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Understanding of cortical encoding of itch is limited. Injection of pruritogens and algogens into the skin of the cheek produces distinct behaviors, making the rodent cheek a useful model for understanding mechanisms of itch and pain. We examined responses of neurons in the primary somatosensory cortex by applying of mechanical stimuli (brush, pressure, and pinch) and stimulations by intradermal injections of pruritic and algesic chemical of receptive fields located on the skin of the cheek in urethane-anesthetized rats. Stimuli included chloroquine, serotonin, beta-alanine, histamine, capsaicin, and mustard oil. All 33 neurons studied were excited by noxious mechanical stimuli applied to the cheek. Based on mechanical stimulation most neurons were functionally classified as high threshold. Of 31 neurons tested for response to chemical stimuli, 84% were activated by one or more pruritogens/partial pruritogens. No cells were activated by all five substances. Histamine activated the greatest percentage of neurons and evoked the greatest mean discharge. Importantly, no cells were excited exclusively by pruritogens or partial pruritogens. The recording sites of all neurons that responded to chemical stimuli applied to the cheek were located in the dysgranular zone (DZ) and in deep laminae of the medial border of the vibrissal barrel fields (VBF). Therefore, neurons in the DZ/VBF of rats encode mechanical and chemical pruritogens and algogens. This cortical region appears to contain primarily nociceptive neurons as defined by responses to noxious pinching of the skin. Its role in encoding itch and pain from the cheek of the face needs further study.
Learn More >Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCβ), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCβ-mediated reduction of TRPM8 desensitization. This MOR-PKCβ dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCβ as important targets for opioid-induced cold hypersensitivity.
Learn More >Morphine-induced alterations in sphingolipid metabolism in the spinal cord and increased formation of the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) have been implicated in the development of morphine-induced hyperalgesia (OIH; increased pain sensitivity) and antinociceptive tolerance. These adverse effects hamper opioid use for treating chronic pain and contribute to dependence and abuse. S1P produces distinct effects through five G protein-coupled receptors (S1PR1-5) and several intracellular targets. How S1P exerts its effects in response to morphine remains unknown. Here, we report that S1P contributes to the development of OIH and tolerance through S1P1 receptor subtype 1 (S1PR1) signaling in uninjured male and female rodents that can be blocked by targeting S1PR1 with S1PR1 antagonists or RNA silencing. In mouse neuropathic pain models, S1PR1 antagonists blocked the development of tolerance to the anti-allodynic effects of morphine without altering morphine pharmacokinetics and prevented prolonged morphine-induced neuropathic pain. Targeting S1PR1 reduced morphine-induced neuroinflammatory events in the dorsal horn of the spinal cord: increased glial marker expression, mitogen-activated protein kinase p38 and nuclear factor κB activation and increased inflammatory cytokine expression, such as interleukin-1β, a cytokine central in the modulation of opioid-induced neural plasticity. Our results identify S1PR1 as a critical path for S1P signaling in response to sustained morphine and reveal downstream neuroinflammatory pathways impacted by S1PR1 activation. Our data support investigating S1PR1 antagonists as a clinical approach to mitigate opioid-induced adverse effects and repurposing the functional S1PR1 antagonist FTY720, which is FDA-approved for multiple sclerosis, as an opioid adjunct.
Learn More >Skeletal metastases are frequently accompanied by chronic pain that is mechanoceptive in nature. Mechanistically, cancer-induced bone pain (CIBP) is mediated by peripheral sensory neurones innervating the cancerous site, the cell bodies of which are housed in the dorsal root ganglia (DRG). How these somatosensory neurons encode sensory information in CIBP remains only partly explained. Using a validated rat model, we first confirmed cortical bone destruction in CIBP but not sham-operated rats (day 14 post-surgery, designated 'late' stage bone cancer). This occurred with behavioural mechanical hypersensitivity (Kruskal-Wallis H for independent samples; CIBP vs. Sham-operated, day 14; p < 0.0001). Next, hypothesising that the proportion and phenotype of primary afferents would be altered in the disease state, DRG in vivo imaging of genetically-encoded calcium indicators and Markov Cluster Analysis were employed to analyse 1748 late-stage CIBP (n=10), and 757 sham-operated (n=9), neurons. Distinct clusters of responses to peripheral stimuli were revealed. In CIBP rats, upon knee compression of the leg ipsilateral to the tumour: (1) three times as many sensory afferents responded (RM-ANOVA: p < 0.0001 (vs. sham)); (2) there were significantly more small neurons responding (Kruskal-Wallis for independent samples (vs. sham): p < 0.0001) and (3) approximately 13% of traced tibial cavity afferents responded (no difference observed between CIBP and sham-operated animals). We conclude that an increased sensory afferent response is present in CIBP rats and this is likely to reflect afferent recruitment from outside of the bone rather than increased intraosseous afferent activity.
Learn More >While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: RelmαMgl1 snMacs in the epineurium and RelmαMgl1 snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.
Learn More >δ-opioid receptor (DOPr) agonists have analgesic efficacy in chronic pain models but development of tolerance limits their use for long-term pain management. Although agonist potential for inducing acute analgesic tolerance has been associated with distinct patterns of DOPr internalization, the association between trafficking and chronic tolerance remains ill-defined. In a rat model of streptozotocin (STZ)-induced diabetic neuropathy, deltorphin II and TIPP produced sustained analgesia following daily (intrathecal) i.t. injections over six days, whereas similar treatment with SNC-80 or SB235863 led to progressive tolerance and loss of the analgesic response. Trafficking assays in murine neuron cultures showed no association between the magnitude of ligand-induced sequestration and development of chronic tolerance. Instead, ligands that supported DOPr recycling were also the ones producing sustained analgesia over 6-day treatment. Moreover, endosomal endothelin-converting enzyme 2 (ECE2) blocker 663444 prevented DOPr recycling by deltorphin II and TIPP and precipitated tolerance by these ligands. In conclusion, agonists, which support DOPr recycling, avoid development of analgesic tolerance over repeated administration.
