Pain-sensing Neurons Research Articles

The single-cell transcriptomic atlas iPain identifies senescence of nociceptors as a therapeutical target for chronic pain treatment

Chronic pain remains a significant medical challenge with complex underlying mechanisms, and an urgent need for new treatments. Our research built and utilized the iPain single-cell atlas to study chronic pain progression in dorsal root and trigeminal ganglia. We discovered that senescence of a small subset of pain-sensing neurons may be a driver of chronic pain. This mechanism was observed in animal models after nerve injury and in human patients diagnosed with chronic pain or diabetic painful neuropathy. Notably, treatment with senolytics, drugs that remove senescent cells, reversed pain symptoms in mice post-injury. These findings highlight the role of cellular senescence in chronic pain development, demonstrate the therapeutic potential of senolytic treatments, and underscore the value of the iPain atlas for future pain research.

The Nociceptor Primary Cilium Contributes to Mechanical Nociceptive Threshold and Inflammatory and Neuropathic Pain.

The primary cilium, a single microtubule-based organelle protruding from the cell surface and critical for neural development, also functions in adult neurons. While some dorsal root ganglion neurons elaborate a primary cilium, whether it is expressed by and functional in nociceptors is unknown. Recent studies have shown a role of Hedgehog, whose canonical signaling is primary cilium dependent, in nociceptor sensitization. We establish the presence of primary cilia in soma of rat nociceptors, where they contribute to mechanical threshold, prostaglandin E2 (PGE2)-induced hyperalgesia, and chemotherapy-induced neuropathic pain (CIPN). Intrathecal administration of siRNA targeting Ift88, a primary cilium-specific intra-flagellar transport (IFT) protein required for ciliary integrity, resulted in attenuation of Ift88 mRNA and nociceptor primary cilia. Attenuation of primary cilia was associated with an increase in mechanical nociceptive threshold in vivo, and decrease in nociceptor excitability in vitro, abrogation of hyperalgesia, and nociceptor sensitization induced by both a prototypical pronociceptive inflammatory mediator PGE2 and paclitaxel CIPN, in a sex-specific fashion. siRNA targeting Ift52, another IFT protein, and knockdown of NompB, the Drosophila Ift88 ortholog, also abrogated CIPN and reduced baseline mechanosensitivity, respectively, providing independent confirmation for primary cilia control of nociceptor function. Hedgehog-induced hyperalgesia is attenuated by Ift88 siRNA, supporting a role for primary cilia in Hedgehog-induced hyperalgesia. Attenuation of CIPN by cyclopamine (intradermal and intra-ganglion), which inhibits Hedgehog signaling, supports a role of Hedgehog in CIPN. Our findings support a role of the nociceptor primary cilium in control of mechanical nociceptive threshold and inflammatory and neuropathic pain, the latter Hedgehog-dependent.Significance statement Many neurons have a tiny antenna-like structure, the primary cilium, protruding from their cell body, which processes information about the extracellular environment as well as regulates intracellular signaling. We report experiments aimed at understanding the role of the primary cilium in pain sensory neurons (nociceptors), including in the setting of inflammatory and neuropathic pain. We establish that nociceptors bear a primary cilium and show that this organelle regulates detection of noxious stimuli and contributes to nociceptor sensitization, using both the rat and the fruit fly as model organisms to manipulate primary cilia in pain states. We also identify primary cilium dependence of the contribution of Hedgehog to pain states.

A promising alternative to opioids.

A complex extracted from the amniotic membrane in humans reduces post-surgical pain in mice by directly inhibiting pain-sensing neurons.

State-dependent inhibition of Nav1.8 channels by VX-150 and VX-548.

