Excitability Of Dorsal Root Ganglion Neurons Research Articles

A10 FOOD ANTIGEN-STRESS INTERACTION LEADS TO INCREASE PAIN SIGNALING IN ILEUM AND COLON VIA STAT6 IN AN IBS MODEL

Abstract Background Abdominal pain can be triggered by food ingestion in IBS patients. Previously we have shown that a food antigen induces local release of immune mediators in the colon that increase dorsal root ganglion (DRG) neuron excitability when there is previous antigen exposure in the presence of psychological stress. However, it is unknown if this effect is limited to the colon. Furthermore, the involvement of histamine in the neuronal hyperexcitability suggests that the stress-food antigen interaction evokes a Th2 immune response. Thus, we sought to investigate the role of STAT6, a transcription factor downstream of Th2 cytokines and important for IgE production. Aims 1) Determine if stress-food antigen interaction leads to release of mediators within the small intestine that increase DRG neuron excitability. 2) Determine the involvement of STAT6 on neuronal hyperexcitability induced by the stress-food antigen interaction. Methods BALB/c mice were exposed to water avoidance stress (WAS) or sham stress (SHAM) for 1 hr daily for 10 days. On day 2–10, mice were exposed to ovalbumin (OVA) or saline (SAL). Seven days later, mice were re-exposed to either OVA or SAL every 2 days for 2 weeks yielding 3 groups: WAS/OVA+OVA, WAS/SAL+OVA, and SHAM/OVA+OVA. STAT6 deficient mice were also exposed to WAS/OVA+OVA protocol. Ileum or colonic supernatants were obtained 4 hours after tissue collection. DRG neurons were incubated overnight with supernatants prior to perforated patch clamp recordings. Neuronal excitability was evaluated by measuring the rheobase (minimum current to elicit an action potential, decreased rheobase indicates increased excitability). Mechanosensitivity of extrinsic afferent nerves innervating distal ileum was examined using ex vivo extracellular afferent nerve recordings. Data was analyzed by one or two-way ANOVA with Bonferroni post-hoc test. Results Ileum supernatants from WAS/OVA+OVA mice increased DRG neuron excitability compared to WAS/SAL+OVA and SHAM/OVA+OVA mice (63.3 ± 6.2 pA vs 83.2 ± 5.4 pA, 86.7 ± 4.5 pA, p<0.05). Ileum afferent nerve response to distention was significantly augmented in WAS/OVA+OVA mice compared to WAS/SAL+OVA and SHAM/OVA+OVA (P<0.05, n=4–7). DRG neurons incubated with WAS/OVA+OVA supernatant from STAT6 deficient mice were less excitable compared to neurons incubated with colonic supernatants from wild type mice (86.5 ± 4.1 pA vs 67.6 ± 4.8 pA, p<0.05). Conclusions Stress-food antigen interaction releases mediators in both the small intestine and colon to increase nociceptive signaling, an important finding as IBS can involve both areas. The release of excitatory mediators within the gut appears to involve STAT6. Thus, a stress-food antigen interaction evoking a Th2 immune response in the gut may be a mechanism underlying food induced symptoms in IBS. Funding Agencies Queen’s University, Department of Medicine

Electric Field Stimulation for the Functional Assessment of Isolated Dorsal Root Ganglion Neuron Excitability.

Genetically encoded calcium indicators have proven useful for characterizing dorsal root ganglion neuron excitability in vivo. Challenges persist in achieving high spatial-temporal resolutions in vivo, however, due to deep tissue imaging and motion artifacts that may be limiting technical factors in obtaining measurements. Here we report an ex vivo imaging method, using a peripheral neuron-specific Advillin-GCaMP mouse line and electric field stimulation of dorsal root ganglion tissues, to assess the sensitivity of neurons en bloc. The described method rapidly characterizes Ca2+ activity in hundreds of dorsal root ganglion neurons (221 ± 64 per dorsal root ganglion) with minimal perturbation to the in situ soma environment. We further validate the method for use as a drug screening platform with the voltage-gated sodium channel inhibitor, tetrodotoxin. Drug treatment led to decreased evoked Ca2+ activity; half-maximal response voltage (EV50) increased from 13.4V in untreated tissues to 21.2, 23.3, 51.5 (p < 0.05), and 60.6V (p < 0.05) at 0.01, 0.1, 1, and 10µM doses, respectively. This technique may help improve an understanding of neural signaling while retaining tissue structural organization and serves as a tool for the rapid ex vivo recording and assessment of neural activity.

TRPV1 Channel Activated by the PGE2/EP4 Pathway Mediates Spinal Hypersensitivity in a Mouse Model of Vertebral Endplate Degeneration.

