A molecular basis of analgesic tolerance to cannabinoids

Clinical usage of cannabinoids in chronic pain states is limited by their central side effects and the pharmacodynamic tolerance that sets in after repeated dosage. Analgesic tolerance to cannabinoids in vivo could be caused by agonist-induced downregulation and intracellular trafficking of cannabinoid receptors, but little is known about the molecular mechanisms involved. We show here that the type 1 cannabinoid receptor (CB1) interacts physically with G-protein-associated sorting protein 1 (GASP1), a protein that sorts receptors in lysosomal compartments destined for degradation. CB1 - GASP1 interaction was observed to be required for agonist-induced downregulation of CB1 in spinal neurons ex vivo as well as in vivo. Importantly, uncoupling CB1 from GASP1 in mice in vivo abrogated tolerance toward cannabinoid-induced analgesia. These results suggest that GASP1 is a key regulator of the fate of CB1 after agonist exposure in the nervous system and critically determines analgesic tolerance to cannabinoids

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

Mast cells link immune sensing to antigen-avoidance behaviour

The physiological functions of mast cells remain largely an enigma. In the context of barrier damage, mast cells are integrated in type 2 immunity and, together with immunoglobulin E (IgE), promote allergic diseases. Allergic symptoms may, however, facilitate expulsion of allergens, toxins and parasites and trigger future antigen avoidance1,2,3. Here, we show that antigen-specific avoidance behaviour in inbred mice4,5 is critically dependent on mast cells; hence, we identify the immunological sensor cell linking antigen recognition to avoidance behaviour. Avoidance prevented antigen-driven adaptive, innate and mucosal immune activation and inflammation in the stomach and small intestine. Avoidance was IgE dependent, promoted by Th2 cytokines in the immunization phase and by IgE in the execution phase. Mucosal mast cells lining the stomach and small intestine rapidly sensed antigen ingestion. We interrogated potential signalling routes between mast cells and the brain using mutant mice, pharmacological inhibition, neural activity recordings and vagotomy. Inhibition of leukotriene synthesis impaired avoidance, but overall no single pathway interruption completely abrogated avoidance, indicating complex regulation. Collectively, the stage for antigen avoidance is set when adaptive immunity equips mast cells with IgE as a telltale of past immune responses. On subsequent antigen ingestion, mast cells signal termination of antigen intake. Prevention of immunopathology-causing, continuous and futile responses against per se innocuous antigens or of repeated ingestion of toxins through mast-cell-mediated antigen-avoidance behaviour may be an important arm of immunity

Presynaptically Localized Cyclic GMP-Dependent Protein Kinase 1 Is a Key Determinant of Spinal Synaptic Potentiation and Pain Hypersensitivity

Electrophysiological and behavioral experiments in mice reveal that a cGMP-dependent kinase amplifies neurotransmitter release from peripheral pain sensors, potentiates spinal synapses, and leads to exaggerated pain

The genetics of neuropathic pain from model organisms to clinical application

Neuropathic pain (NeuP) arises due to injury of the somatosensory nervous system and is both common and disabling, rendering an urgent need for non-addictive, effective new therapies. Given the high evolutionary conservation of pain, investigative approaches from Drosophila mutagenesis to human Mendelian genetics have aided our understanding of the maladaptive plasticity underlying NeuP. Successes include the identification of ion channel variants causing hyper-excitability and the importance of neuro-immune signaling. Recent developments encompass improved sensory phenotyping in animal models and patients, brain imaging, and electrophysiology-based pain biomarkers, the collection of large well-phenotyped population cohorts, neurons derived from patient stem cells, and high-precision CRISPR generated genetic editing. We will discuss how to harness these resources to understand the pathophysiological drivers of NeuP, define its relationship with comorbidities such as anxiety, depression, and sleep disorders, and explore how to apply these findings to the prediction, diagnosis, and treatment of NeuP in the clinic

Pain in experimental autoimmune encephalitis: a comparative study between different mouse models

