Nociceptin Receptor Knockout Research Articles

The Nociceptin Receptor (NOP) Agonist AT-312 Blocks Acquisition of Morphine- and Cocaine-Induced Conditioned Place Preference in Mice.

Treatment of drug addiction remains an unmet medical need due to the dearth of approved pharmacotherapies. There are no approved treatments for cocaine addiction, whereas the current opioid crisis has revealed the stark reality of the limited options to treat prescription and illicit opioid abuse. Preclinical studies in rodents and nonhuman primates have shown that orphanin FQ/nociceptin (N/OFQ), the endogenous ligand for the nociceptin opioid receptor (NOP) reduces the rewarding effects of several abused substances, including opioids, psychostimulants and alcohol. A few nonpeptide small-molecule NOP agonists have also shown efficacy in attenuating the rewarding effects of various abused drugs. We previously demonstrated that a high affinity small-molecule NOP agonist AT-312 selectively reduced the rewarding effects of ethanol in the conditioned place preference paradigm in mice. In the present study, we examined if AT-312 (3 mg/kg, i.p. or s.c. respectively), would alter the rewarding action of morphine (7.5 mg/kg, s.c.) or cocaine (15 mg/kg, i.p.). The effect of AT-312 on morphine- and cocaine-induced motor stimulation was also assessed on the conditioning days. The role of the NOP receptor in the effects of AT-312 was further confirmed by conducting the place conditioning experiments in NOP knockout mice and compared to their wild-type controls. Our results showed that AT-312 significantly reduced the acquisition of morphine and cocaine CPP in wild-type mice but not in mice lacking NOP receptors. AT-312 also suppressed morphine-induced and completely abolished cocaine-induced motor stimulation in NOP wild-type mice, but not in NOP knockout mice. These results show that small-molecule NOP receptor agonists have promising efficacy for attenuating the rewarding effects of morphine and cocaine, and may have potential as pharmacotherapy for opioid and psychostimulant addiction or for treating polydrug addiction.

Parsing the hedonic and motivational influences of nociceptin on feeding using licking microstructure analysis in mice.

Opioid peptides are implicated in processes related to reward and aversion; however, how specific opioid peptides are involved remains unclear. We investigated the role of nociceptin (NOC) in voluntary licking for palatable and aversive tastants by studying the effect of intracerebroventricularly administered NOC on licking microstructure in wild-type and NOC receptor knockout (NOP KO) mice. Compared with the wild-type mice, NOP KO mice emitted fewer bouts of licking when training to lick for a 20% sucrose solution. Correspondingly, intracerebroventricular administration of NOC increased the number of licking bouts for sucrose and sucralose in wild-type, but not in NOP KO mice. The ability of NOC to initiate new bouts of licking for sweet solutions suggests that NOC may drive motivational aspects of feeding behavior. Conversely, adulterating a sucrose solution with the aversive tastant quinine reduced licking bout lengths in wild-type and NOP KOs, suggesting that NOC signaling is not involved in driving voluntary consumption of semiaversive tastants. Interestingly, when consuming sucrose following 20 h of food deprivation, NOP KO mice emitted longer bouts of licking than wild types, suggesting that under hungry conditions, NOC may also contribute toward hedonic aspects of feeding. Together, these results suggest differential roles for NOC in the motivational and hedonic aspects of feeding.

Time to first cigarette: A potential clinical screening tool for nicotine dependence and cancer risk

