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After injury, mammalian spinal cords develop scars to confine the lesion and prevent further damage. However, excessive scarring can hinder neural regeneration and functional recovery 1,2 . These competing actions underscore the importance of developing therapeutic strategies to dynamically modulate scar progression. Previous research on scarring has primarily focused on astrocytes, but recent evidence has suggested that ependymal cells also participate. Ependymal cells normally form the epithelial layer encasing the central canal, but they undergo massive proliferation and differentiation into astroglia following certain injuries, becoming a core scar component 3-7 . However, the mechanisms regulating ependymal proliferation in vivo remain unclear. Here we uncover an endogenous κ-opioid signalling pathway that controls ependymal proliferation. Specifically, we detect expression of the κ-opioid receptor, OPRK1, in a functionally under-characterized cell type known as cerebrospinal fluid-contacting neuron (CSF-cN). We also discover a neighbouring cell population that expresses the cognate ligand prodynorphin (PDYN). Whereas κ-opioids are typically considered inhibitory, they excite CSF-cNs to inhibit ependymal proliferation. Systemic administration of a κ-antagonist enhances ependymal proliferation in uninjured spinal cords in a CSF-cN-dependent manner. Moreover, a κ-agonist impairs ependymal proliferation, scar formation and motor function following injury. Together, our data suggest a paracrine signalling pathway in which PDYN + cells tonically release κ-opioids to stimulate CSF-cNs and suppress ependymal proliferation, revealing an endogenous mechanism and potential pharmacological strategy for modulating scarring after spinal cord injury., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Aims: Fibromyalgia patients might experience temporomandibular disorder (TMD) as a comorbidity. However, the connection between these two syndromes is not fully understood. Nociceptin (N/OFQ) and NOP receptors are implicated in both conditions, but their relevance in the comorbidity needs investigation. This study featured a comorbidity model of fibromyalgia plus TMD in mice, attempting to evaluate the significance of the N/OFQ-NOP receptor in this paradigm., Materials and Methods: Female CF-1 mice were submitted to the fibromyalgia model induced by three daily consecutive injections of reserpine (0.25 mg/kg) and received an intra-masseter injection of complete Freund's adjuvant (CFA; 10 μl; diluted 1:1) on day four., Key Findings: There was a rise in nocifensive and depression-like behaviors in the comorbidity group, as evaluated by the Grimace scores and the tail suspension test (TST). This group displayed anxiogenic-like effects in the hole board and the elevated plus maze tests. The comorbidity group showed an increment of c-Fos immunopositivity in the ipsilateral side of CFA injection, in the trigeminal ganglion (TG) and thalamus. The administration of N/OFQ (1 nmol/kg, i.p.) boosted the Grimace scores in the comorbidity group, with no effect for the NOP receptor antagonist UFP-101 (1 nmol/kg, i.p.). Either NOP ligand failed to alter depression or anxiety behavioral changes. Alternatively, pregabalin (30 mg/kg; i.p.) reduced the nociceptive responses and the number of head dips in the hole board., Significance: Data reveal new evidence suggesting that inducing TMD with CFA may worsen fibromyalgia symptoms in reserpine-treated mice, an effect partially regulated by systemic N/OFQ., 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 © 2024 Elsevier Inc. All rights reserved.)

Cold injury or frostbite is a common medical condition that causes serious clinical complications including sensory abnormalities and chronic pain ultimately affecting overall well-being. Opioids are the first-choice drug for the treatment of frostbite-induced chronic pain; however, their notable side effects, including sedation, motor incoordination, respiratory depression, and drug addiction, present substantial obstacle to their clinical utility. To address this challenge, we have exploited peripheral mu-opioid receptors as potential target for the treatment of frostbite-induced chronic pain. In this study, we investigated the effect of dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a peripheral mu-opioid receptor agonist, on frostbite injury and hypersensitivity induced by deep freeze magnet exposure in rats. Animals with frostbite injury displayed significant hypersensitivity to mechanical, thermal, and cold stimuli which was significant ameliorated on treatment with different doses of DALDA (1, 3, and 10 mg/kg) and ibuprofen (100 mg/kg). Further, molecular biology investigations unveiled heightened oxido-nitrosative stress, coupled with a notable upregulation in the expression of TRP channels (TRPA1, TRPV1, and TRPM8), glial cell activation, and neuroinflammation (TNF-α, IL-1β) in the sciatic nerve, dorsal root ganglion (DRG), and spinal cord of frostbite-injured rats. Treatment with DALDA leads to substantial reduction in TRP channels, microglial activation, and suppression of the inflammatory cascade in the ipsilateral L4-L5 DRG and spinal cord of rats. Overall, findings from the present study suggest that activation of peripheral mu-opioid receptors mitigates chronic pain in rats by modulating the expression of TRP channels and suppressing glial cell activation and neuroinflammation., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals., (© 2024. The Author(s).)

Olfactory dysfunctions decrease daily quality of life (QOL) in part by reducing the pleasure of eating. Olfaction plays an essential role in flavor sensation and palatability. The decreased QOL due to olfactory dysfunction is speculated to result from abnormal neural activities in the olfactory and limbic areas of the brain, as well as peripheral odorant receptor dysfunctions. However, the specific underlying neurobiological mechanisms remain unclear. As the olfactory tubercle (OT) is one of the brain's regions with high expression of endogenous opioids, we hypothesize that the mechanism underlying the decrease in QOL due to olfactory dysfunction involves the reduction of neural activity in the OT and subsequent endogenous opioid release in specialized subregions. In this review, we provide an overview and recent updates on the OT, the endogenous opioid system, and the pleasure systems in the brain and then discuss our hypothesis. To facilitate the effective treatment of olfactory dysfunctions and decreased QOL, elucidation of the neurobiological mechanisms underlying the pleasure of eating through flavor sensation is crucial., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Murata, Maegawa, Imoto, Fujieda and Fukazawa.)

