Cellular and Molecular Mechanisms of Pain

The nervous system detects and interprets a wide range of thermal and mechanical stimuli, as well as environmental and endogenous chemical irritants. When intense, these stimuli generate acute pain, and in the setting of persistent injury, both peripheral and central nervous system components of the pain transmission pathway exhibit tremendous plasticity, enhancing pain signals and producing hypersensitivity. When plasticity facilitates protective reflexes, it can be beneficial, but when the changes persist, a chronic pain condition may result. Genetic, electrophysiological, and pharmacological studies are elucidating the molecular mechanisms that underlie detection, coding, and modulation of noxious stimuli that generate pain

Structure-based discovery of opioid analgesics with reduced side effects

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids—which include fatal respiratory depression—are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21—a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids

In Vivo Delta Opioid Receptor Internalization Controls Behavioral Effects of Agonists

GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs

Ensuring transparency and minimization of methodologic bias in preclinical pain research:PPRECISE considerations

Acknowledgements The authors thank Allison Lin, Dan Mellon, and LiSheng Chen for their help throughout the process of writing this article.Peer reviewedPublisher PD

Etude de la fonction du récepteur aux opioïdes delta par modification génétique chez la souris

Le système opioïde est un système neuromodulateur composé des récepteurs mu, delta et kappa et de leurs ligands peptidiques endogènes. Il régule de très nombreuses fonctions centrales et périphériques, notamment le contrôle de la douleur et les réponses émotionnelles. L objectif de ce travail était de contribuer à la compréhension de la fonction du récepteur aux opioïdes delta. La stratégie reposait sur l établissement et l étude de souris génétiquement modifiées.Dans la première partie de ce travail, nous avons analysé le comportement de souris déficientes en récepteur delta dans des modèles de douleur, d anxiété, de dépression et de mémoire. Nous avons pu définir quelle est l implication du récepteur delta dans le contrôle de la douleur aiguë thermique et mettre en évidence sa participation dans les processus de mémorisation contextuelle.Dans la seconde partie de ce travail, nous avons établi une lignée de souris floxées pour le gène codant pour le récepteur delta (dor). Ces animaux expriment des récepteurs delta fonctionnels à un taux physiologique, et permettront, par expression régio-spécifique de la recombinase Cre, d invalider le gène dor puis d étudier la fonction du récepteur dans les différentes sous-populations neuronales où il est exprimé.Enfin, dans la troisième partie de ce travail, nous avons généré et analysé une lignée de souris exprimant un récepteur delta fusionné à la protéine fluorescente verte GFP. La visualisation directe de delta sur des sections de tissu a révélé sa distribution et sa localisation subcellulaire, et permis la caractérisation des neurones dans lesquels il est exprimé. Le trafic intracellulaire du récepteur a également été étudié, à la fois sur des coupes de tissu et en temps réel dans des cultures primaires de neurones. Ceci a permis d évaluer in vivo l incidence des propriétés dynamiques et de la répartition subcellulaire du récepteur sur son activité.The opioid system is a neuromodulator system composed of mu, delta and kappa opioid receptors, and their endogenous peptidic ligands. The opioid system regulates numerous central and peripheral functions such as pain control and emotional responses. The goal of this work was to better understand delta opioid receptor function. The strategy relied on the study of mutant mice.In the first part of this work, we analyzed the behaviour of mice deficient for delta opioid receptors in several models of pain, anxiety, depression and memory. We managed both to clarify the involvement of delta opioid receptors in the control of acute thermal pain, and to reveal its participation to mechanisms underlying processing of contextual memory.In the second part of this work, we have established a mouse line harbouring a floxed delta opioid receptor gene. These animals, which express functional delta opioid receptors at physiological levels, will be useful to study the function of delta opioid receptors expressed in different brain structures through local gene invalidation gene by region-specific expression of the Cre recombinase.Finally, in the third part of this work, we have both generated and analyzed a mouse line expressing delta opioid receptors fused to the green fluorescent protein GFP. Direct visualization of delta on brain slices revealed its distribution and subcellular localization and allowed characterization of delta expressing neurons. Receptor trafficking was also studied, on tissue sections as well as in real-time on primary cultures, to reveal relationship existing between dynamic properties and subcellular localization of delta opioid receptors and their activity in vivo.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Le récepteur aux opioïdes delta dans le contrôle de la douleur et des émotions

STRASBOURG ILLKIRCH-Pharmacie (672182101) / SudocSudocFranceF

An amygdalar neural ensemble that encodes the unpleasantness of pain

Pain is an unpleasant experience. How the brain’s affective neural circuits attribute this aversive quality to nociceptive information remains unknown. By means of time-lapse in vivo calcium imaging and neural activity manipulation in freely behaving mice encountering noxious stimuli, we identified a distinct neural ensemble in the basolateral amygdala that encodes the negative affective valence of pain. Silencing this nociceptive ensemble alleviated pain affective-motivational behaviors without altering the detection of noxious stimuli, withdrawal reflexes, anxiety, or reward. Following peripheral nerve injury, innocuous stimuli activated this nociceptive ensemble to drive dysfunctional perceptual changes associated with neuropathic pain, including pain aversion to light touch (allodynia). These results identify the amygdalar representations of noxious stimuli that are functionally required for the negative affective qualities of acute and chronic pain perception.ISSN:0036-8075ISSN:1095-920

An amygdalar neural ensemble that encodes the unpleasantness of pain

Pain is an unpleasant experience. How the brain’s affective neural circuits attribute this aversive quality to nociceptive information remains unknown. By means of time-lapse in vivo calcium imaging and neural activity manipulation in freely behaving mice encountering noxious stimuli, we identified a distinct neural ensemble in the basolateral amygdala that encodes the negative affective valence of pain. Silencing this nociceptive ensemble alleviated pain affective-motivational behaviors without altering the detection of noxious stimuli, withdrawal reflexes, anxiety, or reward. Following peripheral nerve injury, innocuous stimuli activated this nociceptive ensemble to drive dysfunctional perceptual changes associated with neuropathic pain, including pain aversion to light touch (allodynia). These results identify the amygdalar representations of noxious stimuli that are functionally required for the negative affective qualities of acute and chronic pain perception