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Thienorphine hydrochloride is a new anti-relapse drug for opioid abusers that is currently in phase II clinical trial. In the present study, the antinociception, dependence, and signal transduction induced by thienorphine were examined. Thienorphine showed a potent antinociception effect in acetic acid-induced writhing test and formalin test. In the hot plate test and tail-flick test, thienorphine presented the typical partial opioid agonist character with a ceiling dose-response curve in addition to a bell-shaped curve. The hot plate test revealed that thienorphine induced approximately 50% of antinociception in μ receptor knockout (μ-KO) mice compared to wild-type controls (P < 0.05). The κ, δ selective antagonist nor-binaltorphimine (nor-BNI), and naltrindole decreased approximately 50-60% of theinorphine antinociception in μ-KO mice, respectively. The ORL1 receptor-selective antagonist J113397 did not affect theinorphine antinociception in μ-KO mice. Chronic treatment with thienorphine (1.5 mg/kg) induced some tolerance that was lower compared to buprenorphine or morphine addition. In contrast to buprenorphine or morphine, thienorphine did not lead to psychological dependence by conditioned place preference (CPP). The maximum inhibition of thienorphine on protein kinase A (PKA) activity was about 36%, 100%, 100%, and 12% in CHO-μ/κ/δ/ORL1-PKAcatEGFP cells, respectively. Similar results were observed in cyclic adenosine monophosphate (cAMP) accumulation inhibited by thienorphine in cells. Thienorphine significantly increased pERK1/2 in CHO-κ/δ-PKAcatEGFP cells. These results indicated that thienorphine induced analgesia through activation of κ- and δ-, partial activation of μ- opioid receptor without a bias between G-protein- and β-arrestin-mediated pathways. Thienorphine might be used for antinociception with minimal adverse effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Dynantin is a potent and selective synthetic polypeptide kappa opioid receptor antagonist which has potential antidepressant and anxiolytic-like therapeutic applications, however its clinical development has been hampered by plasma stability issues and poor penetration of the blood brain barrier. Targeted liposome delivery systems represent a promising and non-invasive approach to improving the delivery of therapeutic agents across the blood brain barrier. As part of our work focused on targeted drug delivery, we have developed a novel mannosylated liposome system. Herein, we investigate these glycoliposomes for the targeted delivery of dynantin to the central nervous system. Cholesterol was tested and optimized as a formulation excipient, where it improved particle stability as measured via particle size, entrapment and ex vivo plasma stability of dynantin. The in vitro PRESTO-TANGO assay system was used to confirm that glycoliposomal entrapment did not impact the affinity or activity of the peptide at its receptor. Finally, in vivo distribution studies in mice showed that the mannosylated glycoliposomes significantly improved delivery of dynantin to the brain. Overall, the results clearly demonstrate the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the central nervous system., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Early life stress is suggested to alter behavioral responses during stressful challenges in adulthood and to exacerbate pathological symptoms that reminisce posttraumatic stress disorder (PTSD). These effects are often associated with changes in γ-Aminobutyric acid type A (GABAA) and κ opioid receptor expression and neuromodulation of the limbic system. Anxiety-like and stress coping behaviors were assessed in rats exposed to stress in adulthood on the background of previous exposure to stress in juvenility. Two weeks following behavioral assessment in adulthood, GABAAR α1 and α2 subunits and κ opioid receptor expression levels were measured in the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), amygdala, and periaqueductal gray (PAG). To illustrate changes at the network level, an integrated expression profile was constructed. We found that exposure to juvenile stress affected rats' behavior during adult stress. The combination of juvenile and adult stress significantly affected rats' long term anxious-like behavior. Probabilities predicting model integrating the expression of GABAA α1-α2 and κ opioid receptors in different brain regions yielded highly successful classification rates. This study emphasizes the ability of exposure to stress in juvenility to exacerbate the impact of coping with stress in adulthood. Moreover, the use of integrated receptor expression network profiling was found to effectively characterize the discussed affective styles and their behavioral manifestations.

Activation of κ opioid receptor (KOR) produces analgesia, antipruritic effect, sedation and dysphoria. To characterize neuroanatomy of KOR at high resolutions and circumvent issues of specificity of KOR antibodies, we generated a knock-in mouse line expressing KOR fused at the C terminus with the fluorescent protein tdTomato (KtdT). The selective KOR agonist U50,488H caused anti-scratch effect and hypolocomotion, indicating intact KOR neuronal circuitries. Clearing of brains with CLARITY revealed three-dimensional (3-D) images of distribution of KOR, and any G-protein-coupled receptors, for the first time. 3-D brain images of KtdT and immunohistochemistry (IHC) on brain sections with antibodies against tdTomato show similar distribution to that of autoradiography of [ 3 H]U69,593 binding to KOR in wild-type mice. KtdT was observed in regions involved in reward and aversion, pain modulation, and neuroendocrine regulation. KOR is present in several areas with unknown roles, including the claustrum (CLA), dorsal endopiriform nucleus, paraventricular nucleus of the thalamus (PVT), lateral habenula (LHb), and substantia nigra pars reticulata (SNr), which are discussed. Prominent KtdT-containing fibers were observed to project from caudate putamen (CP) and nucleus accumbens (ACB) to substantia innominata (SI) and SNr. Double IHC revealed co-localization of KtdT with tyrosine hydroxylase (TH) in brain regions, including CP, ACB, and ventral tegmental area (VTA). KOR was visualized at the cellular level, such as co-localization with TH and agonist-induced KOR translocation into intracellular space in some VTA neurons. These mice thus represent a powerful and heretofore unparalleled tool for neuroanatomy of KOR at both the 3-D and cellular levels., (Copyright © 2020 Chen et al.)

Rationale: Cocaine addiction is a chronic brain disease characterized by compulsive drug intake and dysregulation of brain reward systems. Few preclinical studies have modeled the natural longitudinal course of cocaine addiction. Extended access self-administration protocols are powerful tools for modeling the advanced stages of addiction; however, few studies have duration of drug access longer than 12 h/session, potentially limiting their construct validity. Identification of changes in cocaine intake patterns during the development of addictive-like states may allow better treatments for vulnerable subjects. The kappa opioid receptor (KOPr) system has been implicated in the neurobiological regulation of addictive states as well as mood and stress disorders, with selective KOPr antagonists proposed as possible pharmacotherapeutic agents. Chronic cocaine exposure increases the expression of KOPr and its endogenous agonists, the dynorphins, in several brain areas in rodents., Objectives: To examine the behavioral pattern of intake during chronic (14 days) 18 h intravenous cocaine self-administration (0.5 mg/kg/infusion) and the effect of a novel short-acting KOPr antagonist LY2444296 HCl (3 mg/kg) administered during sessions 8 to 14 of chronic 18 h/day cocaine self-administration and prior to a single re-exposure session after 2 cocaine-free withdrawal days., Results: Both daily and hourly cocaine intake patterns changed over 14 days of 18 h self-administration. LY pretreatment affected the pattern of self-administration across the second week of extended access cocaine self-administration and prevented the increase in cocaine intake during re-exposure., Conclusions: Overall, the KOPr antagonist attenuated escalated cocaine consumption in a rat model of extended access cocaine self-administration.

