by the scaffold protein RanBPM

Mu opioid receptors (MOP) are transducers of the pharmacological effects of many opioid drugs, including analgesia and tolerance/dependence. Previously, we observed increased MOP signaling during postnatal development that was not associated with increased MOP or G protein expression. A yeast two-hybrid screen of a human brain cDNA library using the MOP C-terminus as bait identified RanBPM as a potential MOP-interacting protein. RanBPM has been recognized as a multi-functional scaffold protein that interacts with a variety of signaling receptors/proteins. Co-immunoprecipitation studies in HEK293 cells indicated that RanBPM constitutively associates with MOP. Functionally, RanBPM had no effect on MOP-mediated inhibition of adenylyl cyclase, yet reduced agonist-induced endocytosis of MOP. Mechanistically, RanBPM interfered with β arrestin2-GFP translocation stimulated by MOP but not α_1B-adrenergic receptor activation, indicating selectivity of action. Our findings suggest that RanBPM is a novel MOP-interacting protein that negatively regulates receptor internalization without altering MOP signaling through adenylyl cyclase.     

Contrasting effects of mu opioid receptor and delta opioid receptor deletion upon the behavioral and neurochemical effects of cocaine

Conventional brain microdialysis was used to assess basal and cocaine-induced dopamine (DA) levels in the nucleus accumbens of wildtype (WT) C57BL/6J mice and mice with constitutive deletion of ether mu- or delta-opioid receptors (MOR or DOR knockout [KO], respectively). Locomotor activity was assessed in these same animals. Basal locomotor activity of DOR KO was elevated relative to MOR KO, but did not differ from that of WT mice. DOR mice, but not WT or MOR KO, exhibited a significant increase in activity in response to an injection of saline. The acute administration of cocaine produced a dose-related increase in locomotor activity in the three genotypes. The locomotor activating effects of a low dose (10 mg/kg) of cocaine were enhanced in DOR KO mice whereas the locomotor activating effects of both a low and higher (20 mg/kg) dose of cocaine were reduced in MOR KO animals. Microdialysis studies revealed no difference between genotypes in basal DA levels. Acute administration of cocaine, but not saline, increased DA levels in WT and KO animals. Paradoxically, however, the magnitude of this effect was smaller in DOR KO as compared with that in either WT or MOR KO. These data indicate that constitutive deletion of either MOR or DOR results in contrasting effects upon responsiveness to cocaine, which is consistent with the distinct phenotypes previously described for these mutants.     

Role of agonist-dependent receptor internalization in the regulation of mu opioid receptors

Organotypic cultures and ileal neuromuscular preparations were used to determine (i) whether endogenous release of opioids by electrical stimulation induces mu receptor endocytosis, and (ii) whether and under which conditions ligand-induced mu receptor endocytosis influences the responsiveness of neurons expressing native mu receptors. In longitudinal muscle-myenteric plexus preparations, electrical stimulation at 20 Hz induced a prominent endocytosis of mu receptors in enteric neurons, indicating endogenous release of opioids. A similar massive endocytosis was triggered by exogenous application of the mu receptor agonist, [D-Ala(2),MePhe(4), Gly-ol(5)] enkephalin, whereas exogenous application of morphine was ineffective. [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin and morphine induced a concentration-dependent inhibition of neurogenic cholinergic twitch contractions to electrical stimulation at 0.1 Hz. beta-Chlornaltrexamine shifted to the right the inhibitory curve of both agonists with a concentration-dependent reduction of the maximum agonist response, which is consistent with the existence of spare mu opioid receptors. Under these conditions, the induction of mu receptor endocytosis by exogenously applied [D-Ala(2), MePhe(4),Gly-ol(5)] enkephalin diminished the inhibitory effect of this agonist on twitch contractions and tritiated acetylcholine release. In contrast, there was no reduction of the inhibitory effect of morphine, which failed to induce mu receptor endocytosis, on neurogenic cholinergic response.These results provide the first evidence for the occurrence of mu receptor endocytosis in neurons by endogenously released opioids and show that agonist-dependent mu receptor endocytosis could serve as a mechanism to regulate mu opioid receptor responsiveness to ligand stimulation when the opioid receptor reserve is reduced.     

