preproenkephalin and norbinaltorphimine

Given that the κ opioid receptor (KOR) system has been implicated in psychostimulant abuse, we evaluated whether the selective KOR antagonist norbinaltorphimine dihydrochloride (nor-BNI) would attenuate the escalation of methamphetamine (METH) intake in an extended-access self-administration model. Systemic nor-BNI decreased the escalation of intake of long-access (LgA) but not short-access (ShA) self-administration. nor-BNI also decreased elevated progressive-ratio (PR) breakpoints in rats in the LgA condition and continued to decrease intake after 17 d of abstinence, demonstrating that the effects of a nor-BNI injection are long lasting. Rats with an ShA history showed an increase in prodynorphin immunoreactivity in both the nucleus accumbens (NAc) core and shell, but LgA animals showed a selective increase in the NAc shell. Other cohorts of rats received nor-BNI directly into the NAc shell or core and entered into ShA or LgA. nor-BNI infusion in the NAc shell, but not NAc core, attenuated escalation of intake and PR responding for METH in LgA rats. These data indicate that the development and/or expression of compulsive-like responding for METH under LgA conditions depends on activation of the KOR system in the NAc shell and suggest that the dynorphin-KOR system is a central component of the neuroplasticity associated with negative reinforcement systems that drive the dark side of addiction.

Topics: Analysis of Variance; Animals; Central Nervous System Stimulants; Conditioning, Operant; Enkephalins; Male; Methamphetamine; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Protein Precursors; Rats; Rats, Wistar; Receptors, Opioid, kappa; Reinforcement, Psychology; Self Administration

The association between morphine withdrawal and depressive-like symptoms is well documented, however, the role of dynorphin/κ opioid receptor system and the underlying neural substrates have not been fully understood. In the present study, we found that four weeks morphine abstinence after a chronic escalating morphine regimen significantly induced depressive-like behaviors in mice. Prodynorphin mRNA and protein levels were increased in the nucleus accumbens (NAc) after four weeks of morphine withdrawal. Local injection of κ opioid receptor antagonist nor-Binaltorphimine (norBNI) in the NAc significantly blocked the expression of depressive-like behaviors without influencing general locomotor activity. Thus, the present study extends previous findings by showing that prolonged morphine withdrawal-induced depressive-like behaviors are regulated by dynorphin/κ opioid receptor system, and shed light on the κ opioid receptor antagonists as potential therapeutic agents for the treatment of depressive-like behaviors induced by opiate withdrawal.

Topics: Animals; Antidepressive Agents; Depressive Disorder; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalins; Male; Mice, Inbred C57BL; Morphine; Motor Activity; Naltrexone; Narcotic Antagonists; Narcotics; Nucleus Accumbens; Protein Precursors; Receptors, Opioid, kappa; RNA, Messenger; Substance Withdrawal Syndrome

Emotionally arousing events like encounter with an unfamiliar con-species produce strong and vivid memories, whereby the hippocampus and amygdala play a crucial role. It is less understood, however, which neurotransmitter systems regulate the strength of social memories, which have a strong emotional component. It was shown previously that dynorphin signalling is involved in the formation and extinction of fear memories, therefore we asked if it influences social memories as well. Mice with a genetic deletion of the prodynorphin gene Pdyn (Pdyn(-/-)) showed a superior partner recognition ability, whereas their performance in the object recognition test was identical as in wild-type mice. Pharmacological blockade of kappa opioid receptors (KORs) led to an enhanced social memory in wild-type animals, whereas activation of KORs reduced the recognition ability of Pdyn(-/-) mice. Partner recognition test situation induced higher elevation in dynorphin A levels in the central and basolateral amygdala as well as in the hippocampus, and also higher dynorphin B levels in the hippocampus than the object recognition test situation. Our result suggests that dynorphin system activity is increased in emotionally arousing situation and it decreases the formation of social memories. Thus, dynorphin signalling is involved in the formation of social memories by diminishing the emotional component of the experience.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amygdala; Animals; Enkephalins; Hippocampus; Memory; Mice; Mice, Knockout; Naltrexone; Narcotic Antagonists; Protein Precursors; Receptors, Opioid, kappa; Recognition, Psychology; Social Behavior