Low back pain (LBP) is the primary cause of disability globally. There is a close relationship between Modic changes or endplate defects and LBP. Endplates undergo ossification and become highly porous during intervertebral disc (IVD) degeneration. In our study, we used a mouse model of vertebral endplate degeneration by lumbar spine instability (LSI) surgery. Safranin O and fast green staining and μCT scan showed that LSI surgery led to endplate ossification and porosity, but the endplates in the sham group were cartilaginous and homogenous. Immunofluorescent staining demonstrated the innervation of calcitonin gene-related peptide- (CGRP-) positive nerve fibers in the porous endplate of LSI mice. Behavior test experiments showed an increased spinal hypersensitivity in LSI mice. Moreover, we found an increased cyclooxygenase 2 (COX2) expression and an elevated prostaglandin E2 (PGE2) concentration in the porous endplate of LSI mice. Immunofluorescent staining showed the colocalization of E-prostanoid 4 (EP4)/transient receptor potential vanilloid 1 (TRPV1) and CGRP in the nerve endings in the endplate and in the dorsal root ganglion (DRG) neurons, and western blotting analysis demonstrated that EP4 and TRPV1 expression significantly increased in the LSI group. Our patch clamp study further showed that LSI surgery significantly enhanced the current density of the TRPV1 channel in small-size DRG neurons. A selective EP4 receptor antagonist, L161982, reduced the spinal hypersensitivity of LSI mice by blocking the PGE2/EP4 pathway. In addition, TRPV1 current and neuronal excitability in DRG neurons were also significantly decreased by L161982 treatment. In summary, the PGE2/EP4 pathway in the porous endplate could activate the TRPV1 channel in DRG neurons to cause spinal hypersensitivity in LSI mice. L161982, a selective EP4 receptor antagonist, could turn down the TRPV1 current and decrease the neuronal excitability of DRG neurons to reduce spinal pain.

Fibroblast growth factor 13 stabilizes microtubules to promote Na+ channel function in nociceptive DRG neurons and modulates inflammatory pain

IntroductionFibroblast growth factor homologous factors (FHFs), among other fibroblast growth factors, are increasingly found to be important regulators of ion channel functions. Although FHFs have been link to several neuronal diseases and arrhythmia, its role in inflammatory pain still remains unclear. ObjectivesThis study aimed to investigate the role and mechanism of FGF13 in inflammatory pain. MethodsFgf13 conditional knockout mice were generated and CFA-induced chronic inflammatory pain model was established to measure the pain threshold. Immunostaining, western blot and quantitative real-time reverse transcription PCR (qRT-PCR) were performed to detect the expression of FGF13 in CFA-induced inflammatory pain. Whole-cell patch clamp recording was used to record the action potential firing properties and sodium currents of DRG neurons. ResultsConditional knockout of Fgf13 in dorsal root ganglion (DRG) neurons (Fgf13-/Y) led to attenuated pain responses induced by complete Freund's adjuvant (CFA). FGF13 was expressed predominantly in small-diameter DRG neurons. CFA treatment resulted in an increased expression of FGF13 proteins as well as an increased excitability in nociceptive DRG neurons which was inhibited when FGF13 was absent. The role of FGF13 in neuronal excitability of DRG was linked to its modulation of voltage-gated Na+ channels mediated by microtubules. Overexpression of FGF13, but not FGF13 mutant which lacks the ability to bind and stabilize microtubules, rescued the decreased neuronal excitability and Na+ current density in DRG neurons of Fgf13-/Y mice. ConclusionThis study revealed that FGF13 could stabilize microtubules to modulate sodium channel function in DRG neurons and modulate inflammatory pain. This study provides a novel mechanism for FGF13 modulation of sodium channel function and suggests that FGF13 might be a novel target for inflammatory pain treatment.

Sanguinarine is an agonist of TRPA1 channel

Sanguinarine, a benzyl isoquinoline alkaloid extracted from the root of Papaveraceae plants, shows extensive pharmacological activities including anti-microbial, anti-trypanosoma, anti-tumor, anti-platelet, anti-hypertensive effects, as well as inhibition of osteoclast formation. Here we demonstrate that TRPA1 channel (Transient receptor potential cation channel, member A1) is a potential target for sanguinarine. Electrophysiological recordings show that sanguinarine activates TRPA1 channel potently with an EC50 0.09 (0.04–0.13) μM, but has no effects on other examined TRP channels. Sanguinarine increases the intracellular calcium levels and upregulates the excitability of mouse dorsal root ganglion (DRG) neurons in vitro significantly. Plantar injection of sanguinarine evokes nociceptive behaviors similar to that elicited by allyl isothiocyanate (AITC), a classic agonist of TRPA1. Both the enhancement of excitability of DRG neurons and the nociceptive behaviors can be attenuated by treatment of TRPA1 channel antagonist HC030031 or knockout of trpa1 gene. Taken together, our data demonstrate that sanguinarine is a potent and relatively selective agonist of TRPA1 channel.