Rationale: Recently, we demonstrated that the activation of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) signaling facilitates depressive-like behaviors. Additionally, literature findings support the ability of the N/OFQ-NOP system to modulate the hypothalamic-pituitary-adrenal (HPA) axis., Objectives: Considering that dysfunctional HPA axis is strictly related to stress-induced psychopathologies, we aimed to study the role of the HPA axis in the pro-depressant effects of NOP agonists., Methods: Mice were treated prior to stress with the NOP agonist Ro 65-6570, and immobility time in the forced swimming task and corticosterone levels were measured. Additionally, the role of endogenous glucocorticoids and CRF was investigated using the glucocorticoid receptor antagonist mifepristone and the CRF 1 antagonist antalarmin in the mediation of the effects of Ro 65-6570., Results: The NOP agonist in a dose-dependent manner further increased the immobility of mice in the second swimming session compared to vehicle. By contrast, under the same conditions, the administration of the NOP antagonist SB-612111 before stress reduced immobility, while the antidepressant nortriptyline was inactive. Concerning in-serum corticosterone in mice treated with vehicle, nortriptyline, or SB-612111, a significant decrease was observed after re-exposition to stress, but no differences were detected in Ro 65-6570-treated mice. Administration of mifepristone or antalarmin blocked the Ro 65-6570-induced increase in the immobility time in the second swimming session., Conclusions: Present findings suggest that NOP agonists increase vulnerability to depression by hyperactivating the HPA axis and then increasing stress circulating hormones and CRF 1 receptor signaling., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chronic stress causes cognitive deficits, such as impairments in episodic-like hippocampus-dependent memory. Stress regulates an opioid-related neuropeptide named Nociceptin/Orphanin FQ (N/OFQ), the ligand of the G protein-coupled receptor NOP. Since this peptide has deleterious effects on memory, we hypothesized that the N/OFQ system could be a mediator of the negative effects of stress on memory. Chronic stress was mimicked by chronic exposure to corticosterone (CORT). The NOP receptor was either acutely blocked using selective antagonists, or knocked-down specifically in the hippocampus using genetic tools. Long-term memory was assessed in the object recognition (OR) and object location (OL) paradigms. Acute injection of NOP antagonists before learning had a negative impact on memory in naive mice whereas it restored memory performances in the chronic stress model. This rescue was associated with a normalization of neuronal cell activity in the CA3 part of the hippocampus. Chronic CORT induced an upregulation of the N/OFQ precursor in the hippocampus. Knock-down of the NOP receptor in the CA3/Dentate Gyrus region prevented memory deficits in the CORT model. These data demonstrate that blocking the N/OFQ system can be beneficial for long-term memory in a neuroendocrine model of chronic stress. We therefore suggest that NOP antagonists could be useful for the treatment of memory deficits in stress-related disorders., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The endogenous opioid system, which consists of opioid receptors and their ligands, is widely expressed in the nervous system and also found in the immune system. As a part of the body's defense machinery, the immune system is heavily regulated by endogenous opioid peptides. Many types of immune cells, including macrophages, dendritic cells, neutrophils, and lymphocytes are influenced by endogenous opioids, which affect cell activation, differentiation, proliferation, apoptosis, phagocytosis, and cytokine production. Additionally, immune cells also synthesize and secrete endogenous opioid peptides and participate peripheral analgesia. This chapter is structured into two sections. Part one focuses on immunoregulatory functions of central endogenous opioids; and part two describes how opioid peptide-containing immune cells participate in local analgesia., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Endogenous opioids and their associated receptors form a system that maintains survival by positively reinforcing behaviors that are vital to life. Cancer and cancer treatment side effects capitalize on this system pathogenically, leading to maladaptive biological responses (e.g., inflammation), as well as cognitive and emotional consequences, most notably depression. Psychologists who treat people with cancer frequently find depression to be a primary target for intervention. However, in people with cancer, the etiology of depression is unique and complex. This complexity necessitates that psycho-oncologists have a fundamental working knowledge of the biological substrates that underlie depression/cancer comorbidity. Building on other chapters in this volume pertaining to cancer and endogenous opioids, this chapter focuses on the clinical applications of basic scientific findings., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Pleasant emotions take a variety of forms and are a key part of the human experience. Although negative emotions have often been a focus of research, positive emotions, e.g., joy, pleasure, and love, have recently gained more attention. Each of these emotions is rich and complex in its own right. However, positive emotions appear to serve key evolutionary functions, which are mediated by complex biological substrates. This chapter summarizes key research and explores the biological underpinnings of positive emotions, with an emphasis on the roles that endogenous opioids play in the experience, expression, and development of positive emotions. The necessity of emphasizing positive emotions in research is also discussed., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

This second chapter in our trilogy reviews and critically appraises the scientific evidence for the role of endogenous opioid system (EOS) activity in the onset and progression of both obesity and eating disorders. Defining features of normative eating and maladaptive eating behaviors are discussed as a foundation. We review the scientific literature pertaining to the predisposing risk factors and pathophysiology for obesity and eating disorders. Research targeting the association between obesity, disordered eating, and psychiatric comorbidities is reviewed. We conclude by discussing the involvement of endogenous opioids in neurobiological and behavior traits, and the clinical evidence for the role of the EOS in obesity and eating disorders., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

This chapter (part one of a trilogy) summarizes the neurobiological foundations of endogenous opioids in the regulation of energy balance and eating behavior, dysregulation of which translates to maladaptive dietary responses in individuals with obesity and eating disorders, including anorexia, bulimia, and binge eating disorder. Knowledge of these neurobiological foundations is vital to researchers' and clinicians' understanding of pathophysiology as well as the science-based development of multidisciplinary diagnoses and treatments for obesity and eating disorders. We highlight mechanisms of endogenous opioids in both homeostatic and hedonic feeding behavior, review research on the dysregulation of food reward that plays a role in a wide array of obesity and disordered eating, and the clinical implications of neurobiological responses to food for current science-based treatments for obesity and eating disorders., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The neuropeptide nocistatin (NS) is expressed by the nervous system cells and neutrophils as a part of a precursor protein and can undergo stepwise limited proteolysis. Previously, it was shown that rat NS (rNS) is able to activate acid-sensing ion channels (ASICs) and that this effect correlates with the acidic nature of NS. Here, we investigated changes in the properties of rNS in the course of its proteolytic degradation by comparing the effects of the full-size rNS and its two cleavage fragments on the rat isoform 3 ASICs (ASIC3) expressed in X. laevis oocytes and pain perception in mice. The rNS acted as both positive and negative modulator by lowering the steady-state desensitization of ASIC3 at pH 6.8-7.0 and reducing the channel's response to stimuli at pH 6.0-6.9, respectively. The truncated rNSΔ21 peptide lacking 21 amino acid residues from the N-terminus retained the positive modulatory activity, while the C-terminal pentapeptide (rNSΔ30) acted only as a negative ASIC3 modulator. The effects of the studied peptides were confirmed in animal tests: rNS and rNSΔ21 induced a pain-related behavior, whereas rNSΔ30 showed the analgesic effect. Therefore, we have shown that the mode of rNS action changes during its stepwise degradation, from an algesic molecule through a pain enhancer to a pain reliever (rNSΔ30 pentapeptide), which can be considered as a promising drug candidate.

Opioids are excellent analgesics for the clinical treatment of various types of acute and chronic pain, particularly cancer-related pain. Nevertheless, it is well known that opioids have some nasty side effects, including immunosuppression, which is commonly overlooked. As a result, the incidence of opportunistic bacterial and viral infections increases in patients with long-term opioid use. Nowadays, there are no effective medications to alleviate opioid-induced immunosuppression. Understanding the underlying molecular mechanism of opioids in immunosuppression can enable researchers to devise effective therapeutic interventions. This review comprehensively summarized the exogenous opioids-induced immunosuppressive effects and their underlying mechanisms, the regulatory roles of endogenous opioids on the immune system, the potential link between opioid immunosuppressive effect and the function of the central nervous system (CNS), and the future perspectives in this field., 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 © 2023 Elsevier Inc. All rights reserved.)

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of an inhibitory G protein coupled receptor named N/OFQ peptide receptor (NOP). Clinical and preclinical findings suggest that the blockade of the NOP signaling induces antidepressant-like effects. Additionally, the blockade of the NOP receptor during inescapable stress exposure prevented the acquisition of the helplessness phenotype, suggesting that NOP antagonists are able to increase stress resilience. BTRX-246040 (aka LY2940094) is a NOP receptor antagonist with high affinity, potency and selectivity for the NOP over classical opioid receptors. BTRX-246040 is under development for the treatment of depression, eating disorders and alcohol abuse and it already entered clinical trials. In the present study, the antidepressant effects of BTRX-246040 were evaluated in mice subjected to the forced swimming test and to the learned helplessness model of depression. Additionally, the ability of BTRX-246040 to prevent the development of the helpless behavior and to modulate adult hippocampal neurogenesis has been investigated. BTRX-246040 (30 mg/kg, i.p.) produced antidepressant-like effects in the forced swimming test and in the learned helplessness model. More interestingly, when given before the stress induction sessions it was able to prevent the development of the helplessness behavior. Under these experimental conditions, BTRX-246040 did not modulate adult hippocampal neurogenesis, neither in naive nor in stressed mice. This study, performed with a clinically viable ligand, further corroborates growing evidence indicating that the blockade of the NOP signaling may provide an innovative strategy for the treatment of stress related psychopathologies., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Opioids are considered the oldest drugs known by humans and have been used for sedation and pain relief for several centuries. Nowadays, endogenous opioid peptides are divided into four families: enkephalins, dynorphins, endorphins, and nociceptin/orphanin FQ. They exert their action through the opioid receptors (ORs), transmembrane proteins belonging to the super-family of G-protein-coupled receptors, and are expressed throughout the body; the receptors are the δ opioid receptor (DOR), μ opioid receptor (MOR), κ opioid receptor (KOR), and nociceptin/orphanin FQ receptor (NOP). Endogenous opioids are mainly studied in the central nervous system (CNS), but their role has been investigated in other organs, both in physiological and in pathological conditions. Here, we revise their role in stem cell (SC) biology, since these cells are a subject of great scientific interest due to their peculiar features and their involvement in cell-based therapies in regenerative medicine. In particular, we focus on endogenous opioids' ability to modulate SC proliferation, stress response (to oxidative stress, starvation, or damage following ischemia-reperfusion), and differentiation towards different lineages, such as neurogenesis, vasculogenesis, and cardiogenesis.