The abuse of opioid drugs, both illicit and prescription, is a persistent problem in the United States, accounting for >1.2 million users who require treatment each year. Current treatments rely on suppressing immediate withdrawal symptoms and replacing illicit drug use with long-acting opiate drugs. However, the mechanisms that lead to preventing opiate dependence are still poorly understood. We hypothesized that κ opioid receptor (KOR) activation during chronic opioid intake contributes to negative affective states associated with withdrawal and the motivation to take increasing amounts of heroin. Using a 12 h long-access model of heroin self-administration, rats showed escalation of heroin intake over several weeks. This was prevented by a single high dose (30 mg/kg) of the long-acting KOR antagonist norbinaltorphimine (nor-BNI), paralleled by reduced motivation to respond for heroin on a progressive-ratio schedule of reinforcement, a measure of compulsive-like responding. Systemic nor-BNI also significantly decreased heroin withdrawal-associated anxiety-like behavior. Immunohistochemical analysis showed prodynorphin content increased in the nucleus accumbens core in all heroin-exposed rats, but selectively increased in the nucleus accumbens shell in long-access rats. Local infusion of nor-BNI (4 μg/side) into accumbens core altered the initial intake of heroin but not the rate of escalation, while local injection into accumbens shell selectively suppressed increases in heroin intake over time without altering initial intake. These data suggest that dynorphin activity in the nucleus accumbens mediates the increasing motivation for heroin taking and compulsive-like responding for heroin, suggesting that KOR antagonists may be promising targets for the treatment of opioid addiction.

Topics: Animals; Anxiety; Catheterization; Conditioning, Operant; Enkephalins; Heroin Dependence; Immunohistochemistry; Male; Motivation; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Protein Precursors; Rats; Rats, Wistar; Receptors, Opioid, kappa; Reinforcement Schedule; Self Administration; Substance Withdrawal Syndrome

The aim of this study was to determine the role of the endogenous dynorphin/kappa opioid receptor (DYN/KOP) system in ethanol-induced state-dependent conditioned place preference (CPP). To this end, mice lacking the pro-DYN gene and their wild-type littermates/controls were tested for baseline place preference on day 1, received 15-min morning and afternoon conditionings with saline or ethanol (2g/kg) each day for three consecutive days and were then tested for CPP under a drug-free state on day 5 and following a saline or ethanol (1 or 2g/kg) challenge on day 8. Given that compensatory developmental changes may occur in knockout mice, the effect of nor-binaltorphimine (nor-BNI), a KOP antagonist, on state-dependent CPP induced by ethanol was also studied in wild-type mice. On day 1, mice were tested for baseline place preference and, 4h later, treated with saline or nor-BNI (10mg/kg). On days 2-4, mice received 15-min morning and afternoon conditionings and were tested for CPP under a drug-free state on day 5 and following an ethanol (1g/kg) challenge on day 8. A comparable CPP was observed in mice lacking the pro-DYN gene and their wild-type littermates/controls as well as in wild-type mice treated with nor-BNI and their saline-treated controls. However, these mice compared to their respective controls exhibited a greater CPP response following an ethanol (1g/kg) challenge, suggesting that the endogenous DYN/KOP system may negatively regulate ethanol-induced state-dependent CPP.

Topics: Animals; Central Nervous System Depressants; Conditioning, Operant; Drug Administration Schedule; Dynorphins; Enkephalins; Ethanol; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Narcotic Antagonists; Protein Precursors; Sex Factors; Time Factors

Vulnerability to drug abuse disorders is determined not only by environmental but also by genetic factors. A body of evidence suggests that endogenous opioid peptide systems may influence rewarding effects of addictive substances, and thus, their individual expression levels may contribute to drug abuse liability.. The aim of our study was to assess whether basal genotype-dependent brain expression of opioid propeptides genes can influence sensitivity to morphine reward.. Experiments were performed on inbred mouse strains C57BL/6J, DBA/2J, and SWR/J, which differ markedly in responses to morphine administration: DBA/2J and SWR/J show low and C57BL/6J high sensitivity to opioid reward. Proenkephalin (PENK) and prodynorphin (PDYN) gene expression was measured by in situ hybridization in brain regions implicated in addiction. The influence of the kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI), which attenuates effects of endogenous PDYN-derived peptides, on rewarding actions of morphine was studied using the conditioned place preference (CPP) paradigm.. DBA/2J and SWR/J mice showed higher levels of PDYN and lower levels of PENK messenger RNA in the nucleus accumbens than the C57BL/6J strain. Pretreatment with nor-BNI enhanced morphine-induced CPP in the opioid-insensitive DBA/2J and SWR/J strains.. Our results demonstrate that inter-strain differences in PENK and PDYN genes expression in the nucleus accumbens parallel sensitivity of the selected mouse strains to rewarding effects of morphine. They suggest that high expression of PDYN may protect against drug abuse by limiting drug-produced reward, which may be due to dynorphin-mediated modulation of dopamine release in the nucleus accumbens.