Calcium imaging in population of dorsal root ganglion neurons unravels novel mechanisms of visceral pain sensitization and referred somatic hypersensitivity.

Mechanisms of visceral pain sensitization and referred somatic hypersensitivity remain unclear. We conducted calcium imaging in Pirt-GCaMP6s mice to gauge responses of dorsal root ganglion (DRG) neurons to visceral and somatic stimulation in vivo. Intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colonic inflammation and increased the percentage of L6 DRG neurons that responded to colorectal distension above that of controls at day 7. Colorectal distension did not activate L4 DRG neurons. TNBS-treated mice exhibited more Evans blue extravasation than did control mice and developed mechanical hypersensitivity in low-back skin and hind paws, which are innervated by L6 and L4 DRG neurons, respectively, suggesting that colonic inflammation induced mechanical hypersensitivity in both homosegmental and heterosegmental somatic regions. Importantly, the percentage of L4 DRG neurons activated by hind paw pinch and brush stimulation and calcium responses of L6 DRG neurons to low-back brush stimulation were higher at day 7 after TNBS than those in control mice. Visceral irritation from intracolonic capsaicin instillation also increased Evans blue extravasation in hind paws and low-back skin and acutely increased the percentage of L4 DRG neurons responding to hind paw pinch and the response of L6 DRG neurons to low-back brush stimulation. These findings suggest that TNBS-induced colitis and capsaicin-induced visceral irritation may sensitize L6 DRG neurons to colorectal and somatic inputs and also increase the excitability of L4 DRG neurons that do not receive colorectal inputs. These changes may represent a potential peripheral neuronal mechanism for visceral pain sensitization and referred somatic hypersensitivity.

Alpha-lipoic acid downregulates TRPV1 receptor via NF-κB and attenuates neuropathic pain in rats with diabetes.

AimsPainful diabetic neuropathy (PDN) is a refractory complication of diabetes. The study aimed to investigate the role of α‐lipoic acid (ALA) on the regulation of transient receptor potential vanilloid‐1 (TRPV1) in dorsal root ganglion (DRG) neurons of rats with diabetes.MethodsWhole‐cell patch‐clamp recordings were employed to measure neuronal excitability in DiI‐labeled DRG neurons of control and streptozotocin (STZ)‐induced diabetic rats. Western blotting and immunofluorescence assays were used to determine the expression and location of NF‐κBp65 and TRPV1.ResultsSTZ‐induced hindpaw pain hypersensitivity and neuronal excitability in L4‐6 DRG neurons were attenuated by intraperitoneal injection with ALA once a day lasted for one week. TRPV1 expression was enhanced in L4‐6 DRGs of diabetic rats compared with age‐matched control rats, which was also suppressed by ALA treatment. In addition, TRPV1 and p65 colocated in the same DRG neurons. The expression of p65 was upregulated in L4‐6 DRGs of diabetic rats. Inhibition of p65 signaling using recombinant lentiviral vectors designated as LV‐NF‐κBp65 siRNA remarkably suppressed TRPV1 expression. Finally, p65 expression was downregulated by ALA treatment.ConclusionOur findings demonstrated that ALA may alleviate neuropathic pain in diabetes by regulating TRPV1 expression via affecting NF‐κB.

Alcohol-aggravated episodic pain in humans with SCN11A mutation and ALDH2 polymorphism.

Mutations in Nav1.9 encoded by SCN11A have been associated with episodic pain, small-fiber neuropathy, and congenital insensitivity to pain. In this study, we collected and characterized one Chinese family with episodic pain. The SCN11A mutation (c.664C>A/p.Arg222Ser) was identified and cosegregated with the episodic pain phenotype. In addition, we found that alcohol intake triggered intense pain attacks and detected the ALDH2 polymorphism (c.1510G>A/p.Glu504Lys) in 3 patients with episodic pain. The alcohol-aggravated pain symptom and this ALDH2 polymorphism were also reconfirmed in our previously reported episodic pain patient with the Nav1.9 mutation (p.Ala808Gly, patient III-2 in HBBJ family). To assess the pathogenicity of the Nav1.9 mutation and the new trigger, we introduced a mutation (p.Ala796Gly) into the mouse (orthologous mutation in human is p.Ala808Gly). The alteration hyperpolarized channel activation, increased the residual current through noninactivating channels, and induced hyperexcitability of dorsal root ganglion (DRG) neurons in Scn11a mice. The Scn11a mice showed increased sensitivity to mechanical, heat, and cold stimuli, and hypersensitivity to acetaldehyde and formalin, which could account for the alcohol intake-induced pain phenotype in patients. Moreover, acetaldehyde increased the mutant mNav1.9 channel current and excitability of Scn11a mouse DRG neurons. Parecoxib (an anti-inflammatory medication) relieved the heat hypersensitivity in Scn11a mice not receiving inflammatory stimuli and significantly decreased the hyperexcitability of DRG neurons in Scn11a mice. These results indicated that Scn11a mice recapitulated many clinical features of patients and suggested that Nav1.9 channel contributes significantly to the inflammatory pain state.