Opioids targeting mu;μ (MOP) receptors produce analgesia in the peri-operative period and palliative care. They also produce side effects including respiratory depression, tolerance/dependence and addiction. The N/OFQ opioid receptor (NOP) also produces analgesia but is devoid of the major MOP side effects. Evidence exists for MOP-NOP interaction and mixed MOP-NOP ligands produce analgesia with reduced side effects. We have generated a HEKMOP/NOP human expression system and used bivalent MOP-NOP and fluorescent ligands to (i) probe for receptor interaction and (ii) consequences of that interaction. We used HEKMOP/NOP cells and two bivalent ligands; Dermorphin-N/OFQ (MOP agonist-NOP agonist; DeNO) and Dermorphin-UFP101 (MOP agonist-NOP antagonist; De101). We have determined receptor binding profiles, GTPγ[35S] binding, cAMP formation and ERK1/2 activation. We have also probed MOP and NOP receptor interactions in HEK cells and hippocampal neurones using the novel MOP fluorescent ligand, DermorphinATTO488 and the NOP fluorescent ligand N/OFQATTO594. In HEKMOP/NOP MOP ligands displaced NOP binding and NOP ligands displaced MOP binding. Using fluorescent probes in HEKMOP/NOP cells we demonstrated MOP-NOP probe overlap and a FRET signal indicating co-localisation. MOP-NOP were also co-localised in hippocampal tissue. In GTPγ[35S] and cAMP assays NOP stimulation shifted the response to MOP rightwards. At ERK1/2 the response to bivalent ligands generally peaked later. We provide evidence for MOP-NOP interaction in recombinant and native tissue. NOP activation reduces responsiveness of MOP activation; this was shown with conventional and bivalent ligands., Competing Interests: The authors have declared that no competing interests exist.

Opioid peptides exhibit a wide-ranging tissue distribution and control multiple tissue functions not only through reflex mechanisms involving the central nervous system or the modulation of neurotransmitter release, but also by acting directly at the cellular level by targeting selected receptor subtypes (μ, δ, and κ are among the most frequently expressed) [...].

Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, β-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, β-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, β-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of β-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of β-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.

Chronic immobilization stress (CIS) results in sex-dependent changes in opioid peptide levels and receptor subcellular distributions within the rat dorsal hippocampus, which are paralleled with an inability for males to acquire conditioned place preference (CPP) to oxycodone. Here, RNAScope in situ hybridization was used to determine the expression of hippocampal opioid peptides and receptors in unstressed (US) and CIS estrus female and male adult (∼2.5 months old ) Sprague Dawley rats. In all groups, dentate granule cells expressed PENK and PDYN; additionally, numerous interneurons expressed PENK. OPRD1 and OPRM1 were primarily expressed in interneurons, and to a lesser extent, in pyramidal and granule cells. OPRK1-was expressed in sparsely distributed interneurons. There were few baseline sex differences: US females compared to US males had more PENK-expressing and fewer OPRD1-expressing granule cells and more OPRM1-expressing CA3b interneurons. Several expression differences emerged after CIS. Both CIS females and males compared to their US counterparts had elevated: (1) PENK-expressing dentate granule cells and interneurons in CA1 and CA2/3a; (2) OPRD1 probe number and cell expression in CA1, CA2/3a and CA3b and the dentate gyrus; and (3) OPRK1-expressing interneurons in the dentate hilus. Also, CIS males compared to US males had elevated: (1) PDYN expression in granule cells; (2) OPRD1 probe and interneuron expression in CA2/3a; (3) OPRM1 in granule cells; and (4) OPRK1 interneuron expression in CA2/3a. The sex-specific changes in hippocampal opioid gene expression may impact network properties and synaptic plasticity processes that may contribute to the attenuation of oxycodone CPP in CIS males., (© 2021 Wiley Periodicals LLC.)

During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added.

Introduction: A recent study has shown that acetate administration leads to a fourfold increase in the transcription of proopiomelanocortin (POMC) mRNA in the hypothalamus. POMC is cleaved to peptides, including β-endorphin, an endogenous opioid (EO) agonist that binds preferentially to the µ-opioid receptor (MOR). We hypothesised that an acetate challenge would increase the levels of EO in the human brain. We have previously demonstrated that increased EO release in the human brain can be detected using positron emission tomography (PET) with the selective MOR radioligand [ 11 C]carfentanil. We used this approach to evaluate the effects of an acute acetate challenge on EO levels in the brain of healthy human volunteers., Methods: Seven volunteers each completed a baseline [ 11 C]carfentanil PET scan followed by an administration of sodium acetate before a second [ 11 C]carfentanil PET scan. Dynamic PET data were acquired over 90 minutes, and corrected for attenuation, scatter and subject motion. Regional [ 11 C] carfentanil BP ND values were then calculated using the simplified reference tissue model (with the occipital grey matter as the reference region). Change in regional EO concentration was evaluated as the change in [ 11 C]carfentanil BP ND following acetate administration., Results: Following sodium acetate administration, 2.5-6.5% reductions in [ 11 C]carfentanil regional BP ND were seen, with statistical significance reached in the cerebellum, temporal lobe, orbitofrontal cortex, striatum and thalamus., Conclusions: We have demonstrated that an acute acetate challenge has the potential to increase EO release in the human brain, providing a plausible mechanism of the central effects of acetate on appetite in humans.

Background: Nociceptin/orphanin FQ (N/OFQ) has been revealed to play bidirectional roles in orofacial pain modulation. Calcitonin gene-related peptide (CGRP) is a well-known pro-nociceptive molecule that participates in the modulation of orofacial pain. We aimed to determine the effects of N/OFQ on the modulation of orofacial pain and on the release of CGRP., Methods: Orofacial pain model was established by ligating springs between incisors and molars in rats for the simulation of tooth movement. The expression level of N/OFQ was determined and pain level was scored in response to orofacial pain. Both agonist and antagonist of N/OFQ receptor were administered to examine their effects on pain and the expression of CGRP in trigeminal ganglia (TG). Moreover, gene therapy based on the overexpression of N/OFQ was delivered to validate the modulatory role of N/OFQ on pain and CGRP expression., Results: Tooth movement elicited orofacial pain and an elevation in N/OFQ expression. N/OFQ exacerbated orofacial pain and upregulated CGRP expression in TG, while UFP-101 alleviated pain and downregulated CGRP expression. N/OFQ-based gene therapy was successful in overexpressing N/OFQ in TG, which resulted in pain exacerbation and elevation of CGRP expression in TG., Conclusions: N/OFQ exacerbated orofacial pain possibly through upregulating CGRP.