Ligand-induced mu opioid receptor endocytosis and recycling in enteric neurons

Immunohistochemistry and confocal microscopy were used to investigate endocytosis and recycling of the native mu opioid receptor (muOR) in enteric neurons. Isolated segments of the guinea-pig ileum were exposed to increasing concentrations of muOR agonists at 4 degrees C to allow ligand binding and warming to 37 degrees C for 0 min (baseline) to 6 h in ligand-free medium to allow receptor internalization and recycling. The endogenous ligand, [Met]enkephalin, and [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin (DAMGO), an opioid analog, and the alkaloids, etorphine and fentanyl, induced rapid internalization of muOR immunoreactivity in enteric neurons, whereas morphine did not. muOR internalization was prevented by muOR antagonists. Basal levels of muOR immunoreactivity in the cytoplasm were 10.52+/-2.05%. DAMGO (1 nM-100 microM) induced a concentration-dependent increase of muOR immunofluorescence density in the cytoplasm to a maximum of 84.37+/-2.26%. Translocation of muOR immunoreactivity in the cytoplasm was detected at 2 min, reached the maximum at 15-30 min, remained at similar levels for 2 h, began decreasing at 4 h, and was at baseline values at 6 h. A second exposure to DAMGO (100 nM) following recovery of internalized muOR immunoreactivity at the cell surface induced a translocation of muOR immunoreactivity in the cytoplasm comparable to the one observed following the first exposure (46.89+/-3.11% versus 43.31+/-3.80%). muOR internalization was prevented by hyperosmolar sucrose, phenylarsine oxide or potassium depletion, which inhibit clathrin-mediated endocytosis. muOR recycling was prevented by pre-treatment with bafilomycin A1, an acidotropic agent that inhibits endosomal acidification, but not by the protein synthesis inhibitor, cycloheximide.This study shows that native muOR in enteric neurons undergoes ligand-selective endocytosis, which is primarily clathrin-mediated, and recycles following endosomal acidification. Following recycling, muOR is activated and internalized by DAMGO indicating that recycled receptors are functional.     

Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence

To analyze candidate genes and establish complex genotype-phenotype relationships against a background of high natural genome sequence variability, we have developed approaches to (i) compare candidate gene sequence information in multiple individuals; (ii) predict haplotypes from numerous variants; and (iii) classify haplotypes and identify specific sequence variants, or combinations of variants (pattern), associated with the phenotype. Using the human mu opioid receptor gene (OPRM1) as a model system, we have combined these approaches to test a potential role of OPRM1 in substance (heroin/cocaine) dependence. All known functionally relevant regions of this prime candidate gene were analyzed by multiplex sequence comparison in 250 cases and controls; 43 variants were identified and 52 different haplotypes predicted in the subgroup of 172 African-Americans. These haplotypes were classified by similarity clustering into two functionally related categories, one of which was significantly more frequent in substance-dependent individuals. Common to this category was a characteristic pattern of sequence variants [-1793T-->A, -1699Tins, -1320A-->G, -111C-->T, +17C-->T (A6V)], which was associated with substance dependence. This study provides an example of approaches that have been successfully applied to the establishment of complex genotype-phenotype relationships in the presence of abundant DNA sequence variation.     

Stimulation of endogenous opioid release displaces mu receptor binding in rat hippocampus

Physiological release of endogenous opioids in the rat hippocampus was detected by an in vitro radioligand displacement assay using [3H][D-Ala2,N-methyl-Phe4,glyol5]enkephalin ([3H]DAGO), a mu selective opioid agonist. In this assay, radioligand binding to opioid receptors in the in vitro hippocampal slice was reduced by competition with endogenous opioids released following tissue depolarization. Veratridine-induced opioid release caused displacement of [3H]DAGO that could be blocked by either tetrodotoxin addition or calcium removal from the incubation buffer. Maximal displacement of [3H]DAGO also required the presence of peptidase inhibitors in the incubation buffer. None of the buffer composition changes directly affected [3H]DAGO binding to rat brain membranes. Calcium-dependent displacement of [3H]DAGO binding from mu receptor sites elicited by focal electrical stimulation depended on the intensity and frequency of stimulation and positioning of the electrode in the slice. Maximal displacement of [3H]DAGO binding was observed following high intensity (150-300 microA), high frequency (10-50 Hz) stimulation of the perforant path, a major afferent fiber system to the hippocampus previously shown to contain proenkephalin-derived opioids. Low frequency stimulation (0.1-1 Hz) was ineffective. Stimulation of the mossy fibers (containing both dynorphins and enkephalins) also significantly reduced mu receptor binding, but to a lesser extent. Electrical stimulation of the hippocampal slice at sites not containing opioid peptides did not cause mu receptor displacement. These results demonstrate that under physiological conditions, the release of endogenous opioids from the major opioid containing pathways can be detected in a single hippocampal slice following high frequency stimulation.     