Prenatal ethanol exposure (PEE) promotes ethanol consumption in the adolescent offspring accompanied by the transcriptional regulation of kappa opioid receptor (KOR) system genes. This study analysed if environmental enrichment (EE, from gestational day 20 to postnatal day 26) exerts protective effects upon PEE-modulation of gene expression, ethanol intake and anxiety responses. Pregnant rats were exposed to PEE (0.0 or 2.0 g/kg ethanol, gestational days 17-20) and subsequently the dam and offspring were reared under EE or standard conditions. PEE upregulated KOR mRNA levels in amygdala (AMY) and prodynorphin (PDYN) mRNA levels in ventral tegmental area (VTA) with the latter effect associated with lower DNA methylation at the gene promoter. These effects were normalized by exposure to EE. PEE modulated BDNF mRNA levels in VTA and Nucleus accumbens (AcbN), and EE mitigated the changes in AcbN. EE induced a protective effect on ethanol intake and preference, an effect more noticeable in males than in females, and in prenatal vehicle-treated (PV) than in PEE rats. The male offspring drank significantly less ethanol than the female offspring. The latter result suggests that the protective effect of EE on ethanol drinking may only emerge at lower levels of drinking. In the dams, PEE induced an upregulation of PDYN and KOR in AcbN. PDYN gene expression was normalized by exposure to EE. These results suggest that EE is a promising treatment to inhibit the effects of PEE. The results confirm that PEE effects are mediated by alterations in the transcriptional regulation of KOR system genes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Naltrexone is a nonselective opioid receptor antagonist used as a treatment for alcohol use disorder. However, only modest clinical effects have been observed, possibly because of limited knowledge about the biological variables affecting the efficacy of naltrexone. We investigated the potential role of the kappa opioid receptor (KOR) in the therapeutic effect of naltrexone., Methods: A total of 48 non-treatment-seeking heavy drinkers (16 women) who met DSM-IV criteria for alcohol dependence participated in two alcohol drinking paradigms (ADPs) separated by a week of open-label naltrexone (100 mg daily). Craving, assessed with the Alcohol Urge Questionnaire and the Yale Craving Scale, and drinking behavior were recorded in each ADP. Prior to naltrexone initiation, KOR availability was determined in the amygdala, hippocampus, pallidum, striatum, cingulate cortex, and prefrontal cortex using positron emission tomography with [ 11 C]LY2795050., Results: Participants reported lower levels of craving (Yale Craving Scale: -11 ± 1, p < .0001; Alcohol Urge Questionnaire: -6 ± 0.6, p < .0001) and consumed fewer drinks (-3.7 ± 4, p < .0001) during the second ADP following naltrexone therapy. The observed reduction in drinking was negatively associated with baseline KOR availability in the striatum (p = .005), pallidum (p = .023), and cingulate cortex (p = .018). Voxelwise analysis identified clusters in the bilateral insula, prefrontal, and cingulate cortex associated with the reduction in drinking (p < .0001). In addition, KOR availability in all evaluated brain regions was associated with craving measured in both ADPs., Conclusions: The KOR is implicated in drinking and craving following naltrexone therapy in alcohol use disorder., (Copyright © 2019 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Kappa opioid receptors (KORs) have been characterized as an aversive system in the brain and implicated in social behavior in preclinical models. This work investigated the effect of social status on the KOR system in humans using positron emission tomography (PET) imaging with the novel KOR agonist radiotracer [ 11 C]EKAP. Eighteen healthy participants (mean age 41.2 ± 9.3) completed the Barratt Simplified Measure of Social Status (BSMSS), an MRI and an [ 11 C]EKAP PET scan on the High Resolution Research Tomograph. Arterial blood sampling and metabolite analysis were conducted to obtain the input function. Regions of interest were based upon an MR template and included the reward/aversion areas of the brain. The multilinear analysis-1 (MA1) method was applied to the regional time-activity curves (TACs) to calculate [ 11 C]EKAP regional volume of distribution (V T ). Mixed models and Pearson correlation coefficients were used for body mass index (BMI), gender and age, with age being dropped in subsequent analyses because of nonsignificance. An overall effect of primary ROIs (F 7, 112 7.43, p < 0.0001), BSMSS score (F 1, 13 7.45, p = 0.02), BMI (F 1, 13 23.5, p < 0.001), and gender (F 1, 13 23.75, p < 0.001), but not age (F 1, 13 1.12, p = 0.35) was observed. Regional [ 11 C]EKAP V T and BSMSS were found to be negatively correlated in the amygdala (r = -0.69, p < 0.01), anterior cingulate cortex (r = -0.56, p = 0.02), caudate (r = -0.66, p < 0.01), frontal cortex (r = -0.52, p = 0.04), hippocampus (r = -0.60, p = 0.01), pallidum (r = -0.59, p = 0.02), putamen (r = -0.62, p = 0.01), and ventral striatum (r = -0.66, p < 0.01). In secondary (non-reward) regions, correlations of [ 11 C]EKAP V T and BSMSS were nonsignificant with the exception of the insula. There was an inverse correlation between social status and KOR levels that was largely specific to the reward/aversion (e.g., saliency) areas of the brain. This finding suggests the KOR system may act as a mediator for the negative effects of social behaviors in humans.

Animal studies indicate that the kappa-opioid receptor/dynorphin system plays an important role in cocaine binges and stress-induced relapse. Our goal was to investigate changes in kappa-opioid receptor (KOR) availability in the human brain using positron emission tomography (PET), before and after a cocaine binge. We also investigated the correlation between KOR and stress-induced cocaine self-administration. PET imaging was performed with the KOR selective agonist [ 11 C]GR103545. Subjects with cocaine-use disorder (CUD) underwent PET scans and performed two types of cocaine self-administration sessions in the laboratory as follows: (1) choice sessions following a cold pressor test, to induce stress, and (2) binge dosing of cocaine. This allowed us investigate the following: (1) the association between KOR binding and a laboratory model of stress-induced relapse and (2) the change in KOR binding following a 3-day cocaine binge, which is thought to represent a change in endogenous dynorphin. A group of matched healthy controls was included to investigate between group differences in KOR availability. A significant association between [ 11 C]GR103545 binding and cocaine self-administration was seen: greater KOR availability was associated with more choices for cocaine. In addition, the 3-day cocaine binge significantly reduced [ 11 C]GR103545 binding by 18% in the striatum and 14% across brain regions. No difference in [ 11 C]GR103545 binding was found between the CUD subjects and matched controls. In the context of previous studies, these findings add to the growing evidence that pharmacotherapies targeting the KOR have the potential to significantly impact treatment development for cocaine-use disorder.

Opioid receptors are implicated in alcoholism, other addictions, withdrawal, and depression, and are considered potential pharmacological targets for treatment. Our goal in the present study was to compare the availability of kappa opioid receptors (KOR) between an alcohol-dependent cohort (AD) and a healthy control cohort (HC). Sixty-four participants-36 AD and 28 HC-underwent PET scans with [ 11 C]LY2795050, a selective kappa antagonist tracer. Partial-volume correction was applied to all PET data to correct for atrophy. Volume of distribution (V T ) of the tracer was estimated regionally as a measure of KOR availability. V T values of AD versus HC were compared for 15 defined ROIs. Multivariate analysis showed a main effect of group on V T across these 15 ROIs. Post hoc tests showed that AD had significantly lower V T and thus a lower KOR availability than HC in amygdala and pallidum (corrected for multiple comparisons). Exploratory analysis of change in V T with age was conducted; V T was not found to vary significantly with age in any region. Our findings of lower V T in AD versus HC in multiple regions are in contrast to findings in the mu and delta opioid receptor systems of higher V T in AD versus HC. Although age-related decline in receptors has previously been observed in the mu opioid receptor system, we found that KOR availability does not change with age.

Past studies have shown that it has been difficult to discover and develop potent and selective κ opioid receptor antagonists, particularly compounds having potential for clinical development. In this study, we present a structure-activity relationship (SAR) study of a recently discovered new class of tetrahydroisoquinoline κ opioid receptor antagonists which led to (3 R)-7-hydroxy- N-{(1 S)-2-methyl-1-[(-4-methylpiperidine-1-yl)methyl]propyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (12) (4-Me-PDTic). Compound 12 had a K e = 0.37 nM in a [ 35 S]GTPγS binding assay and was 645- and >8100-fold selective for the κ relative to the μ and δ opioid receptors, respectively. Calculated log BB and CNS (central nervous system) multiparameter optimization (MPO) and low molecular weight values all predict that 12 will penetrate the brain, and pharmacokinetic studies in rats show that 12 does indeed penetrate the brain.