Aims: Opioid peptides are implicated in processes related to reward and aversion; however, the how specific opioid peptides are involved remains unclear. Here, we aim to investigate the role nociceptin in behavioral responding for palatable and aversive tastants. Methods: A microstructural licking analysis of the effects of intracerebroventricular administerednociceptinon total licks, total bouts of licking, and lengths of licking bouts was conducted. Additionally, we compared these licking behaviors in wildtype and nociceptin receptor (NOP) knockout mice, to identify the role of endogenous nociceptin in these processes. We hypothesize that NOP knockout mice will show lower levels of licking behavior under normal conditions and will be irresponsive to the appetitestimulating effects of nociceptin administration. Results: Repeated measures ANOVA analyses show that NOP knockout mice generally emit fewer licks than wildtype mice when given access to sucrose solution. Furthermore, results from post-hoc analyses show nociceptin administration increases total number of licking bouts in wildtype mice in both chow sated and hungry states, an effect thatwas absent inNOPknockoutmice. Similar effects were seen for the non-caloric sweetener sucralose, but to a lesser degree. Hunger tended to increase total bouts of licking for sucrose in all mice, but also increased mean bout length in NOPknockoutmiceonly. Finally, adulterating sucrose solutionwith the bitter aversive tastant quinine produced similarly suppressive effects in both wildtype and NOP receptor knockout mice. Conclusions: Together, these results support our hypothesis, suggesting that endogenous nociceptin contributes to the positive incentive properties of palatable tastants. Further understanding of the contributions of opioid neurotransmission to hedonic processing may aid in developing appropriate treatment strategies for pathologies characterized by aberrant reward seeking and taking behaviors. Financial support: Grant #: DA024635, DA09359, and DA05010.

The anxiolytic-like profile of the nociceptin receptor agonist, endo-8-[bis(2-chlorophenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octane-3-carboxamide (SCH 655842): Comparison of efficacy and side effects across rodent species

The endogenous opioid-like peptide, nociceptin, produces anxiolytic-like effects that are mediated via the nociceptin (NOP) receptor. Similarly, synthetic, non-peptide NOP agonists produce robust anxiolytic-like effects although these effects are limited by marked side effects. In the present studies, the effects of a novel NOP receptor agonist, SCH 655842, were examined in rodent models sensitive to anxiolytic drugs and tests measuring potential adverse affects. Oral administration of SCH 655842 produced robust, anxiolytic-like effects in three species, i.e., rat, guinea pig, and mouse. Specifically, SCH 655842 was effective in rat conditioned lick suppression (3–10 mg/kg) and fear-potentiated startle (3–10 mg/kg) tests, a guinea pig pup vocalization test (1–3 mg/kg), as well as in mouse Geller–Seifter (30 mg/kg) and marble burying (30 mg/kg) tests. The anxiolytic-like effect of SCH 655842 in the conditioned lick suppression test was attenuated by the NOP antagonist, J-113397. In mice, SCH 655842 reduced locomotor activity and body temperature at doses similar to the anxiolytic-like dose and these effects were absent in NOP receptor knockout mice. In rats, SCH 655842 did not produce adverse behavioral effects up to doses of 70–100 mg/kg. Pharmacokinetic studies in the rat confirmed dose-related increases in plasma and brain levels of SCH 655842 across a wide oral dose range. Taken together, SCH 655842 may represent a NOP receptor agonist with improved tolerability compared to other members of this class although further studies are necessary to establish whether this extends to higher species.

Reduced degeneration of dopaminergic terminals and accentuated astrocyte activation by high dose methamphetamine administration in nociceptin receptor knock out mice

A major pathology of methamphetamine abuse is loss of dopaminergic function due to destruction of dopaminergic terminals, especially in the striatum. This process is accompanied by gliosis by astrocytes and microglia. Here, we evaluated the function of endogenous nociceptin/orphanin FQ in these events using nociceptin receptor (NOP) knockout mice. Wild-type and knockout mice were injected systemically either saline vehicle or 5mg/kg methamphetamine four times interspersed by 2h intervals. Three days later, brains were immunohistochemically processed to visualize methamphetamine-induced loss of tyrosine hydroxylase (as a marker of damage to dopamine terminals), glial fibrillary acidic protein (GFAP, as a marker of astrocytes), and ionized calcium-binding adapter molecule 1 (lba-1, as a marker of microglia) in the striatum. Methamphetamine treatment induced an approximately 80% loss of tyrosine hydroxylase-immunoreactivity, and this effect was mildly attenuated in NOP receptor knockout mice. There was a large increase (approximately 15-fold) in GFAP-immunoreactivity in methamphetamine-treated wild-type mice, which was almost two times larger still in NOP receptor knockout mice. In contrast, Iba-1 immunostaining was only modestly increased (approximately 30%) by methamphetamine treatment, and there were no difference between genotypes. Finally, there were no genotype-dependent differences in hyperthermic responses to methamphetamine. These results indicate that endogenous nociceptin/orphanin FQ exacerbates the neurotoxic effects of methamphetamine on striatal dopamine neurons, and suggests this is due in part to an astrocyte-mediated event.