There exist three main types of endogenous opioid peptides, enkephalins, dynorphins and β-endorphin, all of which are derived from their precursors. These endogenous opioid peptides act through opioid receptors, including mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR), and play important roles not only in analgesia, but also many other biological processes such as reward, stress response, feeding and emotion. The MOR gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms. One type of these splice variants, the full-length 7 transmembrane (TM) Carboxyl ( C )-terminal variants, has the same receptor structures but contains different intracellular C -terminal tails. The pharmacological functions of several endogenous opioid peptides through the mouse, rat and human OPRM1 7TM C -terminal variants have been considerably investigated together with various mu opioid ligands. The current review focuses on the studies of these endogenous opioid peptides and summarizes the results from early pharmacological studies, including receptor binding affinity and G protein activation, and recent studies of β-arrestin2 recruitment and biased signaling, aiming to provide new insights into the mechanisms and functions of endogenous opioid peptides, which are mediated through the OPRM1 7TM C -terminal splice variants.

Tissue injury results in the release of inflammatory mediators, including a cascade of nociceptive substances, which contribute to development of hyperalgesia. In addition, during this process endogenous analgesic substances are also peripherally released with the aim of controlling the hyperalgesia. Thus, the present study aimed to investigate whether inflammatory mediators TNF-α, IL-1β, CXCL1, norepinephrine (NE) and prostaglandin E2 (PGE2) may be involved in the deflagration of peripheral endogenous modulation of inflammatory pain by activation of the opioid system. Thus, male Swiss mice and the paw withdrawal test were used. All substances were injected by the intraplantar route. Carrageenan, TNF-α, CXCL-1, IL1-β, NE and PGE2 induced hyperalgesia. Selectives μ (clocinamox), δ (naltrindole) and κ (norbinaltorphimine, nor-BNI) and non-selective (naloxone) opioid receptor antagonists potentiated the hyperalgesia induced by carrageenan, TNF-α, CXCL-1 and IL1-β. In contrast, when the enzyme N-aminopeptidase involved in the degradation of endogenous opioid peptides was inhibited by bestatin, the hyperalgesia was significantly reduced. In addition, the western blotting assay indicated that the expression of the opioid δ receptor was increased after intraplantar injection of carrageenan. The data obtained in this work corroborate the hypothesis that TNF-α, CXCL-1 and IL-β cause, in addition to hyperalgesia, the release of endogenous substances such as opioid peptides, which in turn exert endogenous control over peripheral inflammatory pain., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can be protective in inflammatory and neurologic disorders, and can alleviate pain. Classically, IL-4 diminishes pain by blocking the production of proinflammatory cytokines. Here, we uncovered that IL-4 induces acute antinociception by IL-4 receptor α (IL-4Rα)-dependent release of opioid peptides from M1 macrophages at injured nerves. As a model of pathologic pain, we used a chronic constriction injury (CCI) of the sciatic nerve in male mice. A single application of IL-4 at the injured nerves (14 d following CCI) attenuated mechanical hypersensitivity evaluated by von Frey filaments, which was reversed by co-injected antibody to IL-4Rα, antibodies to opioid peptides such as Met-enkephalin (ENK), β-endorphin and dynorphin A 1-17, and selective antagonists of δ-opioid, µ-opioid, and κ-opioid receptors. Injured nerves were predominately infiltrated by proinflammatory M1 macrophages and IL-4 did not change their numbers or the phenotype, assessed by flow cytometry and qRT-PCR, respectively. Macrophages isolated from damaged nerves by immunomagnetic separation (IMS) and stimulated with IL-4 dose dependently secreted all three opioid peptides measured by immunoassays. The IL-4-induced release of ENK was diminished by IL-4Rα antibody, intracellular Ca 2+ chelator, and inhibitors of protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), and ryanodine receptors. Together, we identified a new opioid mechanism underlying the IL-4-induced antinociception that involves PKA-mediated, PI3K-mediated, ryanodine receptor-mediated, and intracellular Ca 2+ -mediated release from M1 macrophages of opioid peptides, which activate peripheral opioid receptors in injured tissue. SIGNIFICANCE STATEMENT Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can ameliorate pain. The IL-4-mediated effects are considered to mostly result from the inhibition of the production of proinflammatory mediators (e.g., IL-1β, tumor necrosis factor, prostaglandin E2). Here, we found that IL-4 injected at the injured nerves attenuates pain by releasing opioid peptides from the infiltrating macrophages in mice. The opioids were secreted by IL-4 in the intracellular Ca 2+ -dependent manner and activated local peripheral opioid receptors. These actions represent a novel mode of IL-4 action, since its releasing properties have not been so far reported. Importantly, our findings suggest that the IL-4-opioid system should be targeted in the peripheral damaged tissue, since this can be devoid of central and systemic side effects., (Copyright © 2021 the authors.)

Energy homeostasis is regulated in coordinate fashion by the brain-gut axis, the homeostatic energy balance circuitry in the hypothalamus and the hedonic energy balance circuitry comprising the mesolimbcortical A 10 dopamine pathway. Collectively, these systems convey and integrate information regarding nutrient status and the rewarding properties of ingested food, and formulate it into a behavioral response that attempts to balance fluctuations in consumption and food-seeking behavior. In this review we start with a functional overview of the homeostatic and hedonic energy balance circuitries; identifying the salient neural, hormonal and humoral components involved. We then delve into how the function of these circuits differs in males and females. Finally, we turn our attention to the ever-emerging roles of nociceptin/orphanin FQ (N/OFQ) and pituitary adenylate cyclase-activating polypeptide (PACAP)-two neuropeptides that have garnered increased recognition for their regulatory impact in energy homeostasis-to further probe how the imposed regulation of energy balance circuitry by these peptides is affected by sex and altered under positive (e.g., obesity) and negative (e.g., fasting) energy balance states. It is hoped that this work will impart a newfound appreciation for the intricate regulatory processes that govern energy homeostasis, as well as how recent insights into the N/OFQ and PACAP systems can be leveraged in the treatment of conditions ranging from obesity to anorexia.

Incubation of eggs is a critical component of parental care in avian species. However, we do not fully understand the neuroendocrine mechanisms underlying this vital behavior. While prolactin is clearly involved, it alone cannot explain the fine-tuning of incubation behavior. The present experiments explored the possibility that incubation is reinforced through a hedonic system in which contact with eggs elicited an opiate-mediated reinforcing state. Blockade of opiate receptors with naloxone reduced time ring neck doves (Streptopelia risoria) spent on the nest, possibly by uncoupling the opiate-receptor mediated hedonic experience of contact with eggs from nest-sitting behavior. Likewise, activation of opiate receptors with morphine also reduced time spent on the nest, possibly by activating an opiate-receptor mediated hedonic experience, hence rendering the eliciting behavior (contact with eggs) unnecessary. Taken together, the results suggest that the opiate system may play a previously unrecognized role in facilitating incubation through reinforcement., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nociceptin receptor (NOP) belongs to the family of opioid receptors but was discovered and characterized much later than the so called classical opioid receptors, μ, δ and κ (or MOP, DOP and KOP, resp.). Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of this receptor and it controls numerous important functions in the central nervous system and in the periphery, so its analogs may be developed as innovative drugs for the treatment of a variety of conditions and pathological states. Availability of potent and selective ligands with high affinity to NOP receptor is essential to fully understand the role of NOP-N/OFQ system in the body, which in turn may lead to designing novel therapeutics. Here, we have focused on reviewing the structure of potent peptide-based agonists, antagonists, biased analogs and bivalent ligands that target NOP receptor., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

This review discusses our current knowledge on the nociceptin/orphanin (N/OFQ) system regarding its role in regulation of brain functions. Nociceptin receptor (NOPr) was identified in 1994 [Bunzow et al., 1994; Mollereau et al., 1994]. In 1995 a 17 amino acid endogenous peptide was found to be the high-affinity ligand for the NOPr [Reinscheid et al., 1995]. N/OFQ has a broad spectrum of activity and can act as on opioid-like as well as an anti-opioid peptide. Considering high level of N/OFQ and NOPr mRNA expression in the limbic brain regions, the N/OFQ/NOP system is suggested to be involved in regulation of emotions, resward, pain sensitivity, stress responsibility, sexual behavior, aggression, drug abuse and addiction. However it is still not well understood whether an increased vulnerability to drugs of abuse may be associated with dysregulation of N/OFQ/NOP system. Current review further highlights a need for further research on N/OFQ/NOP system as it could have clinical utility for substance abuse, depression, and anxiety pharmacotherapy.