Nav1.8 channels are an attractive therapeutic target for pain because they are prominent in primary pain-sensing neurons with little expression in most other kinds of neurons. Recently, two Nav1.8-targeted compounds, VX-150 and VX-548, have shown efficacy in clinical trials for reducing pain. We examined the characteristics of Nav1.8 inhibition by these compounds. The active metabolite form of VX-150 (VX-150m) inhibited human Nav1.8 channels with an IC50 of 15 nM. VX-548 (suzetrigine) was even more potent (IC50 0.27 nM). Both VX-150m and VX-548 had the unusual property of "reverse use-dependence", whereby inhibition could be relieved by repetitive depolarizations, a property seen before with another Nav1.8 inhibitor, A-887826. The relief of VX-548 inhibition by large depolarizations occurred with a time constant of ~40 ms that was not concentration-dependent. Re-inhibition at negative voltages occurred with a rate that was nearly proportional to drug concentration, consistent with the idea that relief of inhibition reflects dissociation of drug from the channel and re-inhibition reflects re-binding. The relief of inhibition by depolarization suggests a remarkably strong and unusual state-dependence for both VX-150m and VX-548, with very weak binding to channels with fully activated voltage sensors despite very tight binding to channels with voltage sensors in the resting state. Significance Statement The Nav1.8 sodium channel is a current target for new drugs for pain. This work describes the potency, selectivity, and state-dependent characteristics of inhibition of Nav1.8 channels by VX-150 and VX-548, compounds that have recently shown efficacy for relief of pain in clinical trials but whose mechanism of interaction with channels has not been described. The results show that the compounds share an unusual property whereby inhibition is relieved by depolarization, demonstrating a state-dependence different than most sodium channel inhibitors.

Rat Sympathetic Neuron Calcium Channels Are Insensitive to Gabapentin.

The gabapentenoids such as gabapentin (GP) and pregabalin are approved for the treatment of chronic pain, but their utility is limited by persistent side effects. These adverse effects result from GPs affecting many types of neurons and muscle cells, not just the pain-sensing neurons that are the intended targets. We have recently discovered a type of peripheral neuron, rat sympathetic neurons from the superior cervical ganglion (SCG), that is uniquely insensitive to GP effects. Currents were measured using whole-cell patch-clamp electrophysiology from cells in primary culture from either the SCG or the Nodose Ganglion (NDG) as a positive control for the effects of GP. We find that the calcium current density was dramatically reduced by GP pretreatment in NDG neurons, but that neurons from the SCG were resistant. Further, when GP was cytoplasmically injected into these neurons, the resistance of SCG neurons to GP treatment persisted. These data demonstrate that rat sympathetic neurons appear to be uniquely resistant to GP treatment. These results may help us to better understand the mechanism of action of, and resistance to, GP in altering calcium channel current density, which may help to develop future treatments with fewer side effects.

50285 Oxytocin receptor is present in keratinocytes near touch and pain sensory neurons

50285 Oxytocin receptor is present in keratinocytes near touch and pain sensory neurons

Irritable bowel syndrome: When food is a pain in the gut.

Irritable bowel syndrome (IBS) is a chronic gastrointestinal condition associated with altered bowel habits and recurrent abdominal pain, often triggered by food intake. Current treatments focus on improving stool pattern, but effective treatments for pain in IBS are still lacking due to our limited understanding of pathophysiological mechanisms. Visceral hypersensitivity (VHS), or abnormal visceral pain perception, underlies abdominal pain development in IBS, and mast cell activation has been shown to play an important role in the development of VHS. Our work recently revealed that abdominal pain in response to food intake is induced by the sensitization of colonic pain-sensing neurons by histamine produced by activated mast cells following a local IgE response to food. In this review, we summarize the current knowledge on abdominal pain and VHS pathophysiology in IBS, we outline the work leading to the discovery of the role of histamine in abdominal pain, and we introduce antihistamines as a novel treatment option to manage chronic abdominal pain in patients with IBS.

ST-2560, a selective inhibitor of the NaV1.7 sodium channel, affects nocifensive and cardiovascular reflexes in non-human primates.