Activation of EphB receptors contributes to primary sensory neuron excitability by facilitating Ca2+ influx directly or through Src kinase-mediated N-methyl-D-aspartate receptor phosphorylation.

EphrinB-EphB receptor tyrosine kinases have been demonstrated to play important roles in pain processing after peripheral nerve injury. We have previously reported that ephrinB-EphB receptor signaling can regulate excitability and plasticity of neurons in spinal dorsal horn, and thus contribute to spinal central sensitization in neuropathic pain. How EphB receptor activation influences excitability of primary neurons in dorsal root ganglion (DRG), however, remains unknown. Here, we report that EphB receptor activation facilitates calcium influx through N-methyl-D-aspartate receptor (NMDAR) dependent and independent manners. In cultured DRG cells from adult rats, EphB1 and EphB2 receptors were expressed in neurons, but not the glial cells. Bath application of EphB receptor agonist ephrinB2-Fc induced NMDAR-independent Ca influx, which was from the extracellular space rather than endoplasmic reticulum. EphB receptor activation also greatly enhanced NMDAR-dependent Ca influx and NR2B phosphorylation, which was prevented by pretreatment of Src kinase inhibitor PP2. In nerve-injured DRG neurons, elevated expression and activation of EphB1 and EphB2 receptors contributed to the increased intracellular Ca concentration and NMDA-induced Ca influx. Repetitive intrathecal administration of EphB2-Fc inhibited the increased phosphorylation of NR2B and Ca-dependent subsequent signals Src, ERK, and CaMKII as well as behaviorally expressed pain after nerve injury. These findings demonstrate that activation of EphB receptors can modulate DRG neuron excitability by facilitating Ca influx directly or through Src kinase activation-mediated NMDA receptor phosphorylation and that EphB receptor activation is critical to DRG neuron hyperexcitability, which has been considered critical to the subsequent spinal central sensitization and neuropathic pain.

A54 DELTA OPIOID RECEPTOR SIGNALING MEDIATES OPIOID INDUCED TOLERANCE AND HYPERALGESIA IN COLONIC SENSORY NERVES

Abstract Background While opioid drugs are widely used for treating abdominal pain, prolonged exposure to opioids can induce tolerance and paradoxically increase pain. We previously showed in colonic afferent nerves that chronic morphine treatment causes tolerance and paradoxical hyperalgesia, however the mechanisms are unknown. Aims To evaluate the role of delta opioid receptor (DOR) signaling in opioid induced tolerance and hyperalgesia in colonic nerves during chronic morphine exposure. Methods C57BL/6 mice were injected (i.p.) twice daily for 7 days with escalating doses of morphine (10, 20, 30 mg/kg for the first 3 days, then 40mg/kg on 4th to 7th day); a subset of mice were also injected with 2.5 mg/kg naltrindole (NTI), a DOR antagonist. The analgesic response in vivo was monitored daily using the tail-flick test. To assess the effect of chronic morphine exposure, mice were euthanized on day 8, and the dorsal root ganglia (DRGs) and colons were harvested. Isolated DRG neurons were incubated with 1μM morphine (30 min) and neuronal excitability was determined by measuring the rheobase (amount of current required to elicit an action potential) using perforated patch clamp. To assess changes in colonic afferent nerve excitability, ex vivo afferent nerve recordings were obtained from flat-sheet colon preparations to determine the effects of 1μM morphine (10 min perfusion) on mechanosensitivity by probing with 1g von Frey hair. Results The tail flick test showed that the DOR antagonist NTI inhibited (30%) the maximal antinociceptive effect of morphine and reduced morphine tolerance. In patch clamp recordings, 1μM morphine paradoxically increased the excitability of small DRG neurons from morphine mice (rheobase decreased 28%; p&amp;lt;0.05, 2-way ANOVA), whereas this excitatory effect was absent on neurons from NTI+morphine mice. Acute application of 1μM morphine had no effect on afferent nerve responses to probing in morphine mice (14.9 vs 16.7 Hz; p=ns, paired t test, n=15) whereas it inhibited the afferent nerve response to probing in NTI+morphine mice (15.2 vs 9.9 Hz; p&amp;lt;0.001, paired t test, n=16). Furthermore, most single units from morphine mice had an increased response to probing following acute morphine application (7/15 vs 2/16 units excited), whereas most units form NTI-morphine mice were inhibited (14/16 vs. 3/15 units inhibited, P&amp;lt;0.001, Chi-square). Conclusions These findings suggest that DOR signaling plays a significant role in the development of opioid tolerance and hyperalgesia in colonic afferent nerves induced by chronic morphine treatment. Thus, antagonists of DOR may help to mitigate these side effects induced by opioids. Funding Agencies CCC

