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Ca2+/calmodulin-stimulated group Ⅰ adenylyl cyclase (AC) isoforms AC1 and AC8 have been involved in nociceptive processing and morphine responses. However, whether AC3, another member of group I ACs, is involved in nociceptive transmission and regulates opioid receptor signaling remain elusive. Here we report that conditional knockout of AC3 (AC3CKO) in L3 and L4 DRGs robustly facilitates the mouse nociceptive responses, decreases voltage-gated potassium (Kv) channel currents and increases neuronal excitability. Also, AC3CKO eliminates the analgesic effect of κ opioid receptor (KOR) agonist and its inhibition on Kv channel by classical Gαi/o signaling or nonclassical direct interaction of KOR and AC3 proteins. Interestingly, significantly upregulated AC1 level and cAMP concentration are detected in AC3 deficient DRGs. Inhibition of AC1 completely reversed cAMP upregulation, neuronal excitability enhancement and nociceptive behavioral hypersensitivity in AC3CKO mice. Our findings suggest a crucial role of peripheral AC3 in nociceptive modulation and KOR opioid analgesia.
Learn More >Anterior Cingulate Cortex Mediates Hyperalgesia and Anxiety Induced by Chronic Pancreatitis in Rats.
Central sensitization is essential in maintaining chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. Here, we examined the role of the anterior cingulate cortex (ACC) in the pathogenesis of abdominal hyperalgesia in a rat model of CP induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). TNBS treatment resulted in long-term abdominal hyperalgesia and anxiety in rats. Morphological data indicated that painful CP induced a significant increase in FOS-expressing neurons in the nucleus tractus solitarii (NTS) and ACC, and some FOS-expressing neurons in the NTS projected to the ACC. In addition, a larger portion of ascending fibers from the NTS innervated pyramidal neurons, the neural subpopulation primarily expressing FOS under the condition of painful CP, rather than GABAergic neurons within the ACC. CP rats showed increased expression of vesicular glutamate transporter 1, and increased membrane trafficking and phosphorylation of the N-methyl-D-aspartate receptor (NMDAR) subunit NR2B and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluR1 within the ACC. Microinjection of NMDAR and AMPAR antagonists into the ACC to block excitatory synaptic transmission significantly attenuated abdominal hyperalgesia in CP rats, which was similar to the analgesic effect of endomorphins injected into the ACC. Specifically inhibiting the excitability of ACC pyramidal cells via chemogenetics reduced both hyperalgesia and comorbid anxiety, whereas activating these neurons via optogenetics failed to aggravate hyperalgesia and anxiety in CP rats. Taken together, these findings provide neurocircuit, biochemical, and behavioral evidence for involvement of the ACC in hyperalgesia and anxiety in CP rats, as well as novel insights into the cortical modulation of painful CP, and highlights the ACC as a potential target for neuromodulatory interventions in the treatment of painful CP.
Learn More >Endometriosis is a chronic and debilitating condition, commonly characterised by chronic pelvic pain (CPP) and infertility. Chronic pelvic pain can be experienced across multiple pelvic organs, with comorbidities commonly effecting the bowel, bladder, and vagina. Despite research efforts into endometriosis pathophysiology, little is known about how endometriosis induces CPP, and as such, therapeutic interventions are lacking. The aim of this study was to characterise a syngeneic mouse model of endometriosis that mimics naturally occurring retrograde menstruation, thought to precede endometriosis development in patients, and determine whether these mice exhibit signs of CPP and altered behaviour. We characterised the development of endometriosis over 10 weeks following uterine tissue inoculation, measured in vivo and ex vivo hypersensitivity to mechanical stimuli across multiple visceral organs, and assessed alterations in animal spontaneous behaviour. We confirmed that inoculated uterine horn tissue formed into endometriosis lesions throughout the peritoneal cavity, with significant growth by 8 to 10 weeks post inoculation. Additionally, we found that mice with fully developed endometriosis displayed hypersensitivity evoked by (1) vaginal distension, (2) colorectal distension, (3) bladder distension, and (4) cutaneous thermal stimulation, compared to their sham counterparts. Moreover, endometriosis mice displayed alterations in spontaneous behaviour indicative of (5) altered bladder function and (6) anxiety. This model creates a foundation for mechanistical studies into the diffuse CPP associated with endometriosis and the development of targeted therapeutic interventions to improve the quality of life of women with endometriosis.
Learn More >Primary sensory neurons in dorsal root ganglia (DRG) are wrapped by satellite glial cells (SGCs), and neuron-SGC interaction may affect somatosensation, especially nociceptive transmission. P2-purinergic receptors (P2Rs) are key elements in the two-way interactions between DRG neurons and SGCs. However, because the cell types are in such close proximity, conventional approaches such as in vitro culture and electrophysiologic recordings are not adequate to investigate the physiologically relevant responses of these cells at a population level. Here, we performed in vivo calcium imaging to survey the activation of hundreds of DRG neurons in Pirt-GCaMP6s mice and to assess SGC activation in GFAP-GCaMP6s mice in situ. By combining pharmacologic and electrophysiologic techniques, we investigated how ganglionic purinergic signaling initiated by α,β-methyleneadenosine 5'-triphosphate (α,β-MeATP) modulates neuronal activity and excitability at a population level. We found that α,β-MeATP induced robust activation of small neurons-likely nociceptors-through activation of P2X3R. Large neurons, which are likely non-nociceptive, were also activated by α,β-MeATP, but with a delay. Blocking pannexin 1 channels attenuated the late phase response of DRG neurons, indicating that P2R stimulation may subsequently induce paracrine ATP release, which could further activate cells in the ganglion. Moreover, ganglionic α,β-MeATP treatment in vivo sensitized small neurons and enhanced responses of spinal wide-dynamic-range neurons to subsequent C-fiber inputs, suggesting that modulation via ganglionic P2R signaling could significantly affect nociceptive neuron excitability and pain transmission. Therefore, targeting functional P2Rs within ganglia may represent an important new strategy for pain modulation.