Enhanced nicotine sensitivity in nociceptin/orphanin FQ receptor knockout mice

The opioid peptide nociceptin (orphanin FQ) has been implicated in reward, reinforcement and addiction. The current study sought evidence of a role of endogenous nociceptin in nicotine responses by studying nociceptin receptor (NOP) knockout mice. The results were: (1) NOP receptor knockout mice showed enhanced anxiety-like behavior on an elevated plus maze. Whereas nicotine (0.05–0.5 mg/kg) tended to be anxiogenic in wild-type mice, NOP receptor KO mice were resistant to this effect, though interpretation was confounded by their stronger anxiety-like behavior. (2) When presented increasing nicotine concentrations (3–50 μg/ml) in a bottle choice drinking paradigm, there were no genotype-dependent differences in nicotine preference. However, NOP receptor knockout mice consumed more 3 μg/ml nicotine solution when considered in absolute terms. (3) NOP receptor knockout mice showed stronger hypothermic responses to nicotine (1 or 2 mg/kg) administration. (4) There was modest evidence that NOP receptor KO mice showed attenuated behavioral sensitization to a low dose of nicotine (0.05 mg/kg) during repeated daily treatment. (5) NOP receptor knockout mice more rapidly tolerated the sedative effect of nicotine (1 mg/kg), due partially to slightly lower locomotion on first treatment. (6) NOP receptor knockout mice, unlike wild-type mice, showed a significant mecamylamine (2.5 mg/kg) induced conditioned place aversion to nicotine (24 mg/kg/day) withdrawal. These results show that mice lacking the influence of endogenous N/OFQ mice are hypersensitive to nicotine in most measures, showing a role of endogenous nociceptin in modulating or mediating the acute effects of nicotine, and possibly nicotine addiction.

Endogenous nociceptin modulates diet preference independent of motivation and reward

Previous studies show that the opioid peptide nociceptin stimulates food intake. Here, we studied nociceptin receptor knockout (NOP KO) mice in various behavioral paradigms designed to differentiate psychological and physiological loci at which endogenous nociceptin might control feeding. When presented a choice under food restriction, NOP KO mice displayed reduced preference for high sucrose diet, but lower intake of high fat diet under no-choice conditions. These responses were absent under ad libitum feeding conditions. Conditioned place preference to high fat diet under food-deprived conditions was unaltered in NOP KO mice, suggesting no difference in reward responses. Furthermore, operant food self-administration under a variety of conditions showed no genotype-dependent differences, suggesting no differences in the motivational properties of food. Taste reactivity to sucrose was unchanged in NOP KO mice, though NOP KO mice had altered aversive reactions to quinine solutions under ad libitum feeding, suggesting minor differences in the affective impact of palatable and unpalatable tastants. Although NOP KO mice re-fed following food-deprivation showed normal increases in plasma glucose and insulin, multidimensional scaling analysis showed that the relationship between these measures, body weight and plasma leptin was substantially disrupted in NOP KO, particularly in fasted mice. Additionally, the typical positive relationship between body weight and plasma leptin was considerably weaker in NOP KO mice. Together, these findings suggest that endogenous nociceptin differentially modulates diet preference depending on macronutrient content and homeostatic state, independently of the motivating, rewarding or orosensory properties of food, but may involve metabolic or postingestive processes.