A127 THE ROLE OF ENDOGENOUS OPIOIDS IN MITIGATING OPIOID TOLERANCE IN A PRE-CLINICAL MODEL OF IBD

Abstract Background While opioid drugs are used to treat pain in IBD patients, escalating doses can induce tolerance and paradoxical increased pain signaling. Endogenous opioids are released by immune cells during chronic colitis and have an analgesic action but it is unknown whether they can mitigate the development of tolerance to opioid drugs in IBD. Aims To evaluate whether endogenous opioids released during chronic inflammation prevent the development of tolerance and/or increased pain signaling following chronic morphine treatment. Methods Three groups of C57BL/6 mice were studied: 1) control, 2) chronic colitis induced with 3 cycles of 2% DSS (cDSS mice) and 3) chronic DSS mice injected with escalating doses of morphine on the last 7 days of DSS (10, 20, 30 mg/kg for the first 3 days, then 40mg/kg on 4 to 7 day) (cDSS-morphine mice). Ex vivo afferent nerve recordings were obtained from flat-sheet distal colon preparations (day 31) to determine their mechanosensitivity by probing with a 1g von Frey hair before and after acute morphine (1μM, 10 min perfusion). In parallel experiments, supernatants were obtained from the proximal colon and incubated overnight with isolated dorsal root ganglia (DRG) neurons from control mice. Neuronal excitability was then examined by measuring the rheobase using perforated patch clamp. Results In afferent nerve recordings, acute application of 1μM morphine inhibited the colonic afferent response to probing in control mice (17.86 vs 12.31 Hz; p&amp;lt;0.05), whereas no effect of morphine was seen in cDSS mice. In cDSS-morphine mice acute morphine perfusion paradoxically increased the mean afferent response to probing (13.99 vs 17.28 Hz, p&amp;lt;0.05). Single units were analyzed to identify how many mechanosensitive responses were increased, decreased or not affected by acute morphine perfusion. Compared to control mice, cDSS mice exhibited increased numbers of unaffected units (6/18 vs 1/14) whereas cDSS-morphine mice had decreased number of inhibited units (1/17 vs 11/14) and increased number of excited units (8/17 vs. 2/14) (Figure 1). In patch clamp recordings, colonic supernatants from cDSS mice reduced the excitability of DRG neurons (rheobase increased 28%; p&amp;lt;0.05), however, this effect was lost in neurons incubated with cDSS-morphine supernatants. Conclusions Endogenous opioids released during cDSS colitis do not mitigate tolerance to chronic morphine exposure and may diminish the response to acute morphine. Moreover, the paradoxical hyperexcitable response to acute morphine induced by chronic morphine was not blocked by endogenous opioids. Our patch clamp data suggest that endogenous opioid actions are lost following chronic morphine exposure and could result from inhibition of their release from immune cells. Funding Agencies CCC

Pharmacological characterization of a rat Nav1.7 loss-of-function model with insensitivity to pain.

Sodium channel Nav1.7, encoded by the SCN9A gene, is a well-validated target that plays a key role in controlling pain sensation. Loss-of-function mutations of Nav1.7 can cause a syndrome of profound congenital insensitivity to pain in humans. Better understanding of how the loss of Nav1.7 leads to loss of pain sensibility would help to decipher the fundamental mechanisms of nociception and inform strategies for development of novel analgesics. Using a recently described rat Nav1.7 loss-of-function model with deficient nociception but intact olfactory function, we investigated the involvement of endogenous opioid and cannabinoid systems in this rodent model of Nav1.7-related congenital insensitivity to pain. We found that both the opioid receptor antagonist naloxone and cannabinoid receptor blockers SR141716A (rimonabant) and SR144528 fail to restore acute pain sensitivity in Nav1.7 loss-of-function rats. We observed, however, that after rimonabant administration, Nav1.7 loss-of-function but not WT rats displayed abnormal behaviours, such as enhanced scratching, caudal self-biting, and altered facial expressions; the underlying mechanism is still unclear. Dorsal root ganglion neurons from Nav1.7 loss-of-function rats, although hypoexcitable compared with WT neurons, were still able to generate action potentials in response to noxious heat and capsaicin. Our data indicate that complete loss of dorsal root ganglion neuron excitability is not required for insensitivity to pain and suggest that endogenous opioid and cannabinoid systems are not required for insensitivity to pain in the absence of Nav1.7 channels in this rat Nav1.7 loss-of-function model.