The voltage-gated sodium channel isoform NaV1.7 is a high-interest target for the development of non-opioid analgesics due to its preferential expression in pain-sensing neurons. NaV1.7 is also expressed in autonomic neurons, yet its contribution to involuntary visceral reflexes has received limited attention. The small molecule inhibitor ST-2560 was advanced into pain behaviour and cardiovascular models to understand the pharmacodynamic effects of selective inhibition of NaV1.7. Potency of ST-2560 at NaV1.7 and off-target ion channels was evaluated by whole-cell patch-clamp electrophysiology. Effects on nocifensive reflexes were assessed in non-human primate (NHP) behavioural models, employing the chemical capsaicin and mechanical stimuli. Cardiovascular parameters were monitored continuously in freely-moving, telemetered NHPs following administration of vehicle and ST-2560. ST-2560 is a potent inhibitor (IC50 = 39 nM) of NaV1.7 in primates with ≥1000-fold selectivity over other isoforms of the human NaV1.x family. Following systemic administration, ST-2560 (0.1-0.3 mg·kg-1, s.c.) suppressed noxious mechanical- and chemical-evoked reflexes at free plasma concentrations threefold to fivefold above NaV1.7 IC50. ST-2560 (0.1-1.0 mg·kg-1, s.c.) also produced changes in haemodynamic parameters, most notably a 10- to 20-mmHg reduction in systolic and diastolic arterial blood pressure, at similar exposures. Acute pharmacological inhibition of NaV1.7 is antinociceptive, but also has the potential to impact the cardiovascular system. Further work is merited to understand the role of NaV1.7 in autonomic ganglia involved in the control of heart rate and blood pressure, and the effect of selective NaV1.7 inhibition on cardiovascular function.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Pharmacologic Characterization of LTGO-33, a Selective Small Molecule Inhibitor of the Voltage-Gated Sodium Channel NaV1.8 with a Unique Mechanism of Action.

Discovery and development of new molecules directed against validated pain targets is required to advance the treatment of pain disorders. Voltage-gated sodium channels (NaVs) are responsible for action potential initiation and transmission of pain signals. NaV1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of NaV1.8 can ameliorate pain while minimizing effects on other NaV isoforms essential for cardiac, respiratory, and central nervous system physiology. Here we present the pharmacology, interaction site, and mechanism of action of LTGO-33, a novel NaV1.8 small molecule inhibitor. LTGO-33 inhibited NaV1.8 in the nM potency range and exhibited over 600-fold selectivity against human NaV1.1-NaV1.7 and NaV1.9. Unlike prior reported NaV1.8 inhibitors that preferentially interacted with an inactivated state via the pore region, LTGO-33 was state-independent with similar potencies against closed and inactivated channels. LTGO-33 displayed species specificity for primate NaV1.8 over dog and rodent NaV1.8 and inhibited action potential firing in human dorsal root ganglia neurons. Using chimeras combined with mutagenesis, the extracellular cleft of the second voltage-sensing domain was identified as the key site required for channel inhibition. Biophysical mechanism of action studies demonstrated that LTGO-33 inhibition was relieved by membrane depolarization, suggesting the molecule stabilized the deactivated state to prevent channel opening. LTGO-33 equally inhibited wild-type and multiple NaV1.8 variants associated with human pain disorders. These collective results illustrate LTGO-33 inhibition via both a novel interaction site and mechanism of action previously undescribed in NaV1.8 small molecule pharmacologic space. SIGNIFICANCE STATEMENT: NaV1.8 sodium channels primarily expressed in peripheral pain-sensing neurons represent a validated target for the development of novel analgesics. Here we present the selective small molecule NaV1.8 inhibitor LTGO-33 that interdicts a distinct site in a voltage-sensor domain to inhibit channel opening. These results inform the development of new analgesics for pain disorders.

Reduced capsaicin-induced mechanical allodynia and neuronal responses in the dorsal root ganglion in the presence of protein tyrosine phosphatase non-receptor type 6 overexpression.