Global cerebral ischemia induced by cardiac arrest usually leads to poor neurological outcomes. Numerous studies have focused on ways to prevent ischemic damage in the brain, however clinical therapies are still limited. Our previous studies revealed that delta opioid receptor (DOR) activation with [d-Ala2, d-Leu5] enkephalin (DADLE), a DOR agonist, not only significantly promotes neuronal survival on day 3, but also improves spatial memory deficits on days 5-9 after ischemia. However, the neurological mechanism underlying DADLE-induced cognitive recovery remains unclear. This study first examined the changes in neuronal survival in the CA1 region at the advanced time point (day 7) after ischemia/reperfusion (I/R) injury and found a significant amelioration of damaged CA1 neurons in the rats treated with DADLE (2.5 nmol) when administered at the onset of reperfusion. The structure and function of CA1 neurons on days 3 and 7 post-ischemia showed significant improvements in both the density of the injured dendritic spines and the basic transmission of the impaired CA3-CA1 synapses following DADLE treatment. The molecular changes involved in DADLE-mediated synaptic modulation on days 3 and 7 post-ischemia implied the time-related differential regulation of PKCα-MARCKS on the dendritic spine structure and of BDNF- ERK1/2-synapsin I on synaptic function, in response to ischemic/reperfusion injury as well as to DADLE treatment. Importantly, all the beneficial effects of DADLE on ischemia-induced cellular, synaptic, and molecular deficits were eliminated by the DOR inhibitor naltrindole (2.5 nmol). Taken together, this study suggested that DOR activation-induced protective signaling pathways of PKCα-MARCKS involved in the synaptic morphology and BDNF-ERK-synapsin I in synaptic transmission may be engaged in the cognitive recovery in rats suffering from advanced cerebral ischemia.

Background: Dermorphin is a heptapeptide with an analgesic potential higher than morphine that does not present the same risk for the development of tolerance. These pharmacological features make dermorphin a potential doping agent in competitive sports and it is already prohibited for racehorses. For athletes, the development of an efficient strategy to monitor for its abuse necessitates an investigation of the metabolism of dermorphin in humans., Methods: Here, human liver microsomes and zebrafish were utilized as model systems of human metabolism to evaluate the presence and kinetics of metabolites derived from dermorphin. Five hours after its administration, the presence of dermorphin metabolites could be detected in both models by liquid chromatography coupled to highresolution mass spectrometry., Results: Although the two models showed common results, marked differences were also observed in relation to the formed metabolites. Six putative metabolites, based on their exact masses of m/z 479.1915, m/z 501.1733, m/z 495.1657, m/z 223.1073, m/z 180.1017 and m/z 457.2085, are proposed to represent the metabolic pattern of dermorphin. The major metabolite generated from the administration of dermorphin in both models was YAFG-OH (m/z 457.2085), which is the N-terminal tetrapeptide previously identified from studies on rats., Conclusion: Its extensive characterization and commercial availability suggest that it could serve as a primary target analyte for the detection of dermorphin misuse. The metabolomics approach also allowed the assignment of other confirmatory metabolites., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Integrated behavioral paradigms such as nociceptive processing coupled to anti-nociceptive responsiveness include systemically-mediated states of alertness, vigilance, motivation, and avoidance. Within a historical and cultural context, opium and its biologically active compounds, codeine and morphine, have been widely used as frontline anti-nociceptive agents. In eukaryotic cells, opiate alkaloids and opioid peptides were evolutionarily fashioned as regulatory factors in neuroimmune, vascular immune, and systemic immune communication and auto-immunoregulation. The significance of opioidergic regulation of immune function was validated by the identification of novel μ and δ opioid receptors on circulating leukocytes. The novel μ3 opioid receptor subtype has been characterized as an opioid peptide-insensitive and opiate alkaloid-selective G protein-coupled receptor (GPCR) that is functionally linked to the activation of constitutive nitric oxide synthase (cNOS). Opioid peptides stimulate granulocyte and immunocyte activation and chemotaxis via activation of a novel leukocyte δ2 receptor subtype. However, opiate alkaloid μ3 receptor agonists inhibit these same cellular activities. Opiate coupling to cNOS and subsequent production and release of mitochondrial nitric oxide (NO) suggests an evolutionary linkage to similar physiological events in prokaryotic cells. A subpopulation of immunocytes from Mytilus edulis and Leucophaea maderae and human granulocytes respond to low opioid concentrations, mediated by the adherence-promoting role of (D-Ala2-D-Met5)-enkephalinamide (DAMA), which is blocked by naloxone in a dose-dependent manner. Neutral endopeptidase 24.11 (NEP), or enkephalinase (CD10), is present on both human and invertebrate immunocytes. Alkaloids, including morphine, are found in both prokaryotic and eukaryotic cells and may have evolved much later in evolution through horizontal gene transfer. It is possible that opioid-mediated regulatory activities were conserved and elaborated during evolution as the central nervous system (CNS) became immunologically isolated by the blood-brain barrier. Thus, opioid receptor coupling became significant for cognitive and behavioural processes. Although opioid peptides and alkaloids work synergistically to suppress nociception, they mediate different actions in immune surveillance. Increased understanding of the evolutionary development of opioid receptors, nociceptive and anti-nociceptive pathways, and immunomodulation may help in the understanding of the development of tolerance to the clinical use of opiates for pain management. The significance of endogenous morphine's importance to evolution can be ascertained by the number of physiological tissues and systems that can be affected by this chemical messenger mechanism, which transcends pain. An integrated review is presented of opioid and opiate receptors, immunomodulation, and pain associated with inflammation, from an evolutionary perspective., (Copyright © 2020. Published by Elsevier B.V.)

This brief review covers concepts in opioid pharmacology that were promoted during the period leading up to the establishment of the International Narcotics Research Conference (INRC) in the early 1970s and the discovery of endogenous opioid peptides in 1975. The founders of INRC, meeting together during the International Union of Pharmacology meeting in Basel in 1969, recognized that the time was ripe for the creation of an international society that would provide a venue for the discussion of research across disciplines in this rapidly expanding area of science. The emphasis here is on studies leading to the demonstration that specific receptors for morphine-like analgesics exist, the search for endogenous ligands for these receptors, and early attempts to elucidate the mechanisms underlying opiate drug tolerance, dependence, and addiction. SIGNIFICANCE STATEMENT: Research on opioids in the 20th century was driven by the search for nonaddicting analgesics. This review discusses the development of the "analgesic" receptor concept, the demonstration that such receptors existed, and the search for an endogenous ligand. Conceptual models were proposed to explain tolerance to the actions of opiate drugs and the development of dependence and addiction. This review explains these models and indicates how they foreshadowed more recent discoveries on the acute and chronic actions of opiate drugs., (U.S. Government work not protected by U.S. copyright.)