Endogenous kappa opioids mediate pathological responses to stress in animal models. However, the relationship of the kappa opioid receptor (KOR) to life stress and to psychopathology in humans is not well described. This pilot study sought, for the first time, to quantify KOR in major depressive disorder (MDD) in vivo in humans using positron emission tomography (PET). KOR binding was quantified in vivo by PET imaging with the [ 11 C]GR103545 radiotracer in 13 healthy volunteers and 10 participants with current MDD. We examined the relationship between regional [ 11 C]GR103545 total volume of distribution (V T ) and diagnosis, childhood trauma, recent life stress, and, in a subsample, salivary cortisol levels during a modified Trier Social Stress Test (mTSST), amygdala, hippocampus, ventral striatum and raphe nuclei. Whole-brain voxel-wise analyses were also performed. [ 11 C]GR103545 V T did not differ significantly between MDD participants and healthy volunteers in the four a priori ROIs (p = 0.50). [ 11 C]GR103545 V T was unrelated to reported childhood adversity (p = 0.17) or recent life stress (p = 0.56). A trend-level inverse correlation was observed between [ 11 C]GR103545 V T and cortisol area-under-the curve with respect to ground during the mTSST (p = 0.081). No whole-brain voxel-wise contrasts were significant. Regional [ 11 C]GR103545 V T , a measure of in vivo KOR binding, does not differentiate MDD from healthy volunteers in this pilot sample. Future studies may examine KOR binding in subgroups of depressed individuals at increased risk for KOR abnormalities, including co-occurring mood and substance use disorders, as well as depression with psychotic features., (© 2018 Wiley Periodicals, Inc.)

A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Buprenorphine, a mu opioid receptor partial agonist and kappa opioid receptor (KOR) antagonist, is an emerging therapeutic agent for maternal opioid dependence in pregnancy and neonatal abstinence syndrome. However, the endogenous opioid system plays a critical role in modulating neurodevelopment and perinatal buprenorphine exposure may detrimentally influence this. To identify aspects of neurodevelopment vulnerable to perinatal buprenorphine exposure, we defined KOR protein expression and its cellular associations in normal rat brain from embryonic day 16 to postnatal day 23 with double-labelling immunohistochemistry. KOR was expressed on neural stem and progenitor cells (NSPCs), choroid plexus epithelium, subpopulations of cortical neurones and oligodendrocytes, and NSPCs and subpopulations of neurones in postnatal hippocampus. These distinct patterns of KOR expression suggest several pathways vulnerable to perinatal buprenorphine exposure, including proliferation, neurogenesis and neurotransmission. We thus suggest the cautious use of buprenorphine in both mothers and infants until its impact on neurodevelopment is better defined., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Optogenetics and chemogenetics provide the ability to modulate neurons in a type- and region-specific manner. These powerful techniques are useful to test hypotheses regarding the neural circuit mechanisms of general anesthetic end points such as hypnosis and analgesia. With both techniques, a genetic strategy is used to target expression of light-sensitive ion channels (opsins) or designer receptors exclusively activated by designer drugs in specific neurons. Optogenetics provides precise temporal control of neuronal firing with light pulses, whereas chemogenetics provides the ability to modulate neuronal firing for several hours with the single administration of a designer drug. This chapter provides an overview of neuronal targeting and experimental strategies and highlights the important advantages and disadvantages of each technique., (© 2018 Elsevier Inc. All rights reserved.)

Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)-derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real-time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied., (© 2017 Wiley Periodicals, Inc.)

Oxymorphone, one of oxycodone's metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear. This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [ 3 H]oxymorphone revealed high affinity binding sites in mouse brain displaying Kd of 1.7nM and Bmax of 147fmol/mg. Furthermore, we performed quantitative autoradiography binding studies using [ 3 H]oxymorphone in mouse brain. The distribution of [ 3 H]oxymorphone binding sites was found to be similar to the selective MOP agonist [ 3 H]DAMGO in the mouse brain. [ 3 H]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [ 3 H]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP, and not the DOP or the KOP is the main high affinity binding target for oxymorphone., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Numerous animal species display behavioral changes in response to changes in social status or territory possession. For example, in male European starlings only males that acquire nesting sites display high rates of sexual and agonistic behavior. Past studies show that mu and delta opioid receptors regulate behaviors associated with social ascension or defeat. Opioids also act at kappa receptors, with dynorphin binding with the highest affinity; however, the role of these opioids in social behavior has not been well studied. We observed flocks of male starlings during the breeding season and ran quantitative real-time polymerase chain reaction (qPCR) to measure expression of kappa opioid receptors (OPRK1) and prodynorphin (PDYN) in brain regions involved in social behavior and motivation (ventral tegmental area [VTA], medial preoptic nucleus [mPOA]) and vocal behavior (Area X). Males with nesting territories displayed more sexual/agonistic behavior than males without nesting territories. They also had lower OPRK1 expression in VTA and mPOA. OPRK1 expression in VTA correlated negatively with sexual/agonistic behaviors, consistent with past studies showing kappa receptors in VTA to inhibit sociosexual behaviors. PDYN in mPOA correlated negatively with a measure of nesting behavior that may also reflect sexual motivation. PDYN in Area X related positively to song. Distinct patterns of OPRK1 and PDYN expression in VTA, mPOA, and Area X related to gonad volume, suggesting that breeding condition may modify (or be modified by) OPRK1 and PDYN expression. Studies are now needed to further characterize the role of OPRK1 and PDYN in status-appropriate social behaviors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Kappa opioid receptor (KOR) antagonists are potential drug candidates for diseases such as treatment-refractory depression, anxiety, and addictive disorders. PET imaging radiotracers for KOR can be used in occupancy study to facilitate drug development, and to investigate the roles of KOR in health and diseases. We have previously developed two 11 C-labeled antagonist radiotracers with high affinity and selectivity toward KOR. What is limiting their wide applications is the short half-life of 11 C. Herein, we report the synthesis of a first 18 F-labeled KOR antagonist radiotracer and the initial PET imaging study in a nonhuman primate.

JDTic is a potent and selective κ-opioid receptor (KOR) antagonist that reverses U50,488-induced diuresis in rats. It partitions into brain with a duration of action lasting for weeks. In a search for KOR antagonists that do not accumulate in the brain, we compared single doses of five methylated JDTic analogs (RTI-97, -194, -212, -240, and -241) for reversal of U50,488 diuresis and pharmacokinetic (PK) properties. All six compounds showed potent and selective KOR antagonism in a [ 35 S]GTPγS binding assay. Plasma half-lives ranged from 24 to 41 h and brain half-lives from 24 to 76 h. JDTic and RTI-194 showed increasing brain to plasma ratios over time, indicating increasing partitioning into brain and a longer duration of action for reversal of diuresis than did RTI-97. RTI-240 did not show significant brain accumulation. RTI-212 showed no substantive difference between brain and plasma levels and was inactive against diuresis. RTI-241, with a lower brain to plasma ratio than JDTic and RTI-194, formed JDTic as a metabolite, which still reduced diuresis after 9 weeks. The fact that the duration of action was correlated with the brain to blood plasma ratios and area under the concentration-time curves suggests that PK properties could help to predict safety and acceptable duration of action for KOR antagonists.