Nuclear Factor-kappaB Gates Nav1.7 Channels in DRG Neurons via Protein-Protein Interaction.

SummaryIt is well known that nuclear factor-kappaB (NF-κB) regulates neuronal structures and functions by nuclear transcription. Here, we showed that phospho-p65 (p-p65), an active form of NF-κB subunit, reversibly interacted with Nav1.7 channels in the membrane of dorsal root ganglion (DRG) neurons of rats. The interaction increased Nav1.7 currents by slowing inactivation of Nav1.7 channels and facilitating their recovery from inactivation, which may increase the resting state of the channels ready for activation. In cultured DRG neurons TNF-α upregulated the membrane p-p65 and enhanced Nav1.7 currents within 5 min but did not affect nuclear NF-κB within 40 min. This non-transcriptional effect on Nav1.7 may underlie a rapid regulation of the sensibility of the somatosensory system. Both NF-κB and Nav1.7 channels are critically implicated in many physiological functions and diseases. Our finding may shed new light on the investigation into the underlying mechanisms.

Deoxycholic acid activates colonic afferent nerves via 5-HT3 receptor-dependent and -independent mechanisms.

Increased bile acids in the colon can evoke increased epithelial secretion resulting in diarrhea, but little is known about whether colonic bile acids contribute to abdominal pain. This study aimed to investigate the mechanisms underlying activation of colonic extrinsic afferent nerves and their neuronal cell bodies by a major secondary bile acid, deoxycholic acid (DCA). All experiments were performed on male C57BL/6 mice. Afferent sensitivity was evaluated using in vitro extracellular recordings from mesenteric nerves in the proximal colon (innervated by vagal and spinal afferents) and distal colon (spinal afferents only). Neuronal excitability of cultured dorsal root ganglion (DRG) and nodose ganglion (NG) neurons was examined with perforated patch clamp. Colonic 5-HT release was assessed using ELISA, and 5-HT immunoreactive enterochromaffin (EC) cells were quantified. Intraluminal DCA increased afferent nerve firing rate concentration dependently in both proximal and distal colon. This DCA-elicited increase was significantly inhibited by a 5-HT3 antagonist in the proximal colon but not in the distal colon, which may be in part due to lower 5-HT immunoreactive EC cell density and lower 5-HT levels in the distal colon following DCA stimulation. DCA increased the excitability of DRG neurons, whereas it decreased the excitability of NG neurons. DCA potentiated mechanosensitivity of high-threshold spinal afferents independent of 5-HT release. Together, this study suggests that DCA can excite colonic afferents via direct and indirect mechanisms but the predominant mechanism may differ between vagal and spinal afferents. Furthermore, DCA increased mechanosensitivity of high-threshold spinal afferents and may be a mechanism of visceral hypersensitivity.NEW & NOTEWORTHY Deoxycholic acid (DCA) directly excites spinal afferents and, to a lesser extent, indirectly via mucosal 5-HT release. DCA potentiates mechanosensitivity of high-threshold spinal afferents independent of 5-HT release. DCA increases vagal afferent firing in proximal colon via 5-HT release but directly inhibits the excitability of their cell bodies.

Cholecystokinin type B receptor-mediated inhibition of A-type K+ channels enhances sensory neuronal excitability through the phosphatidylinositol 3-kinase and c-Src-dependent JNK pathway

BackgroundCholecystokinin (CCK) is implicated in the regulation of nociceptive sensitivity of primary afferent neurons. Nevertheless, the underlying cellular and molecular mechanisms remain unknown.MethodsUsing patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of CCK-8 on the sensory neuronal excitability and peripheral pain sensitivity mediated by A-type K+ channels.ResultsCCK-8 reversibly and concentration-dependently decreased A-type K+ channel (IA) in small-sized dorsal root ganglion (DRG) neurons through the activation of CCK type B receptor (CCK-BR), while the sustained delayed rectifier K+ current was unaffected. The intracellular subunit of CCK-BR coimmunoprecipitated with Gαo. Blocking G-protein signaling with pertussis toxin or by the intracellular application of anti-Gβ antibody reversed the inhibitory effects of CCK-8. Antagonism of phosphatidylinositol 3-kinase (PI3K) but not of its common downstream target Akts abolished the CCK-BR-mediated IA response. CCK-8 application significantly activated JNK mitogen-activated protein kinase. Antagonism of either JNK or c-Src prevented the CCK-BR-mediated IA decrease, whereas c-Src inhibition attenuated the CCK-8-induced p-JNK activation. Application of CCK-8 enhanced the action potential firing rate of DRG neurons and elicited mechanical and thermal pain hypersensitivity in mice. These effects were mediated by CCK-BR and were occluded by IA blockade.ConclusionOur findings indicate that CCK-8 attenuated IA through CCK-BR that is coupled to the Gβγ-dependent PI3K and c-Src-mediated JNK pathways, thereby enhancing the sensory neuronal excitability in DRG neurons and peripheral pain sensitivity in mice.