Transient Receptor Potential Vanilloid 1 (TRPV1) is a nonselective cation channel expressed by pain-sensing neurons and has been an attractive target for the development of drugs to treat pain. Recently, Src homology region two domain-containing phosphatase-1 (SHP-1, encoded by Ptpn6) was shown to dephosphorylate TRPV1 in dorsal root ganglia (DRG) neurons, which was linked with alleviating different pain phenotypes. These previous studies were performed in male rodents only and did not directly investigate the role of SHP-1 in TRPV-1 mediated sensitization. Therefore, our goal was to determine the impact of Ptpn6 overexpression on TRPV1-mediated neuronal responses and capsaicin-induced pain behavior in mice of both sexes. Twelve-week-old male and female mice overexpressing Ptpn6 (Shp1-Tg) and their wild type (WT) littermates were used. Ptpn6 overexpression was confirmed in the DRG of Shp1-Tg mice by RNA in situ hybridization and RT-qPCR. Trpv1 and Ptpn6 were found to be co-expressed in DRG sensory neurons in both genotypes. Functionally, this overexpression resulted in lower magnitude intracellular calcium responses to 200nM capsaicin stimulation in DRG cultures from Shp1-Tg mice compared to WTs. In vivo, we tested the effects of Ptpn6 overexpression on capsaicin-induced pain through a model of capsaicin footpad injection. While capsaicin injection evoked nocifensive behavior (paw licking) and paw swelling in both genotypes and sexes, only WT mice developed mechanical allodynia after capsaicin injection. We observed similar level of TRPV1 protein expression in the DRG of both genotypes, however, a higher amount of tyrosine phosphorylated TRPV1 was detected in WT DRG. These experiments suggest that, while SHP-1 does not mediate the acute swelling and nocifensive behavior induced by capsaicin, it does mediate a protective effect against capsaicin-induced mechanical allodynia in both sexes. The protective effect of SHP-1 might be mediated by TRPV1 dephosphorylation in capsaicin-sensitive sensory neurons of the DRG.

Mapping the neuroethological signatures of pain, analgesia, and recovery in mice

Ongoing pain is driven by the activation and modulation of pain-sensing neurons, affecting physiology, motor function, and motivation to engage in certain behaviors. The complexity of the pain state has evaded a comprehensive definition, especially in non-verbal animals. Here, in mice, we used site-specific electrophysiology to define key time points corresponding to peripheral sensitivity in acute paw inflammation and chronic knee pain models. Using supervised and unsupervised machine learning tools, we uncovered sensory-evoked coping postures unique to each model. Through 3D pose analytics, we identified movement sequences that robustly represent different pain states and found that commonly used analgesics do not return an animal's behavior to a pre-injury state. Instead, these analgesics induce a novel set of spontaneous behaviors that are maintained even after resolution of evoked pain behaviors. Together, these findings reveal previously unidentified neuroethological signatures of pain and analgesia at heightened pain states and during recovery.

The role of protein kinases in diabetic neuropathic pain: an update review.

Diabetic neuropathic pain (DNP) is a debilitating symptom of diabetic neuropathy which seriously impairs patient's quality of life. Currently, there is no specific therapy for DNP except for duloxetine and gabapentin that show limited utility in alleviating DNP. The present review aims to discuss the central role of protein kinases in the pathogenesis of DNP and their therapeutic modulation. Scopus, PubMed, and Google scholar were searched up to January 2022 to find relevant studies with English language in which the roles of proteins kinases in DNP were examined. DNP is associated with hyperactivity in pain sensory neurons and therapies aim to specifically suppress redundant discharges in these neurons without affecting the activity of other sensory and motor neurons. Transient receptor potential vanilloid 1 (TRPV1) and purinergic 2 × 7 receptors (P2 × 7R) are two receptor channels, highly expressed in pain sensory neurons and their blockade produces remarkable analgesic effects in DNP. The activities of receptor channels are mainly regulated by the protein kinases whose modulation provides remarkable analgesic effects in DNP models. Capsaicin, TRPV1 modulator, is the only agent successfully examined in clinical trials with promising effects in patients with DNP. Current data suggest that blocking calcium calmodulin dependent protein kinase II (CaMKII) is superior to other approaches, considering its pivotal role in regulating the pain neuron potentials. By this means, DNP alleviation is achievable without affecting the activity of other sensory or motor neurons.