Abdominal surgery induces mu opioid receptor endocytosis in enteric neurons of the guinea-pig ileum

Immunohistochemistry and confocal microscopy were used to investigate mu opioid receptor (muOR) internalization in enteric neurons of the guinea-pig ileum following abdominal surgery. The following surgical procedures were performed under halothane or isofluorane anesthesia: a) midline abdominal skin incision, b) laparotomy or c) laparotomy with intestinal manipulation. Gastrointestinal transit was evaluated by using a non-absorbable marker and measuring fecal pellet output. In neurons from normal and control (anesthesia alone) animals, muOR was predominantly at the cell surface. muOR endocytosis following skin incision was not significantly different from controls (21.2+/-3.5% vs. 13.7+/-2.1%, mean+/-S.E.M.), whereas it was significantly increased by laparotomy (46.5+/-6.1%; P<0.01 vs. controls) or laparotomy plus intestinal manipulation (40.5+/-6.1%; P<0.01 vs. controls) 30 min following surgery compared with controls. muOR endocytosis remained elevated at 4 h (38.6+/-1.2%; P<0.01 vs. controls), whereas it was similar to controls at 6 and 12 h (17.5+/-5.8% and 11.2+/-3.0%). muOR endocytosis occurred in cholinergic and nitrergic neurons. Gastrointestinal transit was significantly delayed by laparotomy or laparotomy plus intestinal manipulation (12.8+/-1.2 and 13.8+/-0.6 h vs. 7.0+/-0.5 in controls; P<0.01), but was not significantly changed by skin incision (8.2+/-0.6 h).The findings of the present study support the concept that the noxious stimulation caused by abdominal surgery induces release of endogenous opioids thus resulting in muOR endocytosis in neurochemically distinct enteric neurons. muOR internalization can serve as indirect evidence of opioid release and as a means to visualize neuronal pathways activated by opioids.     

Knockout of the mu opioid receptor enhances the survival of adult-generated hippocampal granule cell neurons

Recent evidence suggests that mu opioid receptors (MOR) are key regulators of hippocampal structure and function. For example, exogenous MOR agonists morphine and heroin negatively impact hippocampal function and decrease adult hippocampal neurogenesis. Here we explored the role of MOR in the birth and survival of hippocampal progenitor cells by examining adult neurogenesis in mice that lack MOR. Adult male mice lacking exon 1 of MOR were injected with the S phase marker bromodeoxyuridine (BrdU) and killed either 2 hours or 4 weeks later to evaluate proliferating and surviving BrdU-immunoreactive (IR) cells, respectively, in the adult hippocampal granule cell layer. Wild-type (WT), heterozygote, and homozygote mice did not differ in the number of BrdU-IR cells at a proliferation time point. However, 4 weeks after BrdU injection, heterozygote and homozygote mice had 57% and 54% more surviving BrdU-IR cells in the hippocampal granule cell layer as compared with WT mice. A decrease in apoptosis in the heterozygote and homozygote mice did not account for the difference in number of surviving BrdU-IR cells since there were no alterations in number of pyknotic, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive, or activated caspase 3-IR cells compared with WT. In concordance with the increased numbers of granule cells maturing into neurons, heterozygote and homozygote mice had larger hippocampal granule cell layers and increased numbers of granule cells. These findings indicate that MOR may play a role in regulating progenitor cell survival and more generally encourage further exploration of how MOR activation can influence hippocampal structure and function.     

Cocaine reward and hyperactivity in the rat: sites of mu opioid receptor modulation

Opioid receptor agonists and antagonists have profound effects on cocaine-induced hyperactivity and conditioned reward. Recently, the role specifically of the mu opioid receptor has been demonstrated based on the finding that i.c.v. administration of the selective mu opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), can attenuate cocaine-induced behaviors. The purpose of the present study was to determine the location of mu opioid receptors that are critical for cocaine-induced reward and hyperactivity. Adult male Sprague-Dawley rats received injections of CTAP into the caudate putamen, the rostral or caudal ventral tegmental area (VTA) or the medial shell or core of the nucleus accumbens prior to cocaine to determine the role of mu opioid receptors in cocaine-induced reward and hyperactivity. Cocaine-induced reward was assessed using an unbiased conditioned place preference procedure. Results demonstrate that animals pre-treated with CTAP into the nucleus accumbens core or rostral VTA, but not the caudal VTA, caudate putamen or medial nucleus accumbens shell, during conditioning with cocaine showed an attenuation of the development of cocaine-induced place preference. In contrast, CTAP injected into the nucleus accumbens shell but not the core attenuated the expression of cocaine place preference. Intra-nucleus accumbens core, caudate putamen or caudal VTA CTAP significantly attenuated cocaine-induced hyperactivity. In addition, the number of cFos positive cells was increased in the motor cortex, medial and ventromedial aspects of the nucleus accumbens shell, basolateral amygdala and caudal VTA during the expression of cocaine place preference, and this increase was attenuated in the animals that received intra-accumbens core CTAP during daily cocaine conditioning. These results demonstrate the importance of mu opioid receptors in the nucleus accumbens and VTA in cocaine-induced reward and hyperactivity and suggest that some aspects of the behavioral effects of cocaine are mediated by endogenous activation of mu opioid receptors in these brain regions.     