Topics: Animals; Behavior, Addictive; Behavior, Animal; Conditioning, Psychological; Cues; Enkephalins; Gene Expression Regulation; Genotype; In Situ Hybridization; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Morphine Dependence; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Phenotype; Protein Precursors; Receptors, Opioid, kappa; Reward; RNA, Messenger; Species Specificity

Exposure to inescapable stressors increases both the rewarding properties and self-administration of cocaine through the signaling of the kappa-opioid receptor (KOR), but the effect of this signaling on other reinforcing agents remains unclear.. The objective of this study is to test the hypothesis that signaling of the KOR mediates the forced swim stress (FSS)-induced potentiation of ethanol reward and self-administration.. Male C57Bl/6J mice were tested in a biased ethanol-conditioned place preference (CPP) procedure, and both C57Bl/6J and prodynorphin gene-disrupted (Dyn -/-) mice were used in two-bottle free choice (TBC) assays, with or without exposure to FSS. To determine the role of the KOR in the resulting behaviors, the KOR agonist U50,488 (10 mg/kg) and antagonist nor-binaltorphimine (nor-BNI, 10 mg/kg) were administered prior to parallel testing.. C57Bl/6J mice exposed to repeated FSS 5 min prior to daily place conditioning with ethanol (0.8 g/kg) demonstrated a 4.4-fold potentiation of ethanol-CPP compared to unstressed mice that was prevented by nor-BNI pretreatment. Likewise, pretreatment with U50,488 90 min prior to daily ethanol place conditioning resulted in a 2.8-fold potentiation of ethanol-CPP. In the TBC assay, exposure to FSS significantly increased the consumption of 10% (v/v) ethanol by 19.3% in a nor-BNI-sensitive manner. Notably, Dyn -/- mice consumed a similar volume of ethanol as wild-type littermates and C57Bl/6J mice, but did not demonstrate significant stress-induced increases in consumption.. These data demonstrated a stress-induced potentiation of the rewarding effects and self-administration of ethanol mediated by KOR signaling.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Conditioning, Psychological; Enkephalins; Ethanol; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Protein Precursors; Receptors, Opioid, kappa; Reward; Self Administration; Signal Transduction; Stress, Physiological

Stress and anxiety are mainly regulated by amygdala and hypothalamic circuitries involving several neurotransmitter systems and providing physiological responses to peripheral organs via the hypothalamic-pituitary-adrenal axis and other pathways. The role of endogenous opioid peptides in this process is largely unknown. Here we show for the first time that anxiolytic parameters of explorative behavior in mice lacking prodynorphin were increased 2-4-fold in the open field, the elevated plus maze and the light-dark test. Consistent with this, treatment of wild-type mice with selective kappa-opioid receptor antagonists GNTI or norbinaltorphimine showed the same effects. Furthermore, treatment of prodynorphin knockout animals with U-50488H, a selective kappa-opioid receptor agonist, fully reversed their anxiolytic phenotype. These behavioral data are supported by an approximal 30% reduction in corticotropin-releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus and central amygdala and an accompanying 30-40% decrease in corticosterone serum levels in prodynorphin knockout mice. Although stress-induced increases in corticosterone levels were attenuated in prodynorphin knockout mice, they were associated with minor increases in depression-like behavior in the tail suspension and forced swim tests. Taken together, our data suggest a pronounced impact of endogenous prodynorphin-derived peptides on anxiety, but not stress coping ability and that these effects are mediated via kappa-opioid receptors. The delay in the behavioral response to kappa-opioid receptor agonists and antagonist treatment suggests an indirect control level for the action of dynorphin, probably by modulating the expression of CRH or neuropeptide Y, and subsequently influencing behavior.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amygdala; Animals; Anxiety; Brain Stem; Corticosterone; Corticotropin-Releasing Hormone; Dynorphins; Enkephalins; Exploratory Behavior; Female; Guanidines; Hypothalamus; Male; Maze Learning; Mesencephalon; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphinans; Naltrexone; Neuropeptide Y; Neuropeptides; Protein Precursors; Raphe Nuclei; Receptors, Opioid, kappa; Stress, Psychological