Endogenous Nociceptin (Orphanin FQ) Suppresses Basal Hedonic State and Acute Reward Responses to Methamphetamine and Ethanol, but Facilitates Chronic Responses

The opioid peptide nociceptin (orphanin FQ) suppresses drug reward, drug self-administration, and impedes some of the processes believed to underlie the transition to addiction. As virtually all previous studies have used administration of nociceptin receptor agonists to evaluate the role of nociceptin on addiction-like behavior, the current study used a pharmacological (nociceptin receptor antagonist) and genetic (nociceptin receptor knockout mice) approach to elucidate the role of endogenous nociceptin. The nociceptin receptor antagonist UFP-101 induced a modest place preference, and enhanced the conditioned place preference induced by methamphetamine. In agreement with this, nociceptin receptor knockout mice had slightly enhanced methamphetamine and ethanol conditioned place preferences compared to wild-type mice. This effect did not appear to depend on differences in learning ability, as nociceptin receptor knockout mice had slightly weaker-conditioned place aversions to lithium chloride, the kappa-opioid receptor agonist, U50488H, and the general opiate antagonist, naloxone. The development of behavioral sensitization to methamphetamine was lower in nociceptin receptor knockout mice, and attenuated by UFP-101 administration to wild-type mice. Additionally, ethanol consumption and preference in a two-bottle choice test was lower in nociceptin receptor knockout mice, though ethanol-stimulated locomotion was stronger. Whereas the rewarding effect of methamphetamine and ethanol following chronic treatment, as measured by place conditioning, strengthened in wild-type mice, this effect was absent in nociceptin receptor knockout mice. These results suggest that endogenous N/OFQ suppresses basal and drug-stimulated increases in hedonic state, and plays either a permissive or facilitatory role in the development of addiction.

The Involvement of the Nociceptin Receptor in the Antinociceptive Action of Nitrous Oxide

Nociceptin and its receptor are widely expressed in the central nervous system and are involved in the modulation of nociception. We have previously reported that the minimum anesthetic alveolar concentrations for volatile anesthetics do not differ between nociceptin receptor knockout (NOP-/-) mice and wild-type (NOP+/+) mice. In the present study, we investigated whether the nociceptin system is involved in the antinociceptive action of nitrous oxide. Using the acetic acid-induced writhing test, we showed that nitrous oxide had significantly less analgesic action in NOP-/- mice than in NOP+/+ mice. Furthermore, when anesthetized with a mixture of halothane and nitrous oxide (70%), intraperitoneal injection of acetic acid resulted in an increase of plasma adrenocorticotropic hormone concentrations in NOP-/- mice but not in NOP+/+ mice. An immunohistochemical study showed that nitrous oxide exposure induced c-Fos expression in the spinal cords of NOP+/+ mice but not in those of NOP-/- mice. These results together suggest that the antinociceptive action of nitrous oxide is, at least partly, mediated by the nociceptin system.

Defective place cell activity in nociceptin receptor knockout mice with elevated NMDA receptor-dependent long-term potentiation.

There is growing evidence that NMDA receptor-dependent long-term potentiation (LTP) in the hippocampus mediates the synaptic plasticity that underlies spatial learning and memory. LTP deficiencies correlate well with spatial memory deficits and LTP enhancements may improve spatial memory. In addition, LTP deficiencies are associated with abnormal place cells as expected from the spatial mapping hypothesis of hippocampal function. In contrast, nothing is known on how enhanced NMDA receptor-dependent LTP affects place cells. To address this question we recorded place cells from mice lacking the nociceptin receptor (NOP1/ORL1/OP4) that have enhanced hippocampal LTP. We found that the enhanced LTP was mediated by NMDA receptors, did not require L-type calcium channels, and occurred only when high frequency tetanizing stimulus trains were used. Place cells in nociceptin receptor knockout mice were abnormal in several ways: they were less stable, had noisier positional firing patterns, larger firing fields and higher discharge rates inside and outside the firing fields. Our results suggest that excessive LTP can cause subnormal hippocampal place cell function. The effects of LTP enhancement on place cell function may therefore also depend on molecular details of synaptic plasticity, including the relationship between stimulus frequency and synaptic strength, and not merely on the magnitude of synaptic strength increases. The data have important clinical implications on development of strategies to improve cognitive function.