κ Opioid receptor system is widely implicated in the regulation of emotion. However, the findings about the role on anxiety in rodents are highly controversial, since both anxiogenic- and anxiolytic-like effects have been reported with κ opioid receptor activation. The mechanism and the underlying neuroanatomical substrates are unexplored. In the present study, we first investigated the effects of κ agonist U50,488H on anxiety-related behaviors over a wide range of doses, and we found that U50,488H produced dual effects in anxiety, with low dose being anxiogenic and high dose being anxiolytic. To assess the potential neuroanatomical substrates, we used phosphorylation of extracellular signal-related kinase1/2 (pERK1/2) to map the underlying neural circuits. We found that the anxiogenic effect of U50,488H was paralleled by an increase of pERK1/2 in the nucleus accumbens, whereas the anxiolytic effect was paralleled by an increase of pERK1/2 in the lateral septal nucleus. We then examined the behavioral consequences with locally microinjection of U50,488H, and we found that microinjection of U50,488H into the nucleus accumbens exerted anxiogenic-like effects, whereas microinjection of U50,488H into the lateral septal nucleus. Both effects can be abolished by κ antagonist nor-BNI pretreatment. To the best of our knowledge, the present work firstly provides the neuroanatomical sites that mediating the dual anxiogenic- and anxiolytic-like effects of U50,488H in mice. This study may help to explain current controversial role of κ receptor activation in anxiety-related behaviors in rodents, and may open new perspectives in the areas of anxiety disorders and κ receptor function., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pancreatic insulin-secreting β-cells express opioid receptors, whose activation by opioid peptides modulates hormone secretion. Opioid receptors are also expressed in multiple brain regions including the hypothalamus, where they play a role in feeding behavior and energy homeostasis, but their potential role in central regulation of glucose metabolism is unknown. Here, we investigate whether central opioid receptors participate in the regulation of insulin secretion and glucose homeostasis in vivo. C57BL/6J mice were acutely treated by intracerebroventricular (i.c.v.) injection with specific agonists for the three main opioid receptors, kappa (KOR), delta (DOR) and mu (MOR) opioid receptors: activation of KOR and DOR did not alter glucose tolerance, whereas activation of brain MOR with the specific agonist DAMGO blunted glucose-stimulated insulin secretion (GSIS), reduced insulin sensitivity, increased the expression of gluconeogenic genes in the liver and, consequently, impaired glucose tolerance. Pharmacological blockade of α2A-adrenergic receptors prevented DAMGO-induced glucose intolerance and gluconeogenesis. Accordingly, DAMGO failed to inhibit GSIS and to impair glucose tolerance in α2A-adrenoceptor knockout mice, indicating that the effects of central MOR activation on β-cells are mediated via sympathetic innervation. Our results show for the first time a new role of the central opioid system, specifically the MOR, in the regulation of insulin secretion and glucose metabolism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Previous studies have identified potential antidepressant effects of buprenorphine (BPN), a drug with high affinity for mu opioid receptor (MORs) and kappa opioid receptors (KORs) and some affinity at delta opioid receptor (DOR) and opioid receptor-like 1 (ORL-1) receptors. Therefore, these studies examined which opioid receptors were involved in BPN's effects on animal behavior tests sensitive to antidepressant drugs. The acute effects of BPN were tested in the forced swim test (FST) using mice with genetic deletion of individual opioid receptors or after pharmacological blockade of receptors. For evaluating the effects of BPN on chronic stress, separate groups of mice were exposed to unpredictable chronic mild stress (UCMS) for 3 weeks and treated with BPN for at least 7 days before behavioral assessment and subsequent measurement of Oprk1, Oprm1, and Pdyn mRNA expression in multiple brain regions. BPN did not reduce immobility in mice with KOR deletion or after pretreatment with norbinaltorphimine, even though desipramine remained effective. In contrast, BPN reduced immobility in MOR and DOR knockout mice and in mice pretreated with the ORL-1 antagonist JTC-801. UCMS reduced sucrose preference, decreased time in the light side of the light/dark box, increased immobility in the FST and induced region-specific alterations in Oprk1, Oprm1, and PDYN mRNA expression in the frontal cortex and striatum. All of these changes were normalized following BPN treatment. The KOR was identified as a key player mediating the effects of BPN in tests sensitive to antidepressant drugs in mice. These studies support further development of BPN as a novel antidepressant.

Kisspeptin-neurokinin B-dynorphin (KNDy) cells of the hypothalamus are a key component in the neuroendocrine regulation of GnRH secretion. Evidence in sheep and other species suggests that dynorphin released by KNDy cells inhibits pulsatile GnRH secretion by acting upon κ-opioid receptors (KOR). However, the precise anatomical location and neurochemical phenotype of KOR-expressing cells in sheep remain unknown. To this end, we determined the distribution of KOR mRNA and protein in the brains of luteal phase ewes, using an ovine specific KOR mRNA probe for in situ hybridization and an antibody whose specificity we confirmed by Western blot analyses and blocking peptide controls. KOR cells were observed in a number of regions, including the preoptic area (POA); anterior hypothalamic area; supraoptic and paraventricular nuclei; ventromedial, dorsomedial, and lateral hypothalamus; and arcuate nucleus. Next, we determined whether KOR is colocalized in KNDy and/or GnRH cells. Dual-label immunofluorescence and confocal analysis of the KNDy population showed a high degree of colocalization, with greater than 90% of these neurons containing KOR. Surprisingly, GnRH cells also showed high levels of colocalization in sheep, ranging from 74.4% to 95.4% for GnRH cells in the POA and medial basal hypothalamus, respectively. Similarly, 97.4% of GnRH neurons in the POA of ovariectomized, steroid-primed female rats also contained immunoreactive KOR protein. These findings suggest that the inhibitory effects of dynorphin on pulsatile GnRH secretion may occur either indirectly by actions upon KOR within the KNDy population and/or directly via the activation of KOR on GnRH cells.

The κ-opioid receptor (KOR) is thought to play an important therapeutic role in a wide range of neuropsychiatric and substance abuse disorders, including alcohol dependence. LY2456302 is a recently developed KOR antagonist with high affinity and selectivity and showed efficacy in the suppression of ethanol consumption in rats. This study investigated brain penetration and KOR target engagement after single oral doses (0.5-25 mg) of LY2456302 in 13 healthy human subjects. Three positron emission tomography scans with the KOR antagonist radiotracer (11)C-LY2795050 were conducted at baseline, 2.5 hours postdose, and 24 hours postdose. LY2456302 was well tolerated in all subjects without serious adverse events. Distribution volume was estimated using the multilinear analysis 1 method for each scan. Receptor occupancy (RO) was derived from a graphical occupancy plot and related to LY2456302 plasma concentration to determine maximum occupancy (rmax) and IC50. LY2456302 dose dependently blocked the binding of (11)C-LY2795050 and nearly saturated the receptors at 10 mg, 2.5 hours postdose. Thus, a dose of 10 mg of LY2456302 appears well suited for further clinical testing. Based on the pharmacokinetic (PK)-RO model, the rmax and IC50 of LY2456302 were estimated as 93% and 0.58 ng/ml to 0.65 ng/ml, respectively. Assuming that rmax is 100%, IC50 was estimated as 0.83 ng/ml., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Prairie voles are unusual mammals in that, like humans, they are capable of forming socially monogamous pair bonds, display biparental care, and engage in alloparental behaviors. Both mu and kappa opioid receptors are involved in behaviors that either establish and maintain, or result from pair bond formation in these animals. Mu and kappa opioid receptors both utilize inhibitory G-proteins in signal transduction mechanisms, however the efficacy by which these receptor subtypes stimulate G-protein signaling across the prairie vole neuraxis is not known. Utilizing [(35)S]GTPγS autoradiography, we characterized the efficacy of G-protein stimulation in coronal sections throughout male and female prairie vole brains by [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin (DAMGO) and U50,488H, selective mu and kappa opioid agonists, respectively. DAMGO stimulation was highest in the forebrain, similar to that found with other rodent species. U-50,488H produced greater stimulation in prairie voles than is typically seen in mice and rats, particularly in select forebrain areas. DAMGO produced higher stimulation in the core versus the shell of the nucleus accumbens (NAc) in females, while the distribution of U-50,488H stimulation was the opposite. There were no gender differences for U50,488H stimulation of G-protein activity across the regions examined, while DAMGO stimulation was greater in sections from females compared to those from males for NAc core, entopeduncular nucleus, and hippocampus. These data suggest that the kappa opioid system may be more sensitive to manipulation in prairie voles compared to mice and rats, and that female prairie voles may be more sensitive to mu agonists in select brain regions than males., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Monitoring changes in opioid receptor binding with positron emission tomography (PET) could lead to a better understanding of tolerance and addiction because altered opioid receptor dynamics following agonist exposure has been linked to tolerance mechanisms. We have studied changes in kappa opioid receptor (KOR) binding availability in vivo with PET following kappa opioid agonist administration. Male Sprague-Dawley rats (n=31) were anesthetized and treated with the (KOR) agonist salvinorin A (0.01-1.8 mg/kg, i.v.) before administration of the KOR selective radiotracer [(11)C]GR103545. When salvinorin A was administered 1 min prior to injection of the radiotracer, [(11)C]GR103545 binding potential (BPND) was decreased in a dose-dependent manner, indicating receptor binding competition. In addition, the unique pharmacokinetics of salvinorin A (half-life ~8 min in non-human primates) allowed us to study the residual impact on KOR after the drug had eliminated from the brain. Salvinorin A was administered up to 5 h prior to [(11)C]GR103545, and the changes in BPND were compared with baseline, 2.5 h, 1 h, and 1 min pretreatment times. At lower doses (0.18 mg/kg and 0.32 mg/kg) we observed no prolonged effect on KOR binding but at 0.60 mg/kg salvinorin A induced a sustained decrease in KOR binding (BPND decreased by 40-49%) which persisted up to 2.5 h post administration, long after salvinorin A had been eliminated from the brain. These data point towards an agonist-induced adaptive response by KOR, the dynamics of which have not been previously studied in vivo with PET.

