Animal Studies

Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel Na1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). When not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of Na1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2) female mice have reduced Na1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2 female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male CRMP2 mice precipitated mechanical allodynia in mice otherwise resistant to developing persistent pain. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2 mice. Finally, silencing these proteins in DRG neurons from female CRMP2 mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non-SUMOylated CRMP2-mediated internalization of sodium channels in vivo.

Nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are essential for neuronal development and survival in embryo. However, after birth they play pivotal roles in generation of hyperalgesia in many painful conditions. Both factors are believed to act on different groups of primary afferents, and no interaction has been studied. Here we show a synergism of both factors. Intramuscular injection of a mixture of both factors of low concentration, each of which alone had no effect, induced a significant muscular mechanical hyperalgesia in rats. We show synergism occurs in the primary afferent neurons and find about 25 % primary afferents innervating the muscle express both TrkA (NGF receptor) and GFRα1 (GDNF receptor). We show by pharmacological means that afferent neurons with TrkA and GFRα1 express both TRPV1 and ASICs. Our data establish a basis for synergism of NGF and GDNF.

After surgery, acute pain is still managed insufficiently and may lead to short- and long-term complications including chronic postsurgical pain and an increased prescription of opioids. Thus, identifying new targets specifically implicated in postoperative pain is of utmost importance to develop effective and non-addictive analgesics. Here, we employed an integrated and multimethod workflow to reveal unprecedented insights into proteome dynamics in dorsal root ganglia (DRG) of mice after plantar incision (INC). Based on a detailed characterization of INC-associated pain-related behavior profiles, including a novel paradigm for non-evoked pain (NEP), we performed quantitative mass-spectrometry-based proteomics in DRG 1 day after INC. Our data revealed a hitherto unknown INC-regulated protein signature in DRG with changes in distinct proteins and cellular signaling pathways. In particular, we show the differential regulation of 44 protein candidates, many of which are annotated with pathways related to immune and inflammatory responses such as MAPK/ERK signaling. Subsequent orthogonal assays comprised multiplex western blotting, bioinformatic protein network analysis, and immunolabeling in independent mouse cohorts to validate (i) the INC-induced regulation of immune/inflammatory pathways and (ii) the high priority candidate Annexin A1 (Anxa1). Taken together, our results propose novel potential targets in the context of incision and, therefore, represent a highly valuable resource for further mechanistic and translational studies of postoperative pain.

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aβ fibers. However, the mechanism by which Aβ fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP) and show that specific ablation or silencing of AAV-NpyP SDH interneurons converted touch-sensing Aβ fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP neurons received excitatory inputs from Aβ fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP neurons exhibited deeper resting membrane potentials, and their excitation by Aβ fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP neurons in nerve-injured rats reversed Aβ fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aβ fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.

Pain is caused by tissue injury, inflammatory disease, pathogen invasion, or neuropathy. The perception of pain is attributed to the neuronal activity in the brain. However, the dynamics of neuronal activity underlying pain perception are not fully known. Herein, we examined theta-oscillation dynamics of local field potentials in the primary somatosensory cortex of a mouse model of formalin-induced pain, which usually shows a bimodal behavioral response interposed between pain-free periods. We found that formalin injection exerted a reversible shift in the theta-peak frequency toward a slower frequency. This shift was observed during nociceptive phases but not during the pain-free period and was inversely correlated with instantaneous pain intensity. Furthermore, instantaneous oscillatory analysis indicated that the probability of slow theta oscillations increased during nociceptive phases with an association of augmented slow theta power. Finally, cross-frequency coupling between theta and gamma oscillations indicated that the coupling peak frequency of theta oscillations was also shifted toward slower oscillations without affecting coupling strength or gamma power. Together, these results suggest that the dynamic changes in theta oscillations in the mouse primary somatosensory cortex represent the ongoing status of pain sensation.

P2X7 receptors are responsible for triggering inflammatory responses contributing to processes of pain in articular tissues. This study aimed to investigate whether the activation of the P2X7 receptor located in the temporomandibular joint (TMJ) tissues induces nociception through an inflammatory mechanisms and/or the activation of C-fibers (small-diameter primary afferents) of rats' TMJ.