This paper is the forty-first consecutive installment of the annual anthological review of research concerning the endogenous opioid system, summarizing articles published during 2018 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides and receptors as well as effects of opioid/opiate agonists and antagonists. The review is subdivided into the following specific topics: molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors (2), the roles of these opioid peptides and receptors in pain and analgesia in animals (3) and humans (4), opioid-sensitive and opioid-insensitive effects of nonopioid analgesics (5), opioid peptide and receptor involvement in tolerance and dependence (6), stress and social status (7), learning and memory (8), eating and drinking (9), drug abuse and alcohol (10), sexual activity and hormones, pregnancy, development and endocrinology (11), mental illness and mood (12), seizures and neurologic disorders (13), electrical-related activity and neurophysiology (14), general activity and locomotion (15), gastrointestinal, renal and hepatic functions (16), cardiovascular responses (17), respiration and thermoregulation (18), and immunological responses (19)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rationale: Evaluation of pharmacotherapies for acute stress disorder (ASD) or post-traumatic stress disorder (PTSD) is challenging due to robust heterogeneity of trauma histories and limited efficacy of any single candidate to reduce all stress-induced effects. Pursuing novel mechanisms, such as the nociceptin/orphanin FQ (NOP) system, may be a viable path for therapeutic development and of interest as it is involved in regulation of relevant behaviors and recently implicated in PTSD and ASD., Objectives: First, we evaluated NOP receptor antagonism on general behavioral performance and again following a three-species predator exposure model (Experiment 1). Then, we evaluated effects of NOP antagonism on fear memory expression (Experiment 2)., Methods: Adult, male rats underwent daily administration of NOP antagonists (J-113397 or SB-612,111; 0-20 mg/kg, i.p.) and testing in acoustic startle, elevated plus maze, tail-flick, and open field tests. Effects of acute NOP antagonism on behavioral performance following predator exposure were then assessed. Separately, rats underwent fear conditioning and were later administered SB-612,111 (0-3 mg/kg, i.p.) prior to fear memory expression tests., Results: J-113397 and SB-612,111 did not significantly alter most general behavioral performance measures alone, suggesting minimal off-target behavioral effects of NOP antagonism. J-113397 and SB-612,111 restored performance in measures of exploratory behavior (basic movements on the elevated plus maze and total distance in the open field) following predator exposure. Additionally, SB-612,111 significantly reduced freezing behavior relative to control groups across repeated fear memory expression tests, suggesting NOP antagonism may be useful in dampening fear responses. Other measures of general behavioral performance were not significantly altered following predator exposure., Conclusions: NOP antagonists may be useful as pharmacotherapeutics for dampening fear responses to trauma reminders, and the present results provide supporting evidence for the implication of the NOP system in the neuropathophysiology of dysregulations in fear learning and memory processes observed in trauma- and stress-related disorders.

Electrophysiological approaches provide powerful tools to further our understanding of how different opioids affect signaling through opioid receptors; how opioid receptors modulate circuitry involved in processes such as pain, respiration, addiction, and feeding; and how receptor signaling and circuits are altered by physiologic challenges, such as injury, stress, and chronic opioid treatment. The use of genetic manipulations to alter or remove μ -opioid receptors (MORs) with anatomic and cell type specificity and the ability to activate or inhibit specific circuits through opto- or chemogenetic approaches are being used in combination with electrophysiological, pharmacological, and systems-level physiology experiments to expand our understanding of the beneficial and maladaptive roles of opioids and opioid receptor signaling. New approaches for studying endogenous opioid peptide signaling and release and the dynamics of these systems in response to chronic opioid use, pain, and stress will add another layer to our understanding of the intricacies of opioid modulation of brain circuits. This understanding may lead to new targets or approaches for drug development or treatment regimens that may affect both acute and long-term effects of manipulating the activity of circuits involved in opioid-mediated physiology and behaviors. This review will discuss recent advancements in our understanding of the role of phosphorylation in regulating MOR signaling, as well as our understanding of circuits and signaling pathways mediating physiologic behaviors such as respiratory control, and discuss how electrophysiological tools combined with new technologies have and will continue to advance the field of opioid research. SIGNIFICANCE STATEMENT: This review discusses recent advancements in our understanding of μ -opioid receptor (MOR) function and regulation and the role of electrophysiological approaches combined with new technologies in pushing the field of opioid research forward. This covers regulation of MOR at the receptor level, adaptations induced by chronic opioid treatment, sites of action of MOR modulation of specific brain circuits, and the role of the endogenous opioid system in driving physiology and behavior through modulation of these brain circuits., (Copyright © 2020 by The Author(s).)

The opioid receptors are members of the G-protein-coupled receptor (GPCR) family and are known to modulate a variety of biological functions, including pain perception. Despite considerable advances, the mechanisms by which opioid agonists and antagonists interact with their receptors and exert their effect are still not completely understood. In this report, six new hybrids of the Dmt-Tic pharmacophore and cyclic peptides, which were shown before to have a high affinity for the µ-opioid receptor (MOR) were synthesized and characterized pharmacologically in calcium mobilization functional assays. All obtained ligands turned out to be selective antagonists of the δ-opioid receptor (DOR) and did not activate or block the MOR. The three-dimensional structural determinants responsible for the DOR antagonist properties of these analogs were further investigated by docking studies. The results indicate that these compounds attach to the DOR in a slightly different orientation with respect to the Dmt-Tic pharmacophore than Dmt-TicΨ[CH 2 -NH]Phe-Phe-NH 2 (DIPP-NH 2 [Ψ]), a prototypical DOR antagonist peptide. Key pharmacophoric contacts between the DOR and the ligands were maintained through an analogous spatial arrangement of pharmacophores, which could provide an explanation for the predicted high-affinity binding and the experimentally observed functional properties of the novel synthetic ligands.