Kappa opioid receptors (KORs) in the central nervous system have been known to be important regulators of a variety of psychiatry illnesses, including anxiety and addiction, but their precise involvement in these disorders is complex and has yet to be fully elucidated. Here, we briefly review the pharmacology of KORs in the brain, including KOR's involvement in anxiety, depression, and drug addiction. We also review the known neuronal circuitry impacted by KOR signaling, and interactions with corticotrophin-releasing factor (CRF), another key peptide in anxiety-related illnesses, as well as the role of glucocorticoids. We suggest that KORs are a promising therapeutic target for a host of neuropsychiatric conditions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The opioid system is involved in infant-mother bonds and adult-adult bonds in many species. We have previously shown that μ opioid receptors (MORs) and κ opioid receptors (KORs) are involved in regulating the adult attachment of the monogamous titi monkey. The present study sought to determine the distribution of MOR and KOR in the titi monkey brain using receptor autoradiography. We used [(3)H][D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) to label MORs and [(3)H]U69,593 to label KORs. MOR binding was heterogeneous throughout the titi monkey brain. Specifically, MOR binding was observed in the cingulate gyrus (CG), striatum, septal regions, diagonal band, amygdala, hypothalamus, hippocampus, and thalamus. Binding was particularly dense in the septum, medial amygdala, paraventricular nucleus of the hypothalamus, mediodorsal thalamus with moderate binding in the nucleus accumbens. Consistent with other primate species, MOR were also observed in "neurochemically unique domains of the accumbens and putamen" (NUDAPs). In general KOR binding was more homogenous. KORs were primarily found in the CG, striatum, amygdala and hippocampus. Dense KOR binding was observed in the claustrum. Relative MOR and KOR binding in the titi monkey striatum was similar to other humans and primates, but was much lower compared to rodents. Relative MOR binding in the titi monkey hypothalamus was much greater than that found in rodents. This study was the first to examine MOR and KOR binding in a monogamous primate. The location of these receptors gives insight into where ligands may be acting to regulate social behavior and endocrine function., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Rationale: Low-efficacy mu opioid receptor agonists may be useful for some clinical indications, but clinically available low-efficacy mu agonists also have low selectivity for mu vs. kappa opioid receptors. NAQ (17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamido]morphinan) is a novel opioid receptor ligand with low-efficacy at mu receptors and greater mu-receptor selectivity than existing low-efficacy agonists., Objectives: This study examined behavioral effects of NAQ in rats using an intracranial self-stimulation (ICSS) procedure that has been used previously to examine other opioids. NAQ effects were examined before, during, and after chronic morphine treatment, and effects of NAQ were compared to effects of nalbuphine and naltrexone., Methods: Adult male Sprague-Dawley rats were trained to respond for electrical brain stimulation delivered via electrodes implanted in the medial forebrain bundle. A range of brain stimulation frequencies maintained a wide range of baseline ICSS rates. Effects of NAQ (0.32-10 mg/kg), nalbuphine (1.0 mg/kg), and naltrexone (0.1 mg/kg) were determined before morphine treatment and during treatment with 3.2 and 18 mg/kg/day morphine. NAQ effects were also redetermined beginning 2 weeks after termination of morphine treatment., Results: NAQ produced weak ICSS facilitation in morphine-naïve rats but more robust ICSS facilitation during and after morphine treatment and also reversed morphine withdrawal-associated depression of ICSS. These effects were similar to effects of nalbuphine., Conclusions: These results agree with the in vitro characterization of NAQ as a low-efficacy mu agonist. Opioid exposure may enhance abuse-related effects of NAQ, but NAQ may also serve as a low-efficacy and relatively safe option for treatment of opioid withdrawal or dependence.

It is generally believed that NMDA receptor antagonism accounts for most of the anesthetic and analgesic effects of ketamine, however, it interacts at multiple sites in the central nervous system, including NMDA and non-NMDA glutamate receptors, nicotinic and muscarinic cholinergic receptors, and adrenergic and opioid receptors. Interestingly, it was shown that at supraspinal sites, ketamine interacts with the μ-opioid system and causes supraspinal antinociception. In this study, we investigated the involvement of endogenous opioids in ketamine-induced central antinociception. The nociceptive threshold for thermal stimulation was measured in Swiss mice using the tail-flick test. The drugs were administered via the intracerebroventricular route. Our results demonstrated that the opioid receptor antagonist naloxone, the μ-opioid receptor antagonist clocinnamox and the δ-opioid receptor antagonist naltrindole, but not the κ-opioid receptor antagonist nor-binaltorphimine, antagonized ketamine-induced central antinociception in a dose-dependent manner. Additionally, the administration of the aminopeptidase inhibitor bestatin significantly enhanced low-dose ketamine-induced central antinociception. These data provide evidence for the involvement of endogenous opioids and μ- and δ-opioid receptors in ketamine-induced central antinociception. In contrast, κ-opioid receptors not appear to be involved in this effect., (Copyright © 2014 Elsevier B.V. All rights reserved.)

It is eastablished that kappa-opioid receptors have complicated structure. These receptors may play the important role in different processes in CNS.

Rationale: Accumulating evidence indicates that brain kappa-opioid receptors (KORs) and dynorphin, the endogenous ligand that binds at these receptors, are involved in regulating states of motivation and emotion. These findings have stimulated interest in the development of KOR-targeted ligands as therapeutic agents. As one example, it has been suggested that KOR antagonists might have a wide range of indications, including the treatment of depressive, anxiety, and addictive disorders, as well as conditions characterized by co-morbidity of these disorders (e.g., post-traumatic stress disorder) A general effect of reducing the impact of stress may explain how KOR antagonists can have efficacy in such a variety of animal models that would appear to represent different disease states., Objective: Here, we review evidence that disruption of KOR function attenuates prominent effects of stress. We will describe behavioral and molecular endpoints including those from studies that characterize the effects of KOR antagonists and KOR ablation on the effects of stress itself, as well as on the effects of exogenously delivered corticotropin-releasing factor, a brain peptide that mediates key effects of stress., Conclusion: Collectively, available data suggest that KOR disruption produces anti-stress effects and under some conditions can prevent the development of stress-induced adaptations. As such, KOR antagonists may have unique potential as therapeutic agents for the treatment and even prevention of stress-related psychiatric illness, a therapeutic niche that is currently unfilled.

Unlabelled: (11)C-LY2795050 is a novel κ-selective antagonist PET tracer. The in vitro binding affinities (Ki) of LY2795050 at the κ-opioid (KOR) and μ-opioid (MOR) receptors are 0.72 and 25.8 nM, respectively. Thus, the in vitro KOR/MOR binding selectivity is about 36:1. Our goal in this study was to determine the in vivo selectivity of this new KOR antagonist tracer in the monkey., Methods: To estimate the ED50 value (dose of a compound [or drug] that gives 50% occupancy of the target receptor) of LY2795050 at the MOR and KOR sites, 2 series of blocking experiments were performed in 3 rhesus monkeys using (11)C-LY2795050 and (11)C-carfentanil with coinjections of various doses of unlabeled LY2795050. Kinetic modeling was applied to calculate regional binding potential (BP(ND)), and 1- and 2-site binding curves were fitted to these data to measure (11)C-LY2795050 binding selectivity., Results: The LY2795050 ED50 at MOR was 119 μg/kg based on a 1-site model for (11)C-carfentanil. The 1-site binding model was also deemed sufficient to describe the specific binding of (11)C-LY2795050 at KOR. The ED50 at KOR estimated from the 1-site model was 15.6 μg/kg. Thus, the ED50 ratio for MOR:KOR was 7.6., Conclusion: The in vivo selectivity of (11)C-LY2795050 for KOR over MOR is 7.6. (11)C-LY2795050 has 4.7-fold-lower selectivity at KOR over MOR in vivo as compared with in vitro. Nevertheless, on the basis of our finding in vivo, 88% of the PET-observed specific binding of (11)C-LY2795050 under baseline conditions will be due to binding of the tracer at the KOR site in a region with similar prevalence of KOR and MOR. (11)C-LY2795050 is sufficiently selective for KOR over MOR in vivo to be considered an appropriate probe for studying the KOR with PET.