Resilience to Pain: A Peripheral Component Identified Using Induced Pluripotent Stem Cells and Dynamic Clamp.

Pain is a complex process that involves both detection in the peripheral nervous system and perception in the CNS. Individual-to-individual differences in pain are well documented, but not well understood. Here we capitalized on inherited erythromelalgia (IEM), a well characterized human genetic model of chronic pain, and studied a unique family containing related IEM subjects with the same disease-causing NaV1.7 mutation, which is known to make dorsal root ganglion (DRG) neurons hyperexcitable, but different pain profiles (affected son with severe pain, affected mother with moderate pain, and an unaffected father). We show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell (iPSC)-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing (WES) and dynamic clamp, we show that it is possible to pinpoint a specific variant of another gene, KCNQ in this particular kindred, that modulates the excitability of iPSC-derived sensory neurons in this family. While different gene variants may modulate DRG neuron excitability and thereby contribute to interindividual differences in pain in other families, this study shows that subject-specific iPSCs can be used to model interindividual differences in pain. We further provide proof-of-principle that iPSCs, WES, and dynamic clamp can be used to investigate peripheral mechanisms and pinpoint specific gene variants that modulate pain signaling and contribute to interindividual differences in pain.SIGNIFICANCE STATEMENT Individual-to-individual differences in pain are well documented, but not well understood. In this study, we show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing and dynamic clamp, we show that it is possible to pinpoint a specific gene variant that modulates pain signaling and contributes to interindividual differences in pain.

Selective Blockade of HCN1/HCN2 Channels as a Potential Pharmacological Strategy Against Pain

A prominent role of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels has been suggested based on their expression and (dys)function in dorsal root ganglion (DRG) neurons, being likely involved in peripheral nociception. Using HCN blockers as antinociceptive drugs is prevented by the widespread distribution of these channels. However, tissue-specific expression of HCN isoforms varies significantly, HCN1 and HCN2 being considered as major players in DRG excitability. We characterized the pharmacological effect of a novel compound, MEL55A, able to block selectively HCN1/HCN2 isoforms, on DRG neuron excitability in-vitro and for its antiallodynic properties in-vivo. HEK293 cells expressing HCN1, HCN2, or HCN4 isoforms were used to verify drug selectivity. The pharmacological profile of MEL55A was tested on mouse DRG neurons by patch-clamp recordings, and in-vivo in oxaliplatin-induced neuropathy by means of thermal hypersensitivity. Results were compared to the non-isoform-selective drug, ivabradine. MEL55A showed a marked preference toward HCN1 and HCN2 isoforms expressed in HEK293, with respect to HCN4. In cultured DRG, MEL55A reduced Ih amplitude, both in basic conditions and after stimulation by forskolin, and cell excitability, its effect being quantitatively similar to that observed with ivabradine. MEL55A was able to relieve chemotherapy-induced neuropathic pain. In conclusion, selective blockade of HCN1/HCN2 channels, over HCN4 isoform, was able to modulate electrophysiological properties of DRG neurons similarly to that reported for classical Ih blockers, ivabradine, resulting in a pain-relieving activity. The availability of small molecules with selectivity toward HCN channel isoforms involved in nociception might represent a safe and effective strategy against chronic pain.

Tetrodotoxin-sensitive voltage-gated sodium channels regulate bladder afferent responses to distension.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a prevalent, chronic bladder disorder that negatively impacts the quality of life for ∼5% of the western population. Hypersensitivity of mechanosensory afferents embedded within the bladder wall is considered a key component in mediating IC/BPS symptoms. Bladder infusion of voltage-gated sodium (Nav) channel blockers show clinical efficacy in treating IC/BPS symptoms; however, the current repertoire of Nav channels expressed by and contributing to bladder afferent function is unknown. We used single-cell reverse-transcription polymerase chain reaction of retrogradely traced bladder-innervating dorsal root ganglia (DRG) neurons to determine the expression profile of Nav channels, and patch-clamp recordings to characterise the contribution of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Nav channels to total sodium current and neuronal excitability. We determined the TTX-S and TTX-R contribution to mechanosensitive bladder afferent responses ex vivo and spinal dorsal horn activation in vivo. Single-cell reverse-transcription polymerase chain reaction of bladder-innervating DRG neurons revealed significant heterogeneity in Nav channel coexpression patterns. However, TTX-S Nav channels contribute the vast majority of the total sodium current density and regulate the neuronal excitability of bladder DRG neurons. Furthermore, TTX-S Nav channels mediate almost all bladder afferent responses to distension. In vivo intrabladder infusion of TTX significantly reduces activation of dorsal horn neurons within the spinal cord to bladder distension. These data provide the first comprehensive analysis of Nav channel expression within sensory afferents innervating the bladder. They also demonstrate an essential role for TTX-S Nav channel regulation of bladder-innervating DRG neuroexcitability, bladder afferent responses to distension, and nociceptive signalling to the spinal cord.