In humans, alcohol is consumed for its rewarding and anxiolytic effects. The central nucleus of the amygdala (CeA) is considered a neuronal nexus that regulates fear, anxiety, and drug self-administration. Manipulations of the CeA alter ethanol (EtOH) consumption under numerous EtOH self-administration models. The experiments determined whether EtOH is reinforcing/anxiolytic within the CeA, whether selective breeding for high alcohol consumption alters the rewarding properties of EtOH in the CeA, and whether the reinforcing/anxiolytic effects of EtOH in the CeA are mediated by the neuropeptides corticotropin-releasing factor (CRF) and nociceptin. The reinforcing properties of EtOH were determined by having male Wistar and Taconic alcohol-preferring (tP) rats self-administer EtOH directly into the CeA. The expression of anxiety-like behaviors was assessed through multiple behavioral models (social interaction, acoustic startle, and open field). Coadministration of EtOH and a CRF1 antagonist (NBI35965) or nociceptin on self-administration into the CeA and anxiety-like behaviors was determined. EtOH was self-administered directly into the lateral CeA, and tP rats self-administered a lower concentration of EtOH than Wistar rats. EtOH microinjected into the lateral CeA reduced the expression of anxiety-like behaviors, indicating an anxiolytic effect. Coadministration of NBI35965 failed to alter the rewarding/anxiolytic properties of EtOH in the CeA. In contrast, coadministration of the nociceptin enhanced both EtOH reward and anxiolysis in the CeA. Overall, the data indicate that the lateral CeA is a key anatomic location that mediates the rewarding and anxiolytic effects of EtOH, and local nociceptin receptors, but not local CRF1 receptors, are involved in these behaviors. SIGNIFICANCE STATEMENT: Alcohol is consumed for the stimulatory, rewarding, and anxiolytic properties of the drug of abuse. The current data are the first to establish that alcohol is reinforcing and anxiolytic within the lateral central nucleus of the amygdala (CeA) and that the nociceptin system regulates these effects of alcohol within the CeA., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Peptides are an important class of molecules with diverse biological activities. Many endogenous peptides, especially neuropeptides and peptide hormones, play critical roles in development and regulating homeostasis. Furthermore, as drug candidates their high receptor selectivity and potent binding leads to reduced off-target interactions and potential negative side effects. However, the therapeutic potential of peptides is severely hampered by their poor stability in vivo and low permeability across biological membranes. Several strategies have been successfully employed over the decades to address these concerns, and one of the most promising strategies is glycosylation. It has been demonstrated in numerous cases that glycosylation is an effective synthetic approach to improve the pharmacokinetic profiles and membrane permeability of peptides. The effects of glycosylation on peptide stability and peptide-membrane interactions in the context of blood-brain barrier penetration will be explored. Numerous examples of glycosylated analogues of endogenous peptides targeting class A and B G-protein coupled receptors (GPCRs) with an emphasis on O-linked glycopeptides will be reviewed. Notable examples of N-, S-, and C-linked glycopeptides will also be discussed. A small section is devoted to synthetic methods for the preparation of glycopeptides and requisite amino acid glycoside building blocks., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: To investigate the effect and mechanism of botulinum neurotoxin type A (BoNT/A) in the modulation of orofacial nociception induced by orthodontic tooth movement in rats., Methods: An orofacial nociception model was established in male Sprague-Dawley rats by ligating closed-coil springs between incisors and ipsilateral molars. There were two group sets of animals. For the first group set, 120 rats were randomly divided into four groups: no-force group (n = 30), force + saline group (n = 30), force + low dose BoNT/A group (1U/6 μL, n = 30), and force + high dose BoNT/A group (1U/6 μL, n = 30). BoNT/A and saline were injected into periodontal ligament to explore the nociceptive effect of BoNT/A. Ipsilateral trigeminal ganglia (TG) were harvested for detecting the expression levels of nociceptin/orphanin-FQ (N/OFQ). For the second group set, 36 rats were randomly divided into three force groups: BoNT/A + saline group (n = 12), BoNT/A + UFP-101 group (n = 12), and saline + UFP-101 group (n = 12). A potent N/OFQ receptor (NOP) antagonist (UFP-101) was used to examine the role of N/OFQ in BoNT/A-induced antinociception. Tooth-movement nociception level of all groups was evaluated by bite force and rat grimace scale (RGS) at baseline, day 1, day 3, day 5, day 7, day 14., Results: The behavioral assessments showed the orofacial nociception level in the force + low dose BoNT/A group and force + high dose BoNT/A group were lower than that in the force + saline group. No significant difference was observed in orofacial nociception among no-force group, force + low dose and force + high dose group. The expression levels of N/OFQ in TG were elevated from day 1 and maintained a high level, presenting in descending order among the force + high dose, force + low dose, force + saline and no-force group, respectively. The nociception level of the BoNT/A + UFP-101 group was higher than that of the BoNT/A + saline group. No significant difference was observed between the BoNT/A + UFP-101 group and the saline + UFP-101 group., Conclusions: BoNT/A can exert an antinociceptive effect on orofacial nociception induced by tooth movement by stimulating the expression of N/OFQ in TG., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Drug-induced phospholipidosis (DIPL) is characterized by phospholipid storage in the lysosomes of affected tissues. Many severe effects and toxicities have been linked to DIPL. The aim of this study was to determine whether the endogenous opioid system is involved in chloroquine-induced phospholipidosis. The effect of naltrexone as an antagonist of opioid receptors in chloroquine-induced phospholipidosis in rat liver was investigated by morphological, biochemical, and molecular modelling studies. Transmission electron microscopy (TEM) showed that morphological characteristic changes of rat liver, including the number of lamellar bodies, grade of vacuolization and cell steatosis, were markedly attenuated in rats treated with naltrexone alone or in combination with chloroquine, in comparison with chloroquine-treated rats. The results of liquid chromatography mass spectrometry (LC/MS) showed that the concentrations of phenylacetylglycine (PAG) and hippuric acid (HA) were significantly decreased and increased, respectively, in target groups. Besides, the concentration ratio of PAG/HA was significantly decreased. Spectrophotometry resulted in a notable decrease in alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities in target groups. The results from the molecular docking and molecular dynamic simulation studies demonstrated clear chloroquine interaction with the active site cavity of the µ opioid receptor. These data suggest that administration of naltrexone alone, or in combination with chloroquine, notably attenuates the side effects of chloroquine-induced phospholipidosis, as well as demonstrating an increased probability of the endogenous opioid system involvement in chloroquine-induced phospholipidosis in rat liver., (© 2020 John Wiley & Sons Australia, Ltd.)

In the mid-1970s, an intense race to identify endogenous substances that activated the same receptors as opiates resulted in the identification of the first endogenous opioid peptides. Since then, >20 peptides with opioid receptor activity have been discovered, all of which are generated from three precursors, proenkephalin, prodynorphin, and proopiomelanocortin, by sequential proteolytic processing by prohormone convertases and carboxypeptidase E. Each of these peptides binds to all three of the opioid receptor types ( μ , δ , or κ ), albeit with differing affinities. Peptides derived from proenkephalin and prodynorphin are broadly distributed in the brain, and mRNA encoding all three precursors are highly expressed in some peripheral tissues. Various approaches have been used to explore the functions of the opioid peptides in specific behaviors and brain circuits. These methods include directly administering the peptides ex vivo (i.e., to excised tissue) or in vivo (in animals), using antagonists of opioid receptors to infer endogenous peptide activity, and genetic knockout of opioid peptide precursors. Collectively, these studies add to our current understanding of the function of endogenous opioids, especially when similar results are found using different approaches. We briefly review the history of identification of opioid peptides, highlight the major findings, address several myths that are widely accepted but not supported by recent data, and discuss unanswered questions and future directions for research. SIGNIFICANCE STATEMENT: Activation of the opioid receptors by opiates and synthetic drugs leads to central and peripheral biological effects, including analgesia and respiratory depression, but these may not be the primary functions of the endogenous opioid peptides. Instead, the opioid peptides play complex and overlapping roles in a variety of systems, including reward pathways, and an important direction for research is the delineation of the role of individual peptides., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Addiction has been proposed as a 'reward deficient' state, which is compensated for with substance use. There is growing evidence of dysregulation in the opioid system, which plays a key role in reward, underpinning addiction. Low levels of endogenous opioids are implicated in vulnerability for developing alcohol dependence (AD) and high mu-opioid receptor (MOR) availability in early abstinence is associated with greater craving. This high MOR availability is proposed to be the target of opioid antagonist medication to prevent relapse. However, changes in endogenous opioid tone in AD are poorly characterised and are important to understand as opioid antagonists do not help everyone with AD. We used [ 11 C]carfentanil, a selective MOR agonist positron emission tomography (PET) radioligand, to investigate endogenous opioid tone in AD for the first time. We recruited 13 abstinent male AD and 15 control participants who underwent two [ 11 C]carfentanil PET scans, one before and one 3 h following a 0.5 mg/kg oral dose of dexamphetamine to measure baseline MOR availability and endogenous opioid release. We found significantly blunted dexamphetamine-induced opioid release in 5 out of 10 regions-of-interest including insula, frontal lobe and putamen in AD compared with controls, but no significantly higher MOR availability AD participants compared with HC in any region. This study is comparable to our previous results of blunted dexamphetamine-induced opioid release in gambling disorder, suggesting that this dysregulation in opioid tone is common to both behavioural and substance addictions.