<p>Abstract</p> <p>Background</p> <p>Pain can be one of the most severe symptoms associated with multiple sclerosis (MS) and develops with varying levels and time courses. MS-related pain is difficult to treat, since very little is known about the mechanisms underlying its development. Animal models of experimental autoimmune encephalomyelitis (EAE) mimic many aspects of MS and are well-suited to study underlying pathophysiological mechanisms. Yet, to date very little is known about the sensory abnormalities in different EAE models. We therefore aimed to thoroughly characterize pain behavior of the hindpaw in SJL and C57BL/6 mice immunized with PLP_139-151 peptide or MOG_35-55 peptide respectively. Moreover, we studied the activity of pain-related molecules and plasticity-related genes in the spinal cord and investigated functional changes in the peripheral nerves using electrophysiology.</p> <p>Methods</p> <p>We analyzed thermal and mechanical sensitivity of the hindpaw in both EAE models during the whole disease course. Qualitative and quantitative immunohistochemical analysis of pain-related molecules and plasticity-related genes was performed on spinal cord sections at different timepoints during the disease course. Moreover, we investigated functional changes in the peripheral nerves using electrophysiology.</p> <p>Results</p> <p>Mice in both EAE models developed thermal hyperalgesia during the chronic phase of the disease. However, whereas SJL mice developed marked mechanical allodynia over the chronic phase of the disease, C57BL/6 mice developed only minor mechanical allodynia over the onset and peak phase of the disease. Interestingly, the magnitude of glial changes in the spinal cord was stronger in SJL mice than in C57BL/6 mice and their time course matched the temporal profile of mechanical hypersensitivity.</p> <p>Conclusions</p> <p>Diverse EAE models bearing genetic, clinical and histopathological heterogeneity, show different profiles of sensory and pathological changes and thereby enable studying the mechanistic basis and the diversity of changes in pain perception that are associated with distinct types of MS.</p

Publisher Correction: Loss of POMC-mediated antinociception contributes to painful diabetic neuropathy.

The original version of the Peer Review File associated with this Article was updated shortly after publication to redact unpublished data

Early-onset treadmill training reduces mechanical allodynia and modulates calcitonin gene-related peptide fiber density in lamina III/IV in a mouse model of spinal cord contusion injury

Abstract: Below-level central neuropathic pain (CNP) affects a large proportion of spinal cord injured individuals. To better define the dynamic changes of the spinal cord neural network contributing to the development of CNP after spinal cord injury (SCI), we characterized the morphological and behavioral correlates of CNP in female C57BL/6 mice after a moderate T11 contusion SCI (50 kdyn) and the influence of moderate physical activity. Compared with sham-operated animals, injured mice developed mechanical allodynia 2 weeks post injury when tested with small-diameter von Frey hair filaments (0.16 g and 0.4 g filament), but presented hyporesponsiveness to noxious mechanical stimuli (1.4 g filament). The mechano-sensory alterations lasted up to 35 days post injury, the longest time point examined. The response latency to heat stimuli already decreased significantly 10 days post injury reaching a plateau 2 weeks later. In contrast, injured mice developed remarkable hyposensitivity to cold stimuli. Animals that underwent moderate treadmill training (2 × 15 minutes; 5 d/wk) showed a significant reduction in the response rate to light mechanical stimuli as early as 6 days after training. Calcitonin gene-related peptide (CGRP) labeling in lamina III-IV of the dorsal horn revealed significant increases in CGRP-labeling density in injured animals compared with sham control animals. Importantly, treadmill training reduced CGRP-labeling density by about 50% (P < 0.01), partially reducing the injury-induced increases. Analysis of IB4-labeled nonpeptidergic sensory fibers revealed no differences between experimental groups. Abnormalities in temperature sensation were not influenced by physical activity. Thus, treadmill training partially resolves signs of below-level CNP after SCI and modulates the density of CGRP-labeled fibers

Nitric oxide synthase (NOS)-interacting protein interacts with neuronal NOS and regulates its distribution and activity.

Mechanisms governing the activity of neuronal nitric oxide synthase (nNOS), the major source of nitric oxide (NO) in the nervous system, are not completely understood. We report here a protein-protein interaction between nNOS and NOSIP (nitric oxide synthase-interacting protein) in rat brain in vivo. NOSIP and nNOS are concentrated in neuronal synapses and demonstrate significant colocalization in various regions of the central and peripheral nervous systems. NOSIP produces a significant reduction in nNOS activity in a neuroepithelioma cell line stably expressing nNOS. Furthermore, overexpression of NOSIP in cultured primary neurons reduces the availability of nNOS in terminal dendrites. These results thus suggest that the interaction between NOSIP and nNOS is functionally involved in endogenous mechanisms regulating NO synthesis. Furthermore, we found that the subcellular distribution and expression levels of NOSIP are dynamically regulated by neuronal activity in vitro as well as in vivo, suggesting that NOSIP may contribute to a mechanism via which neuronal activity regulates the synaptic availability and activity of nNOS