Nociceptin system does not affect MAC of volatile anaesthetics

Nociceptin is the endogenous agonist of the opioid receptor-like (ORL) 1 receptor (NOP), and both nociceptin and NOP are widely expressed in the brain and spinal cord, which are target organs of general anaesthetics. As nociceptin has been reported to be involved in modulating pain mechanisms and stress responses, it is possible that the activity of the nociceptin system affects the anaesthetic potency of general anaesthetics. To address this possibility, we investigated the minimum alveolar concentrations (MACs) of various volatile anaesthetics in nociceptin receptor knockout mice (NOP-/-) and wild-type mice (NOP+/+). We used male NOP-/- mice and NOP+/+ mice. MACs for halothane, isoflurane and sevoflurane were determined by the tail-clamp method. MACs for halothane, isoflurane and sevoflurane in NOP-/- mice were 1.60 (SD 0.06), 1.68 (0.08) and 3.36 (0.07)%, respectively. In NOP+/+ mice, MACs for halothane, isoflurane and sevoflurane were 1.59 (SD 0.07), 1.72 (0.07) and 3.38 (0.09)%, respectively. MACs in NOP-/- mice did not significantly differ from those in NOP+/+ mice for halothane, isoflurane and sevoflurane. This result suggests that the nociceptin system does not affect the anaesthetic potency of volatile anaesthetics.

Methamphetamine sensitization in nociceptin receptor knockout mice: locomotor and c- fos expression

The role of endogenous nociceptin in the development and expression of sensitization to repeated methamphetamine administration in a novel environment was studied in nociceptin receptor knockout mice. No differences in acute or sensitized locomotor responses were found in nociceptin receptor knockout mice. However, analysis of c- fos expression revealed significant interactions between chronic methamphetamine treatment and genotype in the nucleus accumbens and lateral septum. This was due to increased c- fos expression in chronically methamphetamine-treated nociceptin receptor knockout mice contrasted with reduced c- fos expression in chronically vehicle-treated nociceptin receptor knockout mice. Two further regions (nucleus accumbens core and ventromedial caudate putamen) showed significant interactions between genotype, chronic, and acute methamphetamine treatment due to accentuated c- fos expression in nociceptin receptor knockout mice sensitized and challenged with methamphetamine. These findings suggest endogenous nociceptin modulates the response of the central nervous system to repeated psychostimulant administration, although this is little reflected in locomotion.

Lack of nociceptin receptor alters body temperature during resting period in mice

The role of nociceptin (NOC) receptor on body core temperature (Tcore) control was examined using NOC receptor knockout mice. In homozygote NOC receptor-knockout, wild-type, and control C57BL/6J and 129/SV mice, Tcore was continuously recorded under 12:12 h light:dark (LD) and conditions of constant darkness (DD). The Tcore values during the resting period were higher in the NOC receptor-knockout mice than in both wild-type and control mice under both LD and DD conditions. Spontaneous activity during the resting period and plasma cortisol levels were not different between the NOC receptor-knockout and control mice. The findings herein indicate that the NOC receptor is involved in the control of Tcore during the resting period and is independent of light, physical activity and/or cortisol regulation.

Morphine tolerance and dependence in the nociceptin receptor knockout mice.

Here we report the involvement of nociceptin receptor in tolerance to morphine-induced antinociception and in morphine dependence. There was no different nociceptive perception and antinociceptive effects of morphine between wild-type and the nociceptin receptor knockout mice. Tolerance to morphine (10 mg/kg)-induced antinociception was developed in both wild-type and the nociceptin receptor knockout mice after administration of morphine (10 mg/kg) twice a day for 5 days. When naloxone (5 mg/kg) was administered to mice treated with morphine repeatedly on the 6th day, morphine withdrawal syndrome was observed in both wild-type and the nociceptin receptor knockout mice, which were accompanied by the elevation of cyclic AMP levels. While naloxone benzoylhydrazone (1 mg/kg), a putative antagonist for nociceptin receptor/naloxone benzoylhydrazone-sensitive sites, also induced the morphine withdrawal signs in both wild-type and the nociceptin receptor knockout mice, the jumping signs in the nociceptin receptor knockout mice were less severe than those in wild-type mice. Treatment with naloxone benzoylhydrazone in morphine-dependent wild-type mice caused a significant increase in cyclic AMP levels in the thalamus while it had no effect in the nociceptin receptor knockout mice. The analysis of opioid mu-receptor binding showed no difference between wild-type and the nociceptin receptor knockout mice. These results suggest that the nociceptin receptor/naloxone benzoylhydrazone-sensitive sites contribute to the induction of morphine withdrawal syndrome in part. Furthermore, it is demonstrated that morphine withdrawal syndrome excepting jumping can be induced by naloxone benzoylhydrazone without any changes in the cyclic AMP levels in the thalamus.