The generation of fully functional oligodendrocytes, the myelinating cells of the central nervous system, is preceded by a complex maturational process. We previously showed that the timing of oligodendrocyte differentiation and rat brain myelination were altered by perinatal exposure to buprenorphine and methadone, opioid analogs used for the management of pregnant addicts. Those observations suggested the involvement of the μ-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOR). However, it remained to be determined if these receptors and their endogenous ligands could indeed control the timing of myelination under normal physiological conditions of brain development. We now found that the endogenous MOR ligand endomorphin-1 (EM-1) exerts a striking stimulatory action on cellular and morphological maturation of rat pre-oligodendrocytes, but unexpectedly, these effects appear to be restricted to the cells from the female pups. Critically, this stimulation is abolished by coincubation with the endogenous NOR ligand nociceptin. Furthermore, NOR antagonist treatment of 9-day-old female pups results in accelerated brain myelination. Interestingly, the lack of sex-dependent differences in developmental brain levels of EM-1 and nociceptin, or oligodendroglial expression of MOR and NOR, suggests that the observed sex-specific responses may be highly dependent on important intrinsic differences between the male and female oligodendrocytes. The discovery of a significant effect of EM-1 and nociceptin in the developing female oligodendrocytes and brain myelination, underscores the need for further studies investigating brain sex-related differences and their implications in opioid use and abuse, pain control, and susceptibility and remyelinating capacity in demyelinating disease as multiple sclerosis., (© 2020 Wiley Periodicals, Inc.)

Opioid peptides, derived from PENK, POMC, PDYN and PNOC precursors, together with their receptors (DOR, MOR, KOR and ORL1), constitute the opioid system and are suggested to participate in multiple physiological/pathological processes in vertebrates. However, the question whether an opioid system exists and functions in non-mammalian vertebrates including birds remains largely unknown. Here, we cloned genes encoding opioid system from the chicken brain and examined their functionality and tissue expression. As in mammals, 6 opioid peptides encoded by PENK (Met-enkephalin and Leu-enkephalin), POMC (β-endorphin), PDYN (dynorphin-A and dynorphin-B) and PNOC (nociceptin) precursors and four opioid receptors were found to be highly conserved in chickens. Using pGL3-CRE-luciferase and pGL4-SRE-luciferase reporter systems, we demonstrated that chicken opioid receptors (cDOR, cMOR, cKOR and cORL1) expressed in CHO cells, could be differentially activated by chicken opioid peptides, and resulted in the inhibition of cAMP/PKA and activation of MAPK/ERK signaling pathways. cDOR is potently activated by Met-enkephalin and Leu-enkephalin, and cKOR is potently activated by dynorphin-A, dynorphin-B and nociceptin, whereas cORL1 is specifically activated by nociceptin. Unlike cDOR, cKOR and cORL1, cMOR is moderately/weakly activated by enkephalins and other opioid peptides. These findings suggest the ligand-receptor pair in chicken opioid system is similar, but not identical to, that in mammals. Quantitative real-time PCR revealed that the opioid system is mainly expressed in chicken central nervous system including the hypothalamus. Collectively, our data will help to facilitate the better understanding of the conserved roles of opioid system across vertebrates., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The scarcity and limited risk/benefit ratio of painkillers available on the market, in addition to the opioid crisis, warrant reflection on new innovation strategies. The pharmacopoeia of analgesics is based on products that are often old and derived from clinical empiricism, with limited efficacy or spectrum of action, or resulting in an unsatisfactory tolerability profile. Although they are reference analgesics for nociceptive pain, opioids are subject to the same criticism. The use of opium as an analgesic is historical. Morphine was synthesized at the beginning of the 19th century. The efficacy of opioids is limited in certain painful contexts and these drugs can induce potentially serious and fatal adverse effects. The current North American opioid crisis, with an ever-rising number of deaths by opioid overdose, is a tragic illustration of this. It is therefore legitimate to develop research into molecules likely to maintain or increase opioid efficacy while improving their tolerability. Several avenues are being explored including targeting of the mu opioid receptor (MOR) splice variants, developing biased agonists or targeting of other receptors such as heteromers with MOR. Ion channels acting as MOR effectors, are also targeted in order to offer compounds without MOR-dependent adverse effects. Another route is to develop opioid analgesics with peripheral action or limited central nervous system (CNS) access. Finally, endogenous opioids used as drugs or compounds that modify the metabolism of endogenous opioids (Dual ENKephalinase Inhibitors) are being developed. The aim of the present review is to present these various targets/strategies with reference to current indications for opioids, concerns about their widespread use, particularly in chronic non-cancer pains, and ways of limiting the risk of opioid abuse and misuse., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest. We affirm that the manuscript has not been published and is not under consideration for publication elsewhere., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Alzheimer's disease is associated with biochemical and histopathological changes characterized by molecular abnormalities. Due to the lack of effective treatments for Alzheimer's disease, many attempts have been made to find potential therapies to reduce or even return neuronal loss after disease initiation. Alzheimer's disease is also touted as type III diabetes, showing an association with insulin signaling. The large distribution of the insulin receptor on the cell surface and its regulatory role in the central nervous system suggests that the pathogenesis of Alzheimer's disease could be ascribed to insulin signaling. The interference of opioids, such as morphine with insulin signaling pathways, is thought to occur via direct crosstalk between the signaling pathways of the insulin receptor and the mu-opioid receptor. In this review article, we discuss the possible crosstalk between the mu-opioid receptor and insulin signaling pathways. The association of these two signaling pathways with Alzheimer's disease is also debated.

Opioids, such as morphine and fentanyl, are widely used for the treatment of severe pain; however, prolonged treatment with these drugs leads to the development of tolerance and can lead to opioid use disorder. The "Opioid Epidemic" has generated a drive for a deeper understanding of the fundamental signaling mechanisms of opioid receptors. It is generally thought that the three types of opioid receptors (μ, δ, κ) are activated by endogenous peptides derived from three different precursors: Proopiomelanocortin, proenkephalin, and prodynorphin. Posttranslational processing of these precursors generates >20 peptides with opioid receptor activity, leading to a long-standing question of the significance of this repertoire of peptides. Here, we address some aspects of this question using a technical tour de force approach to systematically evaluate ligand binding and signaling properties ([ 35 S]GTPγS binding and β-arrestin recruitment) of 22 peptides at each of the three opioid receptors. We show that nearly all tested peptides are able to activate the three opioid receptors, and many of them exhibit agonist-directed receptor signaling (functional selectivity). Our data also challenge the dogma that shorter forms of β-endorphin do not exhibit receptor activity; we show that they exhibit robust signaling in cultured cells and in an acute brain slice preparation. Collectively, this information lays the groundwork for improved understanding of the endogenous opioid system that will help in developing more effective treatments for pain and addiction., Competing Interests: The authors declare no competing interests.

Deer mice provide a non-pharmacologically induced model for the study of repetitive behaviors. In captivity, these animals develop frequent jumping and rearing that resemble clinical symptoms of obsessive-compulsive behavior (OCB), autism spectrum disorder (ASD), complex motor stereotypies (CMS), and Tourette's syndrome (TS). In this study, we pursue the mechanism of repetitive behaviors by performing stereological analyses and liquid chromatography/ mass spectrometry (LC-MS/MS) measurements of glutamate (Glut), GABA, 3,4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA), leu-enkephalin (leu-enk), and dynorphin-A (dyn-A) in frontal cortex (FC), prefrontal cortex (PFC), and basal ganglia. The only significant stereological alteration was a negative correlation between repetitive behaviors and the cell count in the ventromedial striatum (VMS). Neurochemical analyses demonstrated a significant negative correlation between repetitive behaviors and endogenous opioids (leu-enk and dyn-A) in the FC - the site of origin of habitual behaviors and cortical projections to striatal MSNs participating in direct and indirect pathways. The precise neurochemical process by which endogenous opioids influence synaptic neurotransmission is unknown. One postulated cortical mechanism, supported by our findings, is an opioid effect on cortical interneuron GABA release and a consequent effect on glutamatergic cortical pyramidal cells. Anatomical changes in the VMS could have a role in repetitive behaviors, recognizing that this region influences goal-directed and habitual behaviors., (Copyright © 2019 Elsevier B.V. All rights reserved.)