Functional coupling, desensitization and internalization of virally expressed mu opioid receptors in cultured dorsal root ganglion neurons from mu opioid receptor knockout mice

Although mu opioid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that a system is needed in which functional mu opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged mu opioid receptor is returned to dorsal root ganglia neurons from mu opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged mu opioid receptor in neurons resulted in approximately 1.3x10(6) receptors/cell. Many mu opioid receptor cell lines express a similar density of receptors but this is approximately 7x greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the mu agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag- tagged receptor (71+/-3 vs 29+/-7% respectively; P<0.002), indicating less desensitization in neurons expressing a higher density of receptor. Using flow cytometry to quantify the percentage of receptors remaining on the neuronal cell surface, the flag-tagged receptor internalized by 17+/-1% after 20 min and 55+/-2% after 24 h of DAMGO. These data indicate that this return of function model in neurons recapitulates many of the characteristics of endogenous mu opioid receptor function previously identified in non-neuronal cell lines.     

Haplotype block structure study of the CFTR gene. Most variants are associated with the M470 allele in several European populations

An average of about 1700 CFTR (cystic fibrosis transmembrane conductance regulator) alleles from normal individuals from different European populations were extensively screened for DNA sequence variation. A total of 80 variants were observed: 61 coding SNSs (results already published), 13 noncoding SNSs, three STRs, two short deletions, and one nucleotide insertion. Eight DNA variants were classified as non-CF causing due to their high frequency of occurrence. Through this survey the CFTR has become the most exhaustively studied gene for its coding sequence variability and, though to a lesser extent, for its noncoding sequence variability as well. Interestingly, most variation was associated with the M470 allele, while the V470 allele showed an 'extended haplotype homozygosity' (EHH). These findings make us suggest a role for selection acting either on the M470V itself or through an hitchhiking mechanism involving a second site. The possible ancient origin of the V allele in an 'out of Africa' time frame is discussed.     

Sleep deprivation decreases mu and delta opioid receptor binding in the rat limbic system.

Sleep deprivation induced by the platform technique is considered to be a heavy stressful situation in rats. At the end of the sleep deprivation period (72 h) the rat displayed particular behavior characterized by wakefulness, a high degree of motor and exploratory activity, increased alertness and reactivity to environmental stimuli. Our previous results indicated that this behavior was antagonized by the administration of the opioid receptor antagonist naloxone and increased by opioid agonists. In this paper we show that concomitantly with this behavior, a decreased Bmax of mu and delta opioid receptors is present in the limbic system of these animals. These data suggest an active role of limbic mu and delta receptors in the generation of arousal and insomnia related to sleep deprivation induced stress.     

Immunohistochemical study of the expression of exon11-containing mu opioid receptor variants in mouse brain

The cloned mu opioid receptor MOR-1 undergoes alternative splicing. Extensive 3'-splicing downstream from exon 3 leads to a number of C-terminal splice variants that are differentially expressed within the CNS. Recently, 5'-splicing has been observed with eight additional variants containing exon 11, a new exon located approximately 10 kb upstream from exon 1 that is under the control of a different promoter located even further upstream. Three of these variants generate the same protein as MOR-1 itself, but under the control of the new exon 11 promoter. Three variants in which exon 11 is translated have been identified within the brain, including MOR-1G, MOR-1M and MOR-1N. The present paper defines immunohistochemically the distribution of these variants using an exon 11-specific antiserum. The expression of exon 11-like immunoreactivity (-LI) was seen primarily in the olfactory tubercle, caudate-putamen, globus pallidus and substantia nigra. We did not observe exon 11-LI in a number of regions expressing MOR-1. Within the caudate-putamen, the general pattern of labeling was diffuse, in contrast to the pattern seen with an exon 4-generated antiserum that labels MOR-1 itself. However, we did observe in the caudate-putamen co-expression of exon 4- and exon 11-LI in cells that were apposed to dopaminergic terminals. These results provide new insights regarding the potential physiological significance of these exon 11-containing variants.     