We hypothesized that mice subjected to prolonged stress would demonstrate decreased performance in a learning and memory task attributable to the endogenous activation of the kappa opioid receptor (KOR). C57BL/6J mice were tested using the novel object recognition (NOR) assay at various time points after exposure to repeated forced swim stress (FSS). Unstressed mice demonstrated recognition of the novel object at the end of a procedure using three 10-min object interaction phases, with a recognition index (RI) for the novel object of 71.7+/-3.4%. However, 1 h after exposure to FSS, vehicle-pretreated mice displayed a significant deficit in performance (RI=58.2+/-4.1%) compared with unstressed animals. NOR was still significantly reduced 4 but not 24 h after FSS. Treatment with the KOR-selective antagonist norbinaltorphimine (10 mg/kg, i.p.) prevented the decline in learning and memory performance. Moreover, direct activation of the KOR induced performance deficits in NOR, as exogenous administration of the KOR agonist U50,488 [(+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide] (0.3 mg/kg, i.p.) suppressed NOR (RI=56.0+/-3.9%). The effect of FSS on NOR performance was further examined in mice lacking the gene for the endogenous KOR agonist dynorphin (Dyn). Dyn gene-disrupted mice exposed to FSS did not show the subsequent learning and memory deficits (RI=66.8+/-3.8%) demonstrated by their wild-type littermates (RI=49.7+/-2.9%). Overall, these results suggest that stress-induced activation of the KOR may be both necessary and sufficient to produce subsequent deficits in novel object recognition.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analysis of Variance; Animals; Behavior, Animal; Enkephalins; Gene Expression Regulation; Immobility Response, Tonic; Learning Disabilities; Male; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Naltrexone; Narcotic Antagonists; Protein Precursors; Receptors, Opioid, kappa; Recognition, Psychology; Stress, Psychological; Swimming; Time Factors

Dynorphin is the presumed endogenous ligand for the kappa-opioid receptor. The dynorphin gene may play a role in psychotropic agent-mediated behavioral changes via dopaminergic modulation. Therefore, in this study, possible involvement of the dynorphin gene in nalbuphine-mediated behavioral responses was examined using prodynorphin (Pdyn) gene knock-out (-/-) mice. Pdyn gene deficiency potentiates nalbuphine-induced behavioral sensitization of locomotor activity and accumbal c-Fos expression. Administration of nalbuphine induced a significant increase in the dialysate dopamine level in the nucleus accumbens. This increase was more pronounced in the Pdyn (-/-) mice than in the wild-type (WT) mice. In addition, Pdyn (-/-) mice were more vulnerable to the naloxone-precipitated withdrawal syndrome (i.e., teeth chattering, wet dog shakes, forepaw tremors, jumping, weight loss, and global withdrawal score) after repeated treatment with nalbuphine than the WT mice. Consistently, nor-binaltorphimine, a kappa-opioid receptor antagonist, significantly potentiated nalbuphine-induced behavioral effects in WT mice, whereas U-50488H, a kappa-opioid receptor agonist, significantly attenuated these changes in Pdyn (-/-) mice in a dose-dependent manner. Our data suggest that the kappa-opioid receptor/dynorphin system is specifically modulated in response to behavioral sensitization and withdrawal signs induced by nalbuphine.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Area Under Curve; Blotting, Western; Dopamine; Enkephalins; Gene Expression; Genes, fos; Mice; Mice, Knockout; Microdialysis; Motor Activity; Nalbuphine; Naloxone; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Protein Precursors; Receptors, Opioid, kappa; Reverse Transcriptase Polymerase Chain Reaction; Substance Withdrawal Syndrome