A17 EVIDENCE OF PROTEASE-MEDIATED PRO-NOCICEPTIVE EFFECTS OF FECAL SUPERNATANTS FROM CROHN'S DISEASE PATIENTS

Abstract Background Abdominal pain is a debilitating symptom of Crohn’s disease (CD). Despite the current treatment options for this disease, abdominal pain is an unresolved problem that commonly persists in the absence of active inflammation. This suggests that something other than inflammation is driving the pain during the quiescent phase. We have previously reported that microbial proteases can directly modulate the excitability of dorsal root ganglia (DRG) neurons, many of which are pain-sensing. We hypothesize that luminal proteases of CD patients are contributing to their abdominal pain. Purpose Determine whether luminal mediators in CD fecal samples induce changes in pain signalling. Method The effects of patient (active CD [n = 3] and healthy volunteer (HV) [n = 3]) fecal supernatant (FS) samples on pain-sensing neurons were assessed using ex-vivo single unit afferent nerve recordings from mouse colons. Each sample was tested in colonic preparations from a least 5 mice. To further examine cellular mechanisms, DRG neurons were isolated and incubated overnight in media containing CD FS or HV FS media. Changes in neuronal excitability were recorded by determining the rheobase (lower rheobase=increased excitability) using patch clamp recordings (n ≥ 9 DRG neurons/group). Protease inhibitors were applied in both bioassays to determine whether these inhibited the excitatory effect of FS. Lastly, total proteolytic activity in the CD and HV fecal samples was calculated using a casein colorimetric protease detection assay. Result(s) FS from HV had no effect on afferent nerve excitability (p = 0.8920). FS from active CD patients increased action potential discharge from colonic afferent nerves by 85% (p<0.0001) and selectively increased the activation of high-threshold units, which are putative nociceptors, by 44% (p=0.0074). A protease inhibitor cocktail (1:1000) and protease-activated receptor (PAR)-2 antagonist GB83 (10µM) both blocked the excitatory effects of CD FS (p<0.05). Overnight incubation with CD FS also had an excitatory effect on DRG neurons compared to HV FS (rheobase decreased by 46%, p<0.05). The effect of CD FS was blocked by GB83 (10µM) (p<0.001) and a serine protease inhibitor (FUT175; 100µM) (p<0.05) independently, but the activity was not blocked by E64 (30nM) a cysteine protease inhibitor. A 200-fold increase (p<0.0001) in total proteolytic activity was found in CD FS compared to HV FS. Conclusion(s) Luminal serine proteases, but not cysteine proteases, appear to be driving nociceptive signalling in CD patients. This provides insight into the generation of pain in CD patients and may be a potential target to mitigate this action. Further research is required to elucidate whether these pro-nociceptive proteases are of bacterial or host origin and their effects in the quiescent phase. Disclosure of Interest None Declared

Nav1.9: A unique voltage-gated sodium channel with a role in inflammatory pain.

In inflamed tissue, sensitization of pain-sensing neurons results in exaggerated responses to mild or even innocuous stimuli. A substantial part of this increase in neuronal excitability is due to an effect on NaV1.9, an elusive member of the voltage-gated sodium (NaV) channel family that deviates from other subtypes because of its ultra-slow kinetics and hyperpolarized activation voltage. How these exceptional biophysical characteristics affect neurons at baseline and under stimulated conditions is a matter of debate, and this is likely to remain so until the major hurdles impeding its investigation can be eluded. These hurdles entail the difficulty of expression in heterologous expression systems, a lack of pharmacological tools and the general variability encountered in NaV1.9 experimentation. The goal of our research is to understand the reasons behind these complications and to establish a system that allows robust electrophysiological recordings. This work can be a starting point for future studies focusing on NaV1.9 pharmacology or its involvement in inflammatory pain pathways.

Use-Dependent Relief of Inhibition of Nav1.8 Channels by A-887826.

Sodium channel inhibitors used as local anesthetics, antiarrhythmics, or antiepileptics typically have the property of use-dependent inhibition, whereby inhibition is enhanced by repetitive channel activation. For targeting pain, Nav1.8 channels are an attractive target because they are prominent in primary pain-sensing neurons, with little or no expression in most other kinds of neurons, and a number of Nav1.8-targeted compounds have been developed. We examined the characteristics of Nav1.8 inhibition by one of the most potent Nav1.8 inhibitors so far described, A-887826, and found that when studied with physiologic resting potentials and physiologic temperatures, inhibition had strong "reverse use dependence", whereby inhibition was relieved by repetitive short depolarizations. This effect was much stronger with A-887826 than with A-803467, another Nav1.8 inhibitor. The use-dependent relief from inhibition was seen in both human Nav1.8 channels studied in a cell line and in native Nav1.8 channels in mouse dorsal root ganglion (DRG) neurons. In native Nav1.8 channels, substantial relief of inhibition occurred during repetitive stimulation by action potential waveforms at 5 Hz, suggesting that the phenomenon is likely important under physiologic conditions. SIGNIFICANCE STATEMENT: Nav1.8 sodium channels are expressed in primary pain-sensing neurons and are a prime current target for new drugs for pain. This work shows that one of the most potent Nav1.8 inhibitors, A-887826, has the unusual property that inhibition is relieved by repeated short depolarizations. This "reverse use dependence" may reduce inhibition during physiological firing and should be selected against in drug development.