Haplotype structure of five SNPs within the ACE gene in the Tunisian population

BACKGROUND: The Angiotensin-Converting Enzyme (ACE) is a candidate gene in the aetiology of several common diseases. The study of the haplotype structure of this gene is of interest in diagnosis and in pharmacogenomics. AIM: The study investigated the haplotype profile of single nucleotide polymorphisms (SNPs) within the ACE gene in the Tunisian population and compared it with other populations. SUBJECTS AND METHODS: Five SNPs (rs1800764, rs4291, rs4309, rs4331, rs4340) covering a region of 15.6 kb of the ACE gene were typed by PCR-digestion in a sample of 100 healthy subjects. RESULTS: All SNPs were polymorphic and in Hardy-Weinberg equilibrium. A total of 21 haplotypes were identified but only eight had a frequency of more than 1%. The four most common haplotypes had a cumulative frequency of 87.4%. The 'Yin-Yang' phenomenon (the two major haplotypes are complementary at all sites) was found. Linkage disequilibrium between all pairs of loci was highly significant (p<10-5). A simple and efficient statistical procedure was used to identify three important SNPs. CONCLUSION: The Tunisian population showed a different haplotype structure from the European one for the ACE gene and three important SNPs were identified. These will be very helpful in future association studies in the Tunisian and North African populations.     

Haplotype structure of the beta adrenergic receptor genes in US Caucasians and African Americans

The beta-adrenergic receptors (beta-AR) are G protein-coupled receptors activated by epinephrine and norepinephrine and are involved in a variety of their physiological functions. Previously, three beta-AR genes (ADRB1, ADRB2 and ADRB3) were resequenced, identifying polymorphisms that were used in genetic association studies of cardiovascular and metabolic disorders. These studies have produced intriguing but inconsistent results, potentially because the known functional variants: ADRB1 Arg389Gly and Gly49Ser, ADRB2 Arg16Gly and Gln27Glu, and ADRB3 Arg64Trp provided an incomplete picture of the total functional diversity at these genes. Therefore, we created marker panels for each beta-AR gene that included the known functional markers and also other markers evenly spaced and with sufficient density to identify haplotype block structure and to maximize haplotype diversity. A total of 27 markers were genotyped in 96 US Caucasians and 96 African Americans. In both populations and for each gene, a single block with little evidence of historical recombination was observed. For each gene, haplotype captured most of the information content of each functional locus, even if that locus was not genotyped, and presumably haplotype would capture the signal from unknown functional loci whose alleles are of moderate abundance. This study demonstrates the utility of using beta-AR gene haplotype maps and marker panels as tools for linkage studies on beta-AR function.     

An antisense oligodeoxynucleotide to the mu opioid receptor attenuates cocaine-induced behavioral sensitization and reward in mice

Numerous studies support a role for the endogenous opioid system in cocaine-influenced behavior. Few of these studies, however, selectively delineate a role for the mu opioid receptor (MOR) in this regard. This investigation examined if the MOR modulates cocaine-induced behavior in mice using a 17-base antisense oligodeoxynucleotide (AS ODN) directed against the MOR coding sequence 16-32. Specifically, cocaine-induced behavioral sensitization and conditioned reward were investigated. For the sensitization study, C57BL/6J mice received eight intermittent i.c.v. infusions of saline, mismatch oligodeoxynucleotide (ODN) (20 microg/4 microl) or AS ODN (20 microg/4 microl) over 20 days. Mice also received concomitant once daily i.p. injections of saline (4 ml/kg) or cocaine (15 mg/kg) for 10 days. There was a 7-day withdrawal period, after which all mice were challenged with cocaine (15 mg/kg) to test for behavioral sensitization. For the conditioned place preference (CPP) study, mice received five i.c.v. infusions of mismatch ODN or MOR AS ODN (days 1-5). An unbiased counterbalanced conditioning procedure was used where mice were conditioned with saline (4 ml/kg, i.p.) and cocaine (15 mg/kg, i.p.) on alternate days for four sessions (days 3-6). Mice were tested on day 7 for CPP. Immediately following testing, [3H]DAMGO (D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin) receptor binding to brain homogenates was conducted. MOR AS attenuated cocaine-induced behavioral sensitization and conditioned reward. MOR AS ODN also reduced [3H]DAMGO binding. Collectively, these findings implicate the MOR as playing an important neuromodulatory role in the behavioral effects of cocaine in mice.