Dehydration increases vasopressin (antidiuretic hormone) secretion from the posterior pituitary gland to reduce water loss in the urine. Vasopressin secretion is determined by action potential firing in vasopressin neurones, which can exhibit continuous, phasic (alternating periods of activity and silence), or irregular activity. Autocrine kappa-opioid inhibition contributes to the generation of activity patterning of vasopressin neurones under basal conditions and so we used in vivo extracellular single unit recording to test the hypothesis that changes in autocrine kappa-opioid inhibition drive changes in activity patterning of vasopressin neurones during dehydration. Dehydration increased the firing rate of rat vasopressin neurones displaying continuous activity (from 7.1 +/- 0.5 to 9.0 +/- 0.6 spikes s(1)) and phasic activity (from 4.2 +/- 0.7 to 7.8 +/- 0.9 spikes s(1)), but not those displaying irregular activity. The dehydration-induced increase in phasic activity was via an increase in intraburst firing rate. The selective -opioid receptor antagonist nor-binaltorphimine increased the firing rate of phasic neurones in non-dehydrated rats (from 3.4 +/- 0.8 to 5.3 +/- 0.6 spikes s(1)) and dehydrated rats (from 6.4 +/- 0.5 to 9.1 +/- 1.2 spikes s(1)), indicating that kappa-opioid feedback inhibition of phasic bursts is maintained during dehydration. In a separate series of experiments, prodynorphin mRNA expression was increased in vasopressin neurones of hyperosmotic rats, compared to hypo-osmotic rats. Hence, it appears that dynorphin expression in vasopressin neurones undergoes dynamic changes in proportion to the required secretion of vasopressin so that, even under stimulated conditions, autocrine feedback inhibition of vasopressin neurones prevents over-excitation.

Topics: Action Potentials; Animals; Cholecystokinin; Dehydration; Electrophysiology; Enkephalins; Female; Hypernatremia; Hyponatremia; Immunohistochemistry; In Situ Hybridization; Naltrexone; Narcotic Antagonists; Neurons; Oxytocin; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; RNA, Messenger; Vasopressins

Stress is a complex human experience having both positive and negative motivational properties. When chronic and uncontrollable, the adverse effects of stress on human health are considerable and yet poorly understood. Here, we report that the dysphoric properties of chronic stress are encoded by the endogenous opioid peptide dynorphin acting on specific stress-related neuronal circuits. Using different forms of stress presumed to evoke dysphoria in mice, we found that repeated forced swim and inescapable footshock both produced aversive behaviors that were blocked by a kappa-opioid receptor (KOR) antagonist and absent in mice lacking dynorphin. Injection of corticotropin-releasing factor (CRF) or urocortin III, key mediators of the stress response, produced place aversion that was also blocked by dynorphin gene deletion or KOR antagonism. CRF-induced place aversion was blocked by the CRF2 receptor antagonist antisauvigine-30, but not by the CRF1 receptor antagonist antalarmin. In contrast, place aversion induced by the KOR agonist U50,488 was not blocked by antisauvigine-30. These results suggest that the aversive effects of stress were mediated by CRF2 receptor stimulation of dynorphin release and subsequent KOR activation. Using a phospho-selective antibody directed against the activated KOR to image sites of dynorphin action in the brain, we found that stress and CRF each caused dynorphin-dependent KOR activation in the basolateral amygdala, nucleus accumbens, dorsal raphe, and hippocampus. The convergence of stress-induced aversive inputs on the dynorphin system was unexpected, implicates dynorphin as a key mediator of dysphoria, and emphasizes kappa-receptor antagonists as promising therapeutics.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Behavior, Animal; Conditioning, Operant; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Drug Interactions; Dynorphins; Enkephalins; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Narcotic Antagonists; Odorants; Phosphorylation; Protein Precursors; Receptors, Opioid, kappa; Stress, Physiological; Swimming; Urocortins