Developmental exposure to the pesticide malathion enhances expression of Prdm12, a regulator of nociceptor development, in Xenopus laevis.

The transcription factor Prdm12 exerts important influences on the development of nociceptors, peripheral touch and pain-sensing neurons, and has been implicated in human pain sensation disorders. We examined the consequences of exposing developing Xenopus laevis embryos to the commonly used pesticide malathion on Prdm12 expression. Using qPCR and western blot analysis we observed that malathion treatment for the first six days of tadpole development significantly increased both prdm12 mRNA levels and Prdm12 protein levels compared to controls. Consequently, early exposure to this pesticide has potential to alter nociceptor development.

Gut-innervating nociceptors regulate the intestinal microbiota to promote tissue protection

Nociceptive pain is a hallmark of many chronic inflammatory conditions including inflammatory bowel diseases (IBDs); however, whether pain-sensing neurons influence intestinal inflammation remains poorly defined. Employing chemogenetic silencing, adenoviral-mediated colon-specific silencing, and pharmacological ablation of TRPV1+ nociceptors, we observed more severe inflammation and defective tissue-protectivereparative processes in a murine model of intestinal damage and inflammation. Disrupted nociception led to significant alterations in the intestinal microbiota and a transmissible dysbiosis, while mono-colonization of germ-free mice with Gram+Clostridium spp. promoted intestinal tissue protection through a nociceptor-dependent pathway. Mechanistically, disruption of nociception resulted in decreased levels of substance P, and therapeutic delivery of substance P promoted tissue-protective effects exerted by TRPV1+ nociceptors in a microbiota-dependent manner. Finally, dysregulated nociceptor gene expression was observed in intestinal biopsies from IBD patients. Collectively, these findings indicate an evolutionarily conserved functional link between nociception, the intestinal microbiota, and the restoration of intestinal homeostasis.

Adding data to gut feelings

Pain-sensing neurons detect environmental insults and tissue injury, driving avoidance behavior and the local release of neuropeptides. Two related papers in this issue of Cell report that gut-innervating pain neurons sense bacterial presence to both shape the constituents of the gut microbiome and protect against excessive inflammation.

14-3-3γ mediates the long-term inhibition of peripheral kappa opioid receptor antinociceptive signaling by norbinaltorphimine

Long-term inhibition of kappa opioid receptor (KOR) signaling in peripheral pain-sensing neurons is a potential obstacle for development of peripherally-restricted KOR agonists that produce analgesia. Such a long-term inhibitory mechanism is invoked from activation of c-Jun N-terminal kinase (JNK) that follows a single injection of the KOR antagonist norbinaltorphimine (norBNI). This effect requires protein synthesis of an unknown mediator in peripheral pain-sensing neurons. Using 2D difference gel electrophoresis with tandem mass spectrometry, we have identified that the scaffolding protein 14-3-3γ is upregulated in peripheral sensory neurons following activation of JNK with norBNI. Knockdown of 14-3-3γ by siRNA eliminates the long-term reduction in KOR-mediated cAMP signaling by norBNI in peripheral sensory neurons in culture. Similarly, knockdown of 14-3-3γ in the rat hind paw abolished the norBNI-mediated long-term reduction in peripheral KOR-mediated antinociception. Further, overexpression of 14-3-3γ in KOR expressing CHO cells prevented KOR-mediated inhibition of cAMP signaling. These long-term effects are selective for KOR as heterologous regulation of other receptor systems was not observed. These data suggest that 14-3-3γ is both necessary and sufficient for the long-term inhibition of KOR by norBNI in peripheral sensory neurons.