Background: The actions of endogenous opioid peptides are mediated by 3 main classes of opioid receptors; mu (MOR), kappa (KOR), and delta (DOR)., Methods: We developed an absolute quantitative real-time reverse transcriptase PCR (AQ-rt-RT-PCR) assay to quantify MOR, DOR, and KOR mRNA in 22 human tissues., Results: MOR mRNA was greatly enriched (12-20×10(6)copies/μg) in the cerebellum, nucleus accumbens, and caudate nucleus; moderate (6×10(6)copies/μg) in the dorsal root ganglion, spinal cord, and adrenal gland; low (2×10(4)copies/μg) in the pancreas and small intestine; and absent in the lung, spleen, kidney, heart, skeletal muscle, liver, and thymus. High levels (>8.8×10(6)copies/μg) of DOR mRNA were expressed in the brain and dorsal root ganglion; moderate (1.5×10(6)copies/μg) in the adrenal gland and pancreas; low (2×10(4)-6.5×10(5)copies/μg in the cerebellum, spinal cord, small intestine, skeletal muscle, thymus, lung, and kidney); and very low (3.8×10(3)copies/μg) in the heart. DOR mRNA was not detected in the spleen or liver. KOR mRNA was moderate (1×10(6)copies/μg) in brain regions and dorsal root ganglion, but low (1.6-7×10(5)copies/μg) in the cerebellum, temporal lobe and all other peripheral tissues., Conclusions: Our data demonstrate that the AQ-rt-RT-PCR is a highly reproducible and precise method to study the expression of opioid receptors in various tissues and under different disease conditions., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The Ro1 model of hydrocephalus represents an excellent model for studying the pathogenesis of hydrocephalus due to its complete penetrance and inducibility, enabling the investigation of the earliest cellular and histological changes in hydrocephalus prior to overt pathology. Hematoxylin and eosin staining, immunofluorescence and electron microscopy were used to characterize the histopathological events of hydrocephalus in this model. Additionally, a broad battery of behavioral tests was used to investigate behavioral changes in the Ro1 model of hydrocephalus. The earliest histological changes observed in this model were ventriculomegaly and disorganization of the ependymal lining of the aqueduct of Sylvius, which occurred concomitantly. Ventriculomegaly led to thinning of the ependyma, which was associated with periventricular edema and areas of the ventricular wall void of cilia and microvilli. Ependymal denudation was subsequent to severe ventriculomegaly, suggesting that it is an effect, rather than a cause, of hydrocephalus in the Ro1 model. Additionally, there was no closure of the aqueduct of Sylvius or any blockages within the ventricular system, even with severe ventriculomegaly, suggesting that the Ro1 model represents a model of communicating hydrocephalus. Interestingly, even with severe ventriculomegaly, there were no behavioral changes, suggesting that the brain is able to compensate for the structural changes that occur in the pathogenesis of hydrocephalus if the disorder progresses at a sufficiently slow rate.

We examined whether sex differences in κ-opioid receptor (KOPR) pharmacology exist in guinea pigs, which are more similar to humans in the expression level and distribution of KOPR in the brain than rats and mice. The KOPR agonist trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)benzeneacetamide methanesulfonate (U50,488H) produced a dose-dependent increase in abnormal postures and immobility with more effects in males than females. Males also showed more U50,488H-induced antinociception in the paw pressure test than females. Pretreatment with the KOPR antagonist norbinaltorphimine blocked U50,488H-induced abnormal body postures and antinociception. In contrast, inhibition of cocaine-induced hyperambulation by U50,488H was more effective in females than males. Thus, sex differences in the effects of U50,488H are endpoint-dependent. We then examined whether sex differences in KOPR levels and KOPR-mediated G protein activation in brain regions may contribute to the observed differences using quantitative in vitro autoradiography of [(3)H](5a,7a,8b)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)1-oxaspiro(4,5)dec-8-yl)benzeacetamide ([(3)H]U69,593) binding to the KOPR and U50,488H-stimulated guanosine 5'-O-(3-[(35)S]thiotriphosphate ([(35)S]GTPγS) binding. Compared with females, males exhibited more [(3)H]U69,593 binding in the deep layers of somatosensory and insular cortices, claustrum, endopiriform nucleus, periaqueductal gray, and substantial nigra. Concomitantly, U50,488H-stimulated [(35)S]GTPγS binding was greater in males than females in the superficial and deep layers of somatosensory and insular cortices, caudate putamen, claustrum, medial geniculate nucleus, and cerebellum. In contrast, compared with males, females showed more U50,488H-stimulated [(35)S]GTPγS binding in the dentate gyrus and a trend of higher [(35)S]GTPγS binding in the hypothalamus. These data demonstrate that males and females differ in KOPR expression and KOPR-mediated G protein activation in distinct brain regions, which may contribute to the observed sex differences in KOPR-mediated pharmacology.

Objectives: Repeated morphine treatment results in a decreased analgesic effect or the development of analgesic tolerance. However, we reported that some inflammatory chronic pain may inhibit morphine tolerance via kappa opioid receptor (KOR) activation. In this study, we further investigated the role of KOR in the inhibition of morphine tolerance in a chronic pain condition with a focus on the regulation of protein kinase C (PKC) activity., Methods: Chronic pain was induced by formalin treatment into the dorsal part of the left hind paws of mice. The analgesic effect of morphine was measured by the tail flick method. We analysed the protein expression of PKC and its activity, and G-protein activity of mu opioid receptor (MOR) under repeated morphine treatment with or without formalin treatment., Key Findings: We found that conventional subtypes of PKC (cPKC) were up-regulated by repeated morphine treatment. Also, antisense oligonucleotide (AS-ODN) targeting cPKC completely suppressed the development of morphine tolerance. The disappearance of the repeated morphine-induced up-regulation of cPKC was completely reversed by treatment with AS-ODN targeting KOR. In addition, AS-ODN targeting KOR significantly reversed the chronic pain-induced down-regulation of PKC activity or up-regulation of MOR [(35)S]GTPgammaS binding activity after repeated morphine treatment., Conclusions: These results indicate that KOR plays an important role in the inhibition of repeated morphine-induced cPKC up-regulation under chronic pain condition. Furthermore, this may result in the increase of MOR activity and in the inhibition of morphine tolerance under chronic pain condition.

We investigated D1, D2 receptors and dopamine transporter (DAT) binding levels in mice lacking all three opioid receptors and wild-type (WT) mice on three different genetic backgrounds. Quantitative autoradiography was used to determine the level of radioligand binding to the D1 and D2 receptors and DAT labeled with [(3)H]SCH23390, [(3)H]raclopride, and [(3)H]mazindol, respectively in triple-opioid receptor knockout (KO) and WT maintained on C57BL/6 (B6) and 129/SvEvTac (129) as well as C57BL/6 x 129/SvPas (B6 x 129) strains. No significant genotype effect was observed in D1, D2 receptors and DAT binding in any regions analyzed in any of the strains studied, suggesting that a lack of all three opioid receptors does not influence D1, D2 receptors and DAT expression, irrespective of their genetic strain background. However, strain differences were observed in D1 binding between the three strains of mice studied. Lower levels of D1 binding were observed in the substantia nigra of B6 x 129 WT mice compared with the 129 WT mice and in the olfactory tubercle of B6 x 129 WT compared with B6 WT and 129 WT mice. Lower levels of D1 binding were observed in the caudate putamen of B6 x 129 KO mice compared with 129 KO mice. In contrast, no significant strain differences were observed in D2 and DAT binding between the three strains of mice in any regions analyzed. Overall, these results indicate a lack of modulation of the dopaminergic system by the deletion of all three opioid receptors regardless of different background strains.