Learn More >Recent studies have revealed gender differences in cold perception, and pointed to a possible direct action of testosterone (TST) on the cold-activated TRPM8 (Transient Receptor Potential Melastatin Member 8) channel. However, the mechanisms by which TST influences TRPM8-mediated sensory functions remain elusive. Here, we show that TST inhibits TRPM8-mediated mild-cold perception through the noncanonical engagement of the Androgen Receptor (AR). Castration of both male rats and mice increases sensitivity to mild cold, and this effect depends on the presence of intact TRPM8 and AR. TST in nanomolar concentrations suppresses whole-cell TRPM8-mediated currents and single-channel activity in native dorsal root ganglion (DRG) neurons and HEK293 cells co-expressing recombinant TRPM8 and AR, but not TRPM8 alone. AR cloned from rat DRGs shows no difference from standard AR. However, biochemical assays and confocal imaging reveal the presence of AR on the cell surface and its interaction with TRPM8 in response to TST, leading to an inhibition of channel activity.
Learn More >Psoriasis is inflammatory skin disease associated with itch, a troublesome symptom with few therapeutic options. Transient receptor potential channel 4 (TRPC4) is highly expressed in dorsal root ganglia (DRGs). Recently, we have revealed a previously unknown itch signaling in DRG neurons by which TRPC4 mediates itch to serotonergic antidepressants, and demonstrated the antipruritic effect of the TRPC4 inhibitor ML204. However, the role of TRPC4 in acute and chronic itch is still largely unknown. Here, we have characterized the expression of TRPC4 in peptidergic DRG neurons and showed that acute itch induced by serotonin and histamine was attenuated in TRPC4 knockout (TRPC4 KO) mice and mice treated with ML204. We also showed that silencing TRPC4 in DRG and its inhibition by intradermal injections were also effective in decreasing psoriatic itch after repeated applications of imiquimod (IMQ), a preclinical model of psoriasis. Of clinical relevance, intradermal injections of ML204 in psoriasiform skin significantly reversed IMQ-established chronic itch and cutaneous inflammation. Given that TRPC4 is expressed in human DRGs and a specific inhibitor is in clinical trials, our data not only expand our understanding of itch and psoriasis, but also reveal TRPC4 as a potential therapeutic target with considerable translational benefit.
Learn More >The release of the neuropeptide CGRP from the trigeminal ganglion neurons (TGNs) plays a central role in migraine. Whereas CGRP can activate NO release from ganglionic glial cells, NO in turn enhances CGRP release. However, it remains unclear how NO promotes CGRP release. Here, we report that the NO donor SNAP triggered CGRP release from cultured primary TGNs. This event was associated with GSK-3β activation and Akt inactivation. Immunofluorescent staining revealed that GSK-3β primarily located in neurons. Furthermore, GSK-3β inhibition resulted in a marked reduction in expression of CGRP as well as other migraine-related factors, including substance P, cholecystokinin, and prostaglandin E2. Last, exposure to SNAP also activated NF-κB, while NF-κB inhibition prevented the induction of CGRP by SNAP. Interestingly, this event was blocked by GSK-3β inhibition, in association with inhibition of NF-κB/p65 expression and nuclear translocation. Together, these findings argue that NO could stimulate TGNs to release of CGRP as well as other migraine-related factors, likely by activating GSK-3β, providing a novel mechanism underlying a potential feed-forward loop between NO and CGRP in migraine. They also raise a possibility that GSK-3β might act to trigger migraine through activation of NF-κB, suggesting a link between neuroinflammation and migraine.
Learn More >Heat shock protein 90 (Hsp90) is a ubiquitous signal transduction regulator, and Hsp90 inhibitors are in clinical development as cancer therapeutics. However, there have been very few studies on the impact of Hsp90 inhibitors on pain or analgesia, a serious concern for cancer patients. We previously found that Hsp90 inhibitors injected into the brain block opioid-induced anti-nociception in tail flick, paw incision, and HIV neuropathy pain. This study extended from that initial work to test the cancer-related clinical impact of Hsp90 inhibitors on opioid anti-nociception in cancer-induced bone pain (CIBP) in female BALB/c mice and chemotherapy-induced peripheral neuropathy (CIPN) in male and female CD-1 mice. Mice were treated with Hsp90 inhibitors (17-AAG, KU-32) by the intracerebroventricular, intrathecal, or intraperitoneal routes, and after 24 hours pain behaviors were evaluated following analgesic drug treatment. Hsp90 inhibition in the brain or systemically completely blocked morphine and oxymorphone anti-nociception in CIPN; this effect was partly mediated by decreased ERK and JNK MAPK activation and by increased protein translation, was not altered by chronic treatment, and Hsp90 inhibition had no effect on gabapentin anti-nociception. We also found that the Hsp90 isoform Hsp90α and the co-chaperone Cdc37 were responsible for the observed changes in opioid anti-nociception. In contrast, Hsp90 inhibition in the spinal cord or systemically partially reduced opioid anti-nociception in CIBP. These results demonstrate that Hsp90 inhibitors block opioid anti-nociception in cancer-related pain, suggesting that Hsp90 inhibitors for cancer therapy could decrease opioid treatment efficacy.
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