Enhanced Spinal Nociceptin Receptor Expression Develops Morphine Tolerance and Dependence

The tolerance and dependence after chronic medication with morphine are thought to be representative models for studying the plasticity, including the remodeling of neuronal networks. To test the hypothesis that changes in neuronal plasticity observed in opioid tolerance or dependence are derived from increased activity of the anti-opioid nociceptin system, the effects of chronic treatments with morphine were examined using nociceptin receptor knock-out (NOR(-/-)) mice and a novel nonpeptidic NOR antagonist, J-113397, which shows a specific and potent NOR antagonist activity in in vitro [(35)S]GTPgammaS binding assay and in vivo peripheral nociception test. The NOR(-/-) mice showed marked resistance to morphine analgesic tolerance without affecting morphine analgesic potency in tail-pinch and tail-flick tests. The NOR(-/-) mice also showed marked attenuation of morphine-induced physical dependence, manifested as naloxone-precipitated withdrawal symptoms after repeated morphine treatments. Similar marked attenuation of morphine tolerance was also observed by single subcutaneous (10 mg/kg) or intrathecal (1 nmol) injection of J-113397, which had been given 60 min before the test in morphine-treated ddY mice. However, the intracerebroventricular injection (up to 3 nmol) did not affect the tolerance. On the other hand, morphine dependence was markedly attenuated by J-113397 that had been subcutaneously given 60 min before naloxone challenge. There was also observed a parallel enhancement of NOR gene expression only in the spinal cord during chronic morphine treatments. Together, these findings suggest that the spinal NOR system develops anti-opioid plasticity observed on morphine tolerance and dependence.

Nociceptin system plays a role in the memory retention: involvement of naloxone benzoylhydrazone binding sites.

We investigated the role of nociceptin system in learning and memory in mice. The deficiency of nociceptin receptors and nociceptin itself did not affect the alternation behavior in the Y-maze test. In the passive avoidance test, the step-through latencies of nociceptin receptor knockout mice were longer than those of wild-type mice. Nociceptin shortened the step-through latency in wild-type mice. This impairment on passive avoidance task was reversed by naloxone benzoylhydrazone (NalBzoH), indicating that the amnesic effects of nociceptin may be mediated through the NalBzoH recognition sites. These suggest that nociceptin system plays an important role in the memory retention of passive avoidance task, and NalBzoH-recognized sites are involved in nociceptin-induced impairment of the memory retention.

Loss of Antinociception Induced by Naloxone Benzoylhydrazone in Nociceptin Receptor-Knockout Mice

Nociceptin and nociceptin receptor, which show structural similarities to opioid peptides and opioid receptors, respectively, have been recently found to constitute a novel neuromodulatory system. In the brain, however, the physiological role of the modulation via the nociceptin receptor is still unclear. Administered nociceptin produces hyperalgesia and hypolocomotion, whereas the nociceptin receptor-knockout mice show no significant abnormalities in nociceptive thresholds and locomotion. To clarify possible involvement of the nociceptin receptor in the regulation of nociception and locomotion, we made use of the knockout mice and naloxone benzoylhydrazone (NalBzoH) identified originally as a ligand for opioid receptors. Experiments on the cultured cells transfected with the nociceptin receptor cDNA showed that NalBzoH competed with [3H]nociceptin binding and attenuated the nociceptin-induced inhibition of cAMP accumulation. Furthermore, behavioral studies demonstrated that NalBzoH completely inhibited nociceptin-induced hyperalgesia and hypolocomotion. It is therefore likely that NalBzoH can act as a potent antagonist for the nociceptin receptor in vivo. In wild-type mice, NalBzoH induced antinociception but did not affect locomotor activity. In contrast, in the knockout mice, no significant changes in nociception and locomotion were induced by NalBzoH. These results clearly suggest that the nociceptin system takes part in the physiological regulation of nociceptive thresholds but not in the basal modulation of locomotion.