The Novel Activity of Carbamazepine as an Activation Modulator Extends from NaV1.7 Mutations to the NaV1.8-S242T Mutant Channel from a Patient with Painful Diabetic Neuropathy

Neuropathic pain in patients carrying sodium channel gain-of-function mutations is generally refractory to pharmacotherapy. However, we have shown that pretreatment of cells with clinically achievable concentration of carbamazepine (CBZ; 30 μM) depolarizes the voltage dependence of activation in some NaV1.7 mutations such as S241T, a novel CBZ mode of action of this drug. CBZ reduces the excitability of dorsal root ganglion (DRG) neurons expressing NaV1.7-S241T mutant channels, and individuals carrying the S241T mutation respond to treatment with CBZ. Whether the novel activation-modulating activity of CBZ is specific to NaV1.7, and whether this pharmacogenomic approach can be extended to other sodium channel subtypes, are not known. We report here the novel NaV1.8-S242T mutation, which corresponds to the NaV1.7-S241T mutation, in a patient with neuropathic pain and diabetic peripheral neuropathy. Voltage-clamp recordings demonstrated hyperpolarized and accelerated activation of NaV1.8-S242T. Current-clamp recordings showed that NaV1.8-S242T channels render DRG neurons hyperexcitable. Structural modeling shows that despite a substantial difference in the primary amino acid sequence of NaV1.7 and NaV1.8, the S242 (NaV1.8) and S241 (NaV1.7) residues have similar position and orientation in the domain I S4-S5 linker of the channel. Pretreatment with a clinically achievable concentration of CBZ corrected the voltage dependence of activation of NaV1.8-S242T channels and reduced DRG neuron excitability as predicted from our pharmacogenomic model. These findings extend the novel activation modulation mode of action of CBZ to a second sodium channel subtype, NaV1.8.

A305 THE PRONOCICEPTIVE EFFECT OF HIGH DOSE OPIOIDS ON MOUSE DRG NEURONS IS MEDIATED BY DELTA OPIOID RECEPTORS

AbstractBackgroundOpioid drugs can be efficacious in the treatment of abdominal pain, but escalating doses of these drugs can also induce pronociceptive signalling in patients and paradoxically worsen pain. The analgesic actions of opioid drugs on dorsal root ganglia (DRG) neurons innervating the intestine are largely mediated by mu opioid receptors (MOR) but there is growing evidence that delta opioid receptors (DOR) could be important targets for pain signaling and to prevent opioid tolerance. However, it is unknown whether MOR and DOR are co-expressed on colonic DRG neurons and whether DOR play a role in pain signaling in the gut.AimsThe present study examined whether exposure to low and high concentrations of the opioid receptor agonist DAMGO had differing effects on the excitability of nociceptive DRG neurons, and if so, whether these actions involved MOR and/or DOR signaling.MethodsSingle unit afferent recordings were obtained in situ from mouse colons to determine the effects of the MOR agonist DAMGO (100nM) and the DOR agonist DADLE (100nM) on mechanosensitive responses. To examine the effects of high and low dose opioids, nociceptive mouse DRG neurons were dissociated from control mice and incubated overnight with 10 nM or 10 µM DAMGO or media alone. Changes in neuronal excitability were recorded by measuring the rheobase using patch clamp recordings. To determine whether changes in cell excitability were mediated by MOR or DOR, neurons were incubated with the MOR antagonist CTOP (100 nM) or DOR antagonist SDM25N (1 µM) 30 min prior to the incubation with DAMGO.ResultsSingle unit recordings from mouse colon were inhibited by both MOR and DOR agonists in 38% of afferents (3/8). Overnight incubation with 10 nM DAMGO inhibited neurons (rheobase increased by 43%, p <0.001) whereas 10 µM DAMGO had an excitatory effect (rheobase decreased by 31%, p <0.01). The inhibitory effect of low dose DAMGO was blocked by the MOR antagonist but the DOR antagonist had no effect. In contrast, the excitatory effect of the high dose DAMGO was completely blocked by the DOR antagonist.ConclusionsMany colonic DRG neurons co-express MOR and DOR. High concentrations of opioids paradoxically increase excitability of nociceptive DRG neurons, potentially by signaling to MOR-DOR heterodimers. This could lead to increased pain signaling and targeting DOR may be a potential target to mitigate this action.Funding AgenciesCCC