Grégory Scherrer

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.

Dissociation of the Opioid Receptor Mechanisms that Control Mechanical and Heat Pain

Delta and mu opioid receptors (DORs and MORs) are inhibitory G protein-coupled receptors that reportedly cooperatively regulate the transmission of pain messages by substance P and TRPV1-expressing pain fibers. Using a DOReGFP reporter mouse we now show that the DOR and MOR are, in fact, expressed by different subsets of primary afferents. The MOR is expressed in peptidergic pain fibers, the DOR in myelinated and nonpeptidergic afferents. Contrary to the prevailing view, we demonstrate that the DOR is trafficked to the cell surface under resting conditions, independently of substance P, and internalized following activation by DOR agonists. Finally, we show that the segregated DOR and MOR distribution is paralleled by a remarkably selective functional contribution of the two receptors to the control of mechanical and heat pain, respectively. These results demonstrate that behaviorally relevant pain modalities can be selectively regulated through the targeting of distinct subsets of primary afferent pain fibers.

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

Background 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. Methods and Findings 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. Conclusions 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.

Behavioral indices of ongoing pain are largely unchanged in male mice with tissue or nerve injury-induced mechanical hypersensitivity

&NA; Despite the impact of chronic pain on the quality of life in patients, including changes to affective state and daily life activities, rodent preclinical models rarely address this aspect of chronic pain. To better understand the behavioral consequences of the tissue and nerve injuries typically used to model neuropathic and inflammatory pain in mice, we measured home cage and affective state behaviors in animals with spared nerve injury, chronic constriction injury (CCI), or intraplantar complete Freund’s adjuvant. Mechanical hypersensitivity is prominent in each of these conditions and persists for many weeks. Home cage behavior was continuously monitored for 16 days in a system that measures locomotion, feeding, and drinking, and allows for precise analysis of circadian patterns. When monitored after injury, animals with spared nerve injury and complete Freund’s adjuvant behaved no differently from controls in any aspect of daily life. Animals with CCI were initially less active, but the difference between CCI and controls disappeared by 2 weeks after injury. Further, in all pain models, there was no change in any measure of affective state. We conclude that in these standard models of persistent pain, despite the development of prolonged hypersensitivity, the mice do not have significantly altered “quality of life.” As alteration in daily life activities is the feature that is so disrupted in patients with chronic pain, our results suggest that the models used here do not fully reflect the human conditions and point to a need for development of a murine chronic pain model in which lifestyle changes are manifest. Mice with spared nerve injury, chronic constriction injury, and complete Freund’s adjuvant injection did not alter behavior in their home cage or a battery of affective tests.

A mu-delta opioid receptor brain atlas reveals neuronal co-occurrence in subcortical networks.

Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives.

The delta agonists DPDPE and deltorphin II recruit predominantly mu receptors to produce thermal analgesia: a parallel study of mu, delta and combinatorial opioid receptor knockout mice.

Delta‐selective agonists have been developed to produce potent analgesic compounds with limited side‐effects. DPDPE and deltorphin II are considered prototypes, but their delta‐selectivity in vivo and the true ability of delta receptors to produce analgesia remain to be demonstrated. Here we have performed a parallel analysis of mu, delta and combinatorial opioid receptor knockout mice, in which we found no obvious alteration of G‐protein coupling for remaining opioid receptors. We compared behavioural responses in two models of acute thermal pain following DPDPE and deltorphin II administration by intracerebroventricular route. In the tail‐immersion test, both compounds were fully analgesic in delta knockout mice and totally inactive in mu knockout mice. In the hotplate test, the two compounds again produced full analgesia in delta knockout mice. In mu knockout mice, there was significant, although much lower, analgesia. Furthermore, DPDPE analgesia in the delta knockout mice was fully reversed by the mu selective antagonist CTOP in both tests. Together, this suggests that mu rather than delta receptors are recruited by the two agonists for the tail withdrawal and the hotplate responses. Finally, deltorphin II slightly prolonged jump latencies in double mu/kappa knockout mice (delta receptors only) and this response was abolished in the triple knockout mice, demonstrating that the activation of delta receptors alone can produce weak but significant mu‐independent thermal antinociception.

VGLUT2 expression in primary afferent neurons is essential for normal acute pain and injury-induced heat hypersensitivity

Dorsal root ganglia (DRG) neurons, including the nociceptors that detect painful thermal, mechanical, and chemical stimuli, transmit information to spinal cord neurons via glutamatergic and peptidergic neurotransmitters. However, the specific contribution of glutamate to pain generated by distinct sensory modalities or injuries is not known. Here we generated mice in which the vesicular glutamate transporter 2 (VGLUT2) is ablated selectively from DRG neurons. We report that conditional knockout (cKO) of the Slc17a6 gene encoding VGLUT2 from the great majority of nociceptors profoundly decreased VGLUT2 mRNA and protein in these neurons, and reduced firing of lamina I spinal cord neurons in response to noxious heat and mechanical stimulation. In behavioral assays, cKO mice showed decreased responsiveness to acute noxious heat, mechanical, and chemical (capsaicin) stimuli, but responded normally to cold stimulation and in the formalin test. Strikingly, although tissue injury-induced heat hyperalgesia was lost in the cKO mice, mechanical hypersensitivity developed normally. In a model of nerve injury-induced neuropathic pain, the magnitude of heat hypersensitivity was diminished in cKO mice, but both the mechanical allodynia and the microgliosis generated by nerve injury were intact. These findings suggest that VGLUT2 expression in nociceptors is essential for normal perception of acute pain and heat hyperalgesia, and that heat and mechanical hypersensitivity induced by peripheral injury rely on distinct (VGLUT2 dependent and VGLUT2 independent, respectively) primary afferent mechanisms and pathways.

Pre- and postsynaptic inhibitory control in the spinal cord dorsal horn

Sensory information transmitted to the spinal cord dorsal horn is modulated by a complex network of excitatory and inhibitory interneurons. The two main inhibitory transmitters, GABA and glycine, control the flow of sensory information mainly by regulating the excitability of dorsal horn neurons. A presynaptic action of GABA has also been proposed as an important modulatory mechanism of transmitter release from sensory primary afferent terminals. By inhibiting the release of glutamate from primary afferent terminals, activation of presynaptic GABA receptors could play an important role in nociceptive and tactile sensory coding, while changes in their expression or function could be involved in pathological pain conditions, such as allodynia.

Loss of [mu] opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia

Opioid pain medications have detrimental side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). Tolerance and OIH counteract opioid analgesia and drive dose escalation. The cell types and receptors on which opioids act to initiate these maladaptive processes remain disputed, which has prevented the development of therapies to maximize and sustain opioid analgesic efficacy. We found that μ opioid receptors (MORs) expressed by primary afferent nociceptors initiate tolerance and OIH development. RNA sequencing and histological analysis revealed that MORs are expressed by nociceptors, but not by spinal microglia. Deletion of MORs specifically in nociceptors eliminated morphine tolerance, OIH and pronociceptive synaptic long-term potentiation without altering antinociception. Furthermore, we found that co-administration of methylnaltrexone bromide, a peripherally restricted MOR antagonist, was sufficient to abrogate morphine tolerance and OIH without diminishing antinociception in perioperative and chronic pain models. Collectively, our data support the idea that opioid agonists can be combined with peripheral MOR antagonists to limit analgesic tolerance and OIH.