Previous studies have demonstrated that repeated forced-swim stress-induced behaviors (including analgesia, immobility, and increased drug reward) were mediated by the release of endogenous prodynorphin-derived opioid peptides and subsequent activation of the kappa opioid receptor (KOR). We tested the generality of these effects using a different type of stressful situation: repeated social defeat. C57Bl/6 mice subjected to social defeat stress (SDS) over 3 days showed a characteristic stress-induced immobility and defeated-postural response, as well as stress-induced analgesia (SIA). Daily pretreatment with the KOR antagonist nor-binaltorphimine (nor-BNI, 10 mg/kg, i.p.) blocked the SIA and significantly reduced the stress-induced immobility on the second and third days of SDS exposure. In contrast, prodynorphin gene-disrupted mice showed no significant increase in immobility, socially defeated postures, or SIA following repeated exposure to SDS. Since both stress and the kappa opioid system can modulate the response to drugs of abuse, we tested the effects of SDS on cocaine-conditioned place preference (CPP). SDS-exposed mice conditioned with cocaine (15 mg/kg, s.c.) showed significant potentiation of place-preference for the drug-paired chamber over the responses of unstressed mice. Nor-BNI pretreatment blocked stress-induced potentiation of cocaine-CPP. Consistent with this result, mice lacking the prodynorphin gene did not show stress-induced potentiation of cocaine-CPP, whereas wild-type littermates did. The findings suggest that chronic SDS may activate the kappa opioid system to produce analgesia, immobility, social defeat postures, and resulting in a potentiation of the acute rewarding properties of cocaine.

Topics: Analysis of Variance; Anesthetics, Local; Animals; Behavior, Animal; Cocaine; Conditioning, Operant; Enkephalins; Immobility Response, Tonic; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Narcotic Antagonists; Pain Measurement; Protein Precursors; Reaction Time; Receptors, Opioid, kappa; Social Behavior; Stress, Psychological

Effects of big dynorphin (Big Dyn), a prodynorphin-derived peptide consisting of dynorphin A (Dyn A) and dynorphin B (Dyn B) on memory function, anxiety, and locomotor activity were studied in mice and compared to those of Dyn A and Dyn B. All peptides administered i.c.v. increased step-through latency in the passive avoidance test with the maximum effective doses of 2.5, 0.005, and 0.7 nmol/animal, respectively. Effects of Big Dyn were inhibited by MK 801 (0.1 mg/kg), an NMDA ion-channel blocker whereas those of dynorphins A and B were blocked by the kappa-opioid antagonist nor-binaltorphimine (6 mg/kg). Big Dyn (2.5 nmol) enhanced locomotor activity in the open field test and induced anxiolytic-like behavior both effects blocked by MK 801. No changes in locomotor activity and no signs of anxiolytic-like behavior were produced by dynorphins A and B. Big Dyn (2.5 nmol) increased time spent in the open branches of the elevated plus maze apparatus with no changes in general locomotion. Whereas dynorphins A and B (i.c.v., 0.05 and 7 nmol/animal, respectively) produced analgesia in the hot-plate test Big Dyn did not. Thus, Big Dyn differs from its fragments dynorphins A and B in its unique pattern of memory enhancing, locomotor- and anxiolytic-like effects that are sensitive to the NMDA receptor blockade. The findings suggest that Big Dyn has its own function in the brain different from those of the prodynorphin-derived peptides acting through kappa-opioid receptors.

Topics: Amino Acid Sequence; Animals; Anti-Anxiety Agents; Anxiety; Behavior, Animal; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Hot Temperature; Male; Memory; Mice; Molecular Sequence Data; Motor Activity; Naltrexone; Pain Measurement; Protein Precursors; Reaction Time; Receptors, N-Methyl-D-Aspartate

Self-administration procedures have not yet provided evidence that freely moving mice can reliably acquire and maintain an operant behavior to self-administer cannabinoid agonists. The aim of the present work was to establish a model of cannabinoid operant intravenous self-administration in freely moving mice given the relevance of this species for the use of genetically modified animals. In addition, the possible involvement of the kappa/dynorphin system in cannabinoid self-administration was evaluated by using pro-dynorphin knockout mice. Outbred CD1 wild-type mice as well as pro-dynorphin knockout and wild-type mice were trained to self-administer the cannabinoid receptor agonist WIN 55,212-2 under an FR1 schedule of reinforcement. Two cannabinoid training doses (6.25 and 12.5 microg/kg/infusion) were used in the acquisition studies in outbred mice. Animals acquired a reliable operant responding to self-administer WIN 55,212-2 (12.5 microg/kg/infusion), but required as many as 15 sessions to attain this behavior. Interestingly, when a previous injection of WIN 55,212-2 (0.1 mg/kg, i.p.) was administered in the home-cage 24 h before the first session, mice acquired operant responding for cannabinoid self-administration by the fourth session. When the kappa-opioid agonist antagonist nor-binaltorphimine (5 mg/kg s.c.) was administered 4 h before the first session, the time required to acquire a reliable cannabinoid self-administration was also significantly reduced. Finally, a shift to the left in the dose-intake curve to self-administer WIN 55,212-2 was observed in pro-dynorphin knockout mice when compared to wild-type mice. These results indicate that the activation of the kappa/dynorphin opioid system after WIN 55,212-2 administration could counteract cannabinoid rewarding effects.