The dynorphin-like peptides have profound effects on the state of the brain reward system and human and animal behavior. The dynorphin-like peptides affect locomotor activity, food intake, sexual behavior, anxiety-like behavior, and drug intake. Stimulation of kappa-opioid receptors, the endogenous receptor for the dynorphin-like peptides, inhibits dopamine release in the striatum (nucleus accumbens and caudate putamen) and induces a negative mood state in humans and animals. The administration of drugs of abuse increases the release of dopamine in the striatum and mediates the concomitant release of dynorphin-like peptides in this brain region. The reviewed studies suggest that chronic drug intake leads to an upregulation of the brain dynorphin system in the striatum and in particular in the dorsal part of the striatum/caudate putamen. This might inhibit drug-induced dopamine release and provide protection against the neurotoxic effects of high dopamine levels. After the discontinuation of chronic drug intake these neuroadaptations remain unopposed which has been suggested to contribute to the negative emotional state associated with drug withdrawal and increased drug intake. kappa-Opioid receptor agonists have also been shown to inhibit calcium channels. Calcium channel inhibitors have antidepressant-like effects and inhibit the release of norepinephrine. This might explain that in some studies kappa-opioid receptor agonists attenuate nicotine and opioid withdrawal symptomatology. A better understanding of the role of dynorphins in the regulation of brain reward function might contribute to the development of novel treatments for mood disorders and other disorders that stem from a dysregulation of the brain reward system.

Background and Purpose: Drugs targeting brain kappa-opioid receptors produce profound alterations in mood. In the present study we investigated the possible anxiolytic- and antidepressant-like effects of the kappa-opioid receptor agonist salvinorin A, the main active ingredient of Salvia divinorum, in rats and mice., Experimental Approach: Experiments were performed on male Sprague-Dawley rats or male Albino Swiss mice. The anxiolytic-like effects were tested by using the elevated plus maze, in rats. The antidepressant-like effect was estimated through the forced swim (rats) and the tail suspension (mice) test. kappa-Opioid receptor involvement was investigated pretreating animals with the kappa-opioid receptor antagonist, nor-binaltorphimine (1 or 10 mgxkg(-1)), while direct or indirect activity at CB(1) cannabinoid receptors was evaluated with the CB(1) cannabinoid receptor antagonist, N-(piperidin-1-yl) -5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, 0.5 or 3 mgxkg(-1)), binding to striatal membranes of naïve rats and assay of fatty acid amide hydrolase in prefrontal cortex, hippocampus and amygdala., Key Results: Salvinorin A, given s.c. (0.001-1000 microgxkg(-1)), exhibited both anxiolytic- and antidepressant-like effects that were prevented by nor-binaltorphimine or AM251 (0.5 or 3 mgxkg(-1)). Salvinorin A reduced fatty acid amide hydrolase activity in amygdala but had very weak affinity for cannabinoid CB(1) receptors., Conclusions and Implications: The anxiolytic- and antidepressant-like effects of Salvinorin A are mediated by both kappa-opioid and endocannabinoid systems and may partly explain the subjective symptoms reported by recreational users of S. divinorum.

Neuropeptide VF (NPVF) induces satiety through hypothalamic interactions; however, the central mechanism that mediates these effects is poorly understood. Therefore, this study was conducted to explore some possible opioid receptor associated mechanisms of NPVF-induced satiety using chicks as models. Co-injection of NPVF and a mu opioid receptor antagonist (beta-funaltrexamine, FNA) did not have an additive suppressive effect on food intake compared to NPVF and FNA when injected alone. Contrary, co-injection of NPVF and a delta opioid receptor antagonist (ICI-174,864, ICI) caused a greater reduction in food intake than when both were injected alone. Co-injection of NPVF and a kappa opioid receptor antagonist (nor-binaltorphimine, BNI) did not cause an additive suppressive effect on food intake than when the two were injected alone. A reversal of neuropeptide Y and beta-endorphin induction of food intake occurred when NPVF was co-injected. These results support that NPVF-induced satiety is mediated through mu and kappa but not delta subtypes of opioid receptors, and their ligands including neuropeptide Y and beta-endorphin. Thus, NPVF-associated anorexia may be mediated via modulation of the chick's innate opioid-associated orexigenic system.

Background: Critically ill neonates experience multiple stressors during hospitalization. Opioids are commonly prescribed to ameliorate their pain and stress. However, the enduring effects of stress and opioids are not understood. The kappa opioid system is important in the mediation of stress in adults, but little is known about its function in neonates., Objectives: To characterize kappa opioid receptor (KOR) distribution in the neonatal mouse brain and test whether neonatal exposure to morphine, stress, or both, change KOR signaling., Methods: Five groups of wild-type C57BL/6 or prodynorphin (Pdyn) knockout mice were tested: (1) untreated control (dam-reared, no handling), (2) saline-injected control, (3) morphine-injected control, (4) stressed with saline injections and (5) stressed with morphine injections. Mice were treated from postnatal day 5 to postnatal day 9, after which their brains were immunolabeled with a phospho-specific KOR antibody (KOR-P), glial fibrillary acidic protein or glutamic acid decarboxylase., Results: There were no effects of saline or morphine injection on KOR-P immunoreactivity. Neonatal stress increased KOR-P labeling in wild-type brains (p < 0.05), but not in Pdyn(-/-) animals. Mice exposed to stress and morphine showed region-specific increases in KOR-P immunoreactivity from 38 to 500% (p < 0.05 to p < 0.001), with marked gliosis. In stressed morphine-treated Pdyn(-/-) animals, KOR-P immunoreactivity was absent, but gliosis increased compared to wild-type animals., Conclusions: Neonatal stress increases KOR activation via the dynorphin system. Neonatal stress plus morphine treatment further increased this response and also resulted in hippocampal gliosis. Enhanced gliosis noted in Pdyn(-/-) animals suggests that the endogenous dynorphin may play a role in downregulating this inflammatory response., (Copyright 2009 S. Karger AG, Basel.)

Alterations in the opioidergic system may play a role in the molecular mechanisms underlying neurochemical responses to cerebral ischaemia. The present study aimed to determine the delayed expression of mu, delta and kappa opioid receptors, following 1, 2, 7, and 30 days of middle cerebral artery occlusion (MCAO) in mice. Using quantitative autoradiography, we highlighted significant decreases in mu, delta and kappa opioid receptor expression in ipsilateral cortices from day 1 post-MCAO. Moreover, in contralateral nucleus lateralis thalami pars posterior, ipsi- and contralateral nucleus medialis dorsalis thalami, and ipsilateral substantia nigra, pars reticulata (SNr), kappa receptors were increased; mu receptor densities were decreased in nucleus ventralis thalami, pars posterior (VThP), and SNr. delta-Binding sites were increased in the striatum on day 30 post-MCAO. The alterations in opioid receptors in cortical infarcts were correlated with strong histological damage. Further reductions in opioid receptor densities in cortical infarcts were observed at later time points. In subcortical brain regions, opioid receptor densities were also altered but no histological damage was seen, except in the VThP, in which cell density was increased on day 30. Delayed reductions in opioid receptor densities in the infarct appeared as the continuation of the early processes previously demonstrated. However, changes in subcortical opioid receptor expression may correlate with neuronal alterations in remote brain regions. Changes in opioidergic receptor expression in these regions may be involved in the long-term consequences of stroke and could be used as biomarker of neuronal alteration through the use of imaging techniques in the clinic.

LY255582 is a pan opioid selective receptor antagonist that has been shown to have high affinity for mu, delta, and kappa receptors in vitro. In order to better understand the in vivo opioid receptor selectivity of LY255582, we developed in vivo receptor occupancy assays in the rat for the opioid mu, kappa and delta receptors using the occupancy tracers naltrexone, GR103545 and naltriben respectively. Individual assays for each target were established and then a "triple tracer" assay was created where all three tracers were injected simultaneously, taking advantage of LC/MS/MS technology to selectively monitor brain tracer levels. This is the first report of a technique to concurrently measure receptor specific occupancy at three opioid receptors in the same animal. The opioid subtype selective antagonists cyprodime, JDTic and naltrindole were used to validate selectivity of the assay. Examination of LY255582 in dose-occupancy experiments demonstrated a relative order of potency of mu>kappa>delta, reproducing the previously reported order determined with in vitro binding.

