Accepted

In search of the molecular identities of cold-sensing receptors, we carried out an unbiased genetic screen for cold-sensing mutants in C. elegans and isolated a mutant allele of glr-3 gene that encodes a kainate-type glutamate receptor. While glutamate receptors are best known to transmit chemical synaptic signals in the CNS, we show that GLR-3 senses cold in the peripheral sensory neuron ASER to trigger cold-avoidance behavior. GLR-3 transmits cold signals via G protein signaling independently of its glutamate-gated channel function, suggesting GLR-3 as a metabotropic cold receptor. The vertebrate GLR-3 homolog GluK2 from zebrafish, mouse, and human can all function as a cold receptor in heterologous systems. Mouse DRG sensory neurons express GluK2, and GluK2 knockdown in these neurons suppresses their sensitivity to cold but not cool temperatures. Our study identifies an evolutionarily conserved cold receptor, revealing that a central chemical receptor unexpectedly functions as a thermal receptor in the periphery.

Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. This study was designed to examine the role of anterior insular cortex (aIC) in the pathogenesis of hyperalgesia in a rat model of CP. CP was induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). Abdomen hyperalgesia and anxiety were assessed by von Frey filament and open field tests, respectively. Two weeks after surgery, the activation of aIC was indicated by FOS immunohistochemical staining and electrophysiological recordings. Expressions of VGluT1, NMDAR subunit NR2B and AMPAR subunit GluR1 were analyzed by immunoblottings. The regulatory roles of aIC in hyperalgesia and pain-related anxiety were detected via pharmacological approach and chemogenetics in CP rats. Our results showed that TNBS treatment resulted in long-term hyperalgesia and anxiety-like behavior in rats. CP rats exhibited increased FOS expression and potentiated excitatory synaptic transmission within aIC. CP rats also showed up-regulated expression of VGluT1, and increased membrane trafficking and phosphorylation of NR2B and GluR1 within aIC. Blocking excitatory synaptic transmission significantly attenuated abdomen mechanical hyperalgesia. Specifically inhibiting the excitability of insular pyramidal cells reduced both abdomen hyperalgesia and pain-related anxiety. In conclusion, our findings emphasize a key role for aIC in hyperalgesia and anxiety of painful CP, providing a novel insight into cortical modulation of painful CP and shedding light on aIC as a potential target for neuromodulation interventions in the treatment of CP.

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3 neurons. Whole-cell recordings showed that small-diameter Vglut3 DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3 neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3 neurons. A biophysical analysis revealed voltage-gated sodium channel (Na) currents in menthol-sensitive Vglut3 neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive Nas transcripts between TRPM8-positive and -negative Vglut3 neurons; however, Na1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of Na subunits, with Na1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3 neurons rely primarily on TTX-resistant Na channels. Additionally, when Na1.1 channels were blocked, the remaining Na currents readily entered into slow inactivation in menthol-sensitive Vglut3 neurons. Thus, these data demonstrate that TTX-sensitive Nas drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.Somatosensensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of Na1.1 as a therapeutic target for sensory disorders.

Opioid-induced hyperalgesia (OIH) is a serious adverse event produced by opioid analgesics. Lack of an model has hindered study of its underlying mechanisms. Recent evidence has implicated a role of nociceptors in OIH. To investigate the cellular and molecular mechanisms of OIH in nociceptors, , subcutaneous administration of an analgesic dose of fentanyl (30 μg/kg, s.c.) was performed in male rats. Two days later, when fentanyl was administered intradermally (1 μg, i.d.), in the vicinity of peripheral nociceptor terminals, it produced mechanical hyperalgesia (OIH). Additionally, two days after systemic fentanyl, rats had also developed hyperalgesic priming (opioid-primed rats), long-lasting nociceptor neuroplasticity manifested as prolongation of prostaglandin E (PGE) hyperalgesia. OIH was reversed, , by intrathecal administration of cordycepin, a protein translation inhibitor that reverses priming. When fentanyl (0.5nM) was applied to dorsal root ganglion (DRG) neurons, cultured from opioid-primed rats, it induced a mu-opioid receptor (MOR)-dependent increase in [Ca] in 26% of small-diameter neurons and significantly sensitized (decreased action potential rheobase) weakly IB4-positive and IB4-negative neurons. This sensitizing effect of fentanyl was reversed in weakly IB4-positive DRG neurons cultured from opioid-primed rats after treatment with cordycepin, to reverse of OIH. Thus, administration of fentanyl induces nociceptor neuroplasticity, which persists in culture, providing evidence for the role of nociceptor MOR-mediated calcium signaling and peripheral protein translation, in the weakly IB4-binding population of nociceptors, in OIH.Clinically used mu-opioid receptor agonists such as fentanyl can produce hyperalgesia and hyperalgesic priming. We report on an model of nociceptor neuroplasticity mediating this opioid-induced hyperalgesia (OIH) and priming, induced by fentanyl. Using this model, we have found qualitative and quantitative differences between cultured nociceptors from opioid naïve and opioid primed animals, and provide evidence for the important role of nociceptor MOR-mediated calcium signaling and peripheral protein translation, in the weakly IB4-binding population of nociceptors, in OIH. These findings provide information useful for the design of therapeutic strategies to alleviate OIH, a serious adverse event of opioid analgesics.

Sirtuin1 (SIRT1), which is regulated by microRNA-34a (miR-34a), can modulate pathophysiology processes, including nonalcoholic fatty liver disease and intestinal ischemia/reperfusion injury. We previously reported that SIRT1, an NAD-dependent deacetylase, plays a vital role in the development of neuropathic pain. However, the role of miR-34a/SIRT1 in complete Freund's adjuvant (CFA)-induced inflammatory pain remains unclear. In the present study, we examined miR-34a and SIRT1 in CFA mice. MiR-34a levels increased, while SIRT1 decreased in the spinal cord. Inhibiting miR-34a by intrathecal injection of miR-34a antagomir attenuated CFA-induced pain behavior. Moreover, miR-34a antagomir inhibited the CFA-induced SIRT1 decrease in the spinal cord. Furthermore, the analgesic effect of miR-34a antagomir was abrogated by the SIRT1 inhibitor EX-527. Our data provide support that the underlying mechanisms of miR-34a in promoting inflammatory pain may involve negative regulation of SIRT1.