Learn More >Neuropathic pain is the most common symptom for which patients seek medical attention. Existing treatments to control pain are largely ineffective because of poor understanding the underlying mechanisms. Synaptic plasticity is fundamental to the spinal sensitivity of neuropathic pain. In the present study, we showed that SNL induced significant allodynia and hyperalgesia as well as upregulation of Nwd1 and GluN2B, which were reversed by knockdown of NWD1. Electrophysiological experiments demonstrated that SNL enhanced synaptic transmission, which was prevented by knockdown of NWD1. In vitro experiments showed that knockdown of NWD1 inhibited dendritic growth and synaptogenesis. Taken together, our results suggest that NWD1 enhances synaptic transmission and contributes to the development of neuropathic pain by enhancing GluN2B synaptic expression and anchor and promoting excitatory synaptogenesis.
Learn More >Previous studies have confirmed that the microglial activation and subsequent inflammatory responses in the trigeminal nucleus caudalis (TNC) are involved in the central sensitization of chronic migraine (CM). MicroRNA-155-5p has been shown to modulate the polarization of microglia and participate in inflammatory processes in a variety of neurological diseases. However, its role in CM remains unclear. The purpose of this study was to determine the precise role of miR-155-5p in CM.
Learn More >The σ receptor has attracted intense interest in cancer imaging, psychiatric disease, neuropathic pain and other areas of biology. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone and the tool compound PB28. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ receptor. Crystal structures of two ligands bound to the σ receptor confirmed the docked poses. To investigate the contribution of the σ receptor in pain, two potent σ-selective ligands and one potent σ/σ non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model, suggesting that the σ receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.
Learn More >Obstructive sleep apnea (OSA), characterized by low arterial oxygen saturation during sleep, is associated with an increased risk of orofacial pain. In this study, we simulated chronic intermittent hypoxia (CIH) during the sleep/rest phase (light phase) to determine the role of transient receptor potential vanilloid 1 (TRPV1) in mediating enhanced orofacial nocifensive behavior and trigeminal spinal subnucleus caudalis (Vc) neuronal responses to capsaicin stimulation in a rat model of OSA. Rats were subjected to CIH (nadir O, 5%) during the light phase for 8 or 16 consecutive days. CIH yielded enhanced behavioral responses to capsaicin, a TRPV1 agonist, after application to the ocular surface and intraoral mucosa, which was reversed under normoxic conditions. The percentage of TRPV1-immunoreactive trigeminal ganglion neurons was greater in CIH rats than in normoxic rats and recovered under normoxic conditions after CIH. The ratio of large-sized TRPV1-immunoreactive trigeminal ganglion neurons increased in CIH rats. The density of TRPV1 positive primary afferent terminals in the superficial laminae of Vc was higher in CIH rats. The phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive cells intermingled with central terminal of TRPV1 positive afferents in the Vc. The number of pERK-immunoreactive cells following low-dose capsaicin (0.33 µM) application to the tongue was significantly greater in the middle portion of the Vc of CIH rats than in normoxic rats and recovered under normoxic conditions after CIH. These data suggest that CIH during the sleep (light) phase is sufficient to transiently enhance pain on the ocular surface and intraoral mucosa via TRPV1-dependent mechanisms.
Learn More >The incidence of inflammatory bowel disease with its two main manifestations, colitis ulcerosa and Crohn's disease, is rising globally year after year. There is still a tremendous need to study the underlying pathomechanisms and a well-established tool in order to better understand the disease are colitis models in rodents. Since the concept of the 3Rs was proposed by Russell and Burch, this would include pain medication in animal models of intestinal inflammation as a reduction of suffering. This review argues against pain medication because the administration of pain medication in its current form has an impact on the inflammatory process and the immune response, thus falsifying the results and the reproducibility and therefore leading to misconceptions.
Learn More >High molecular weight hyaluronan (HMWH) is an agonist at cluster of differentiation 44 (CD44), the cognate hyaluronan receptor, on nociceptors, where it acts to induce anti-hyperalgesia in preclinical models of inflammatory and neuropathic pain. In the present experiments we studied the CD44 second messengers that mediate HMWH-induced attenuation of pain associated with oxaliplatin and paclitaxel chemotherapy-induced peripheral neuropathy (CIPN). While HMWH attenuates CIPN only in male rats, following ovariectomy or intrathecal administration of an oligodeoxynucleotide (ODN) antisense to G-protein coupled estrogen receptor (GPR30) mRNA, female rats are also sensitive to HMWH. Intrathecal administration of ODN antisense to CD44 mRNA markedly attenuates HMWH-induced anti-hyperalgesia in male rats with CIPN induced by oxaliplatin or paclitaxel. Intradermal administration of inhibitors of CD44 second messengers, RhoGTPases (RhoA), phospholipase C (PLC) and PI3Kγ attenuates HMWH-induced anti-hyperalgesia, as does intrathecal administration of an oligodeoxynucleotide (ODN) antisense to PI3Kγ. Our results demonstrate that HMWH-induces anti-hyperalgesia in CIPN, mediated by its action at CD44, and downstream signaling by RhoA, PLC and PI3Kγ.
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