Topics: Animals; Benzoxazines; Cannabinoids; Conditioning, Operant; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Male; Mice; Mice, Knockout; Morpholines; Naltrexone; Naphthalenes; Protein Precursors; Receptor, Cannabinoid, CB1; Receptors, Opioid, kappa; Self Administration

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and alpha-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM(-/-) islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM(-/-) islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (approximately 200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate kappa-opioid receptors expressed predominantly in alpha-cells of the islet, we determined the role of dynorphin A-(1-17) in glucagon secretion from the alpha-cell. Stimulation with dynorphin A-(1-17) caused alpha-cell calcium fluctuations and a significant increase in glucagon release. DREAM(-/-) islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of alpha-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Calcium; Cell Line; Cell Nucleus; DNA; Dynorphins; Electrophoretic Mobility Shift Assay; Enkephalins; Gene Expression Regulation; Glucagon; Glucagon-Secreting Cells; Glucose; Humans; Insulin-Secreting Cells; Islets of Langerhans; Kv Channel-Interacting Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Naltrexone; Protein Binding; Protein Precursors; Receptors, Opioid, kappa; Repressor Proteins

Previous studies have demonstrated that stress may increase prodynorphin gene expression, and kappa opioid agonists suppress drug reward. Therefore, we tested the hypothesis that stress-induced release of endogenous dynorphin may mediate behavioral responses to stress and oppose the rewarding effects of cocaine. C57Bl/6 mice subjected to repeated forced swim testing (FST) using a modified Porsolt procedure at 30 degrees C showed a characteristic stress-induced immobility response and a stress-induced analgesia observed with a tail withdrawal latency assay. Pretreatment with the kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI; 10 mg/kg, i.p.) blocked the stress-induced analgesia and significantly reduced the stress-induced immobility. The nor-BNI sensitivity of the behavioral responses suggests an activation of the kappa opioid receptor by a stress-induced release of dynorphin peptides. Supporting this hypothesis, transgenic mice possessing a disrupted prodynorphin gene showed no increase in immobility or stress-induced analgesia after exposure to repeated FST. Because both stress and the kappa opioid system can modulate the response to drugs of abuse, we tested the effects of forced swim stress on cocaine-conditioned place preference (CPP). FST-exposed mice conditioned with cocaine (15 mg/kg, s.c.) showed significant potentiation of place preference for the drug-paired chamber over the responses of unstressed mice. Surprisingly, nor-BNI pretreatment blocked stress-induced potentiation of cocaine CPP. Consistent with this result, mice lacking the prodynorphin gene did not show a stress-induced potentiation of cocaine CPP, whereas wild-type littermates did. The findings suggest that chronic swim stress may activate the kappa opioid system to produce analgesia, immobility, and potentiation of the acute rewarding properties of cocaine in C57Bl/6 mice.