This study was designed to assess the effects of opiate treatment on the expression of Fas-associated protein with death domain (FADD) in the rat brain. FADD is involved in the transmission of Fas-death signals that have been suggested to contribute to the development of opiate tolerance and addiction. Acute treatments with high doses of sufentanil and morphine (mu-agonists), SNC-80 (delta-agonist), and U50488H (kappa-agonist) induced significant decreases (30-60%) in FADD immunodensity in the cerebral cortex, through specific opioid receptor mechanisms (effects antagonized by naloxone, naltrindole, or nor-binaltorphimine). The cannabinoid CB1 receptor agonist WIN 55,212-2 did not alter FADD content in the brain. Chronic (5 days) morphine (10-100 mg/kg), SNC-80 (10 mg/kg), or U50488H (10 mg/kg) was associated with the induction of tachyphylaxis to the acute effects. In morphine- and SNC-80-tolerant rats, antagonist-precipitated (2 h) or spontaneous withdrawal (24-48 h) induced a new and sustained inhibition of FADD (13-50%). None of these treatments altered the densities of caspases 8/3 (including the active cleaved forms) in the brain. Pretreatment of rats with SL 327 (a selective MEK1/2 inhibitor that blocks ERK activation) fully prevented the reduction of FADD content induced by SNC-80 in the cerebral cortex (43%) and corpus striatum (29%), demonstrating the direct involvement of ERK1/2 signaling in the regulation of FADD by the opiate agonist. The results indicate that mu- and delta-opioid receptors have a prominent role in the modulation of FADD (opposite to that of Fas) shortly after initiating treatment. Opiate drugs (and specifically the delta-agonists) could promote survival signals in the brain through inhibition of FADD, which in turn is dependent on the activation of the antiapoptotic ERK1/2 signaling pathway.

Although the neurochemical mechanisms contributing to alcohol withdrawal seizures are poorly understood, withdrawal seizures probably reflect neuronal hyperexcitability resulting from adaptation to chronic alcohol. Altered kappa-Opioid receptor (KOP-R) signaling has been observed in multiple seizure types; however, a role for this system in ethanol withdrawal seizures has not been systematically characterized. We hypothesized that pharmacological manipulations of the KOP-R would alter withdrawal in mice selectively bred for differences in ethanol withdrawal severity. Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were made physically dependent using chronic ethanol vapor inhalation, and the effects of the KOP-R antagonist nor-binaltorphimine or agonist U-50,488H on withdrawal severity were examined. Pretreatment with nor-binaltorphimine significantly increased handling-induced convulsion (HIC) severity in withdrawing WSR mice, with no observable effects in withdrawing WSP mice. In contrast, U-50,488H significantly decreased HIC severity in WSP mice, with no effects in WSR mice. During extended withdrawal (i.e. hours 12+), a rebound hyperexcitability was observed in WSP mice given agonist. Thus, administration of a KOP-R antagonist increased withdrawal severity in mice normally resistant to withdrawal seizures, while a KOP-R agonist reduced convulsion severity in animals susceptible to withdrawal seizures. These observations are consistent with differences in the KOP-R system observed in these lines at the molecular level, and suggest the KOP-R system may be a promising therapeutic target for management of ethanol withdrawal seizures. Finally, these findings underscore the importance of determining the potential for rebound increases in withdrawal severity during later withdrawal episodes.

Agonist stimulation of opioid receptors increases feeding in rodents, while opioid antagonists inhibit food intake. The pan-opioid antagonist, LY255582, produces a sustained reduction in food intake and body weight in rodent models of obesity. However, the specific receptor subtype(s) responsible for this activity is unknown. To better characterize the pharmacology of LY255582, we examined the binding of a radiolabeled version of the molecule, [(3)H]-LY255582, in mouse brain using autoradiography. In mouse brain homogenates, the K(d) and B(max) for [(3)H]-LY255582 were 0.156 +/- 0.07 nM and 249 +/- 14 fmol/mg protein, respectively. [(3)H]-LY255582 bound to slide mounted sections of mouse brain with high affinity and low non-specific binding. High levels of binding were seen in areas consistent with the known localization of opioid receptors. These areas included the caudate putamen, nucleus accumbens, claustrum, medial habenula, dorsal endopiriform nucleus, basolateral nucleus of the amygdala, hypothalamus, thalamus and ventral tegmental area. We compared the binding distribution of [(3)H]-LY255582 to the opioid receptor antagonist radioligands [(3)H]-naloxone (mu), [(3)H]-naltrindole (delta) and [(3)H]-norBNI (kappa). The overall distribution of [(3)H]-LY255582 binding sites was similar to that of the other ligands. No specific [(3)H]-LY255582 binding was noted in sections of mu-, delta- and kappa-receptor combinatorial knockout mice. Therefore, it is likely that LY255582 produces its effects on feeding and body weight gain through a combination of mu-, delta- and kappa-receptor activity.

Abrupt withdrawal from chronic alcohol exposure can produce convulsions that are likely due to ethanol (EtOH) neuroadaptations. While significant efforts have focused on elucidating dependence mechanisms, the alterations contributing to EtOH withdrawal severity are less well characterized. The present studies examined the kappa-opioid receptor (KOP-R) system in Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice, selected lines that display severe and mild convulsions upon removal from chronic EtOH exposure. Previous data demonstrated significant increases in whole brain prodynorphin (Pdyn) mRNA in WSP mice only during EtOH withdrawal. No significant effects of EtOH exposure or withdrawal were observed in WSR mice. The present study characterized Pdyn mRNA and the KOP-R in WSP and WSR mice during EtOH withdrawal using in situ hybridization (ISH) and KOP-R autoradiography. Analyses were performed in brain regions that express Pdyn mRNA and/or KOP-R and that might participate in seizure circuitry: the piriform cortex, olfactory tubercle, nucleus accumbens, caudate-putamen, claustrum, dorsal endopiriform nucleus, and cingulate cortex. ISH analyses confirmed previous findings; EtOH withdrawal increased Pdyn mRNA in multiple brain regions of WSP mice, but not WSR. Basal KOP-R binding was higher in WSR mice than in WSP mice, suggesting an anti-convulsant role for receptor activation. Finally, increased KOP-R density was present during EtOH withdrawal in WSP mice. These data suggest that differences in the KOP-R system among the lines might contribute to their selected difference in EtOH withdrawal severity.

A full-length cDNA that encodes a kappa (kappa) opioid receptor has been isolated from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa. The deduced protein contains 385 amino acids and possesses features commonly attributed to G protein-coupled receptors, such as seven putative transmembrane domains. The newt kappa receptor has 75% sequence identity to kappa opioid receptors cloned from mammals, and 66% sequence identity to the kappa opioid receptor reported for the zebrafish, with the greatest divergence in the extracellular N-terminus, the second and third extracellular loops and the intracellular C-terminus. Membranes isolated from COS-7 cells expressing the newt kappa receptor possessed a single, high-affinity (Kd = 1.5 nM) binding site for the kappa-selective agonist U69593. In competition binding assays, the expressed newt kappa receptor displayed high affinity for the kappa-selective agonists GR89696, dynorphin A(1-13), U69593, U50488 and BRL52537, as well as the kappa-selective antagonist nor-binaltorphimine and the non-selective antagonist naloxone. Rank order potencies and affinity constants were similar in competition binding studies that used either whole brain homogenates or membranes isolated from COS-7 cells expressing the newt kappa receptor. The expressed receptor displayed essentially no affinity for the delta-selective agonist DPDPE ([d-penicillamine, d-penicillamine]enkephalin), but showed moderate affinity for the mu-selective agonist DAMGO ([d-Ala-MePhe, Gly-ol]enkephalin) and moderately high affinity for nociceptin (orphanin FQ), the endogenous ligand for the opioid receptor-like (ORL)1 receptor. These findings support the conclusions that a gene for the kappa opioid receptor is expressed in amphibians and that the pharmacology of the newt kappa receptor closely matches mammalian kappa opioid receptors. However, the functional dichotomy between the classic opioid receptors (kappa, delta, mu) and ORL1 appears less strict in amphibians than in mammals.