Topics: Analgesia; Animals; Behavior, Animal; Cocaine; Drug Synergism; Enkephalins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Naltrexone; Narcotic Antagonists; Pain Threshold; Protein Precursors; Reaction Time; Receptors, Opioid, kappa; Reward; Spatial Behavior; Stress, Physiological; Swimming

The objective of this study was to determine whether prodynorphin-derived opioid peptides could block the spontaneous luteinizing hormone (LH) surge and ovulation, and if so, whether this inhibitory action was mediated through kappa-opioid receptors. Various doses of dynorphin peptides (dynorphin A(1-17), dynorphin A(1-8), dynorphin B, alpha- and beta-neoendorphin) were infused into the brain through third-ventricle cannulae in rats between 1330-1800 h on proestrus. Each dynorphin peptide blocked the LH surge and ovulation in a dose-dependent manner. Dynorphin A(1-17) and A(1-8) were equally effective in producing these actions, and more potent than either dynorphin B or alpha- or beta-neoendorphin. U50,488H, a specific kappa-opioid receptor agonist, also blocked the LH surge and ovulation. When a mixture of five dynorphin peptides was infused intraventricularly, each at a dose that inhibited the LH surge, both the surge and ovulation were blocked. However, when norbinaltorphimine, a specific kappa-opioid receptor antagonist, was coinfused with the mixture of dynorphin peptides, the LH surge and ovulation were fully restored. These results demonstrate that prodynorphin-derived opioid peptides, acting through kappa-opioid receptors, can block the LH surge and ovulation. Dynorphin A(1-17) and A(1-8) are the most potent in this regard.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Brain; Dynorphins; Enkephalins; Female; Luteinizing Hormone; Naltrexone; Ovulation; Peptide Fragments; Proestrus; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

We investigated the effect of long-term administration of opioid antagonists on the regulation of prodynorphin gene expression in rat brain. Intracerebroventricular (i.c.v.) injections for seven days of nor-binaltorphimine (nor-BNI), the highly selective kappa opioid antagonist, naloxone and its longer acting analog naltrexone, both relatively selective antagonists for the mu opioid receptor, markedly raised prodynorphin mRNA levels in rat hypothalamus, hippocampus and striatum. Peptides, namely immunoreactive-dynorphin A (ir-dyn A), were unaffected after chronic treatment with all antagonists, in the same tissues. These results, taken together with our previous observations, suggest that chronic opioid antagonists, acting on kappa and mu opioid receptors, clearly up-regulate prodynorphin gene expression in discrete rat brain regions, activating its biosynthesis. Moreover, our data support the hypothesis that the endogenous opioid system plays a role in the mechanisms underlying the development of opiate tolerance.

Topics: Animals; Brain; Enkephalins; Gene Expression; Male; Naloxone; Naltrexone; Narcotic Antagonists; Protein Precursors; Rats; Rats, Sprague-Dawley; Time Factors

Previous studies have suggested an involvement of enkephalins in regulation of oxytocin (OXT) and vasopressin (AVP) release, which seems to disagree with the very low affinities of Met- and Leu-enkephalin for the kappa opioid receptor. As opioid receptors in the neural lobe exclusively exist of kappa receptors, we studied the binding characteristics of larger pro-enkephalin derived peptides for opioid binding sites in the neural lobe by means of light microscopic receptor autoradiography. In addition, the pharmacological characteristics of opioid binding sites in the neural lobe were compared with those in other parts of the pituitary. In the neural as well as the intermediate lobe both high and low affinity 3H-bremazocine binding sites were present. Binding to these sites was completely displaceable by both naloxone and nor-binaltorphimine suggesting that these sites represent kappa opioid receptors. Also with regard to selectivity and affinity characteristics to other ligands, opioid binding sites in the neural and intermediate lobe were quite similar. In the anterior lobe a very low level of bremazocine binding was present, which could not be displaced by nor-binaltorphimine. Displacement studies with pro-enkephalin and pro-dynorphin derived peptides showed that both groups of peptides could bind to opioid binding sites in the neural and intermediate lobe. Especially the relatively large pro-dynorphin and pro-enkephalin derived peptides, such as dynorphin 1-17 and BAM22, appeared to be very potent ligands for these opioid binding sites and were much more potent than smaller fragments, such as dynorphin 1-8, and Met- and Leu-enkephalin. These results contradict the existence of a mismatch in the neural (and intermediate) lobe with regard to the local type of opioid peptides and receptors present.

Topics: Amino Acid Sequence; Analgesics; Animals; Autoradiography; Benzomorphans; Binding, Competitive; Densitometry; Enkephalins; Ligands; Male; Molecular Sequence Data; Naloxone; Naltrexone; Pituitary Gland; Pituitary Gland, Posterior; Protein Precursors; Rats; Rats, Wistar; Receptors, Opioid; Receptors, Opioid, kappa