Colonic Dorsal Root Ganglia Neurons Research Articles

Melatonin attenuated chronic visceral pain by reducing Nav1.8 expression and nociceptive neuronal sensitization.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, and its specific pathogenesis is still unclear. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized in a rat model of visceral hypersensitivity induced by neonatal colonic inflammation (NCI). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for melatonin (MT) in sensitization of sodium channels in NCI rats. Colorectal distention (CRD) in adult male rats as a measure of visceral hypersensitivity. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. Western blot and Immunofluorescence were employed to detect changes in expression of Nav1.8 and MT2. The results showed that rats exhibited visceral hypersensitivity after NCI treatment. Intrathecal application of melatonin significantly increased the threshold of CRD in NCI rats with a dose-dependent manner, but has no role in the control group. Whole-cell patch clamp recording showed that melatonin remarkably decreased the excitability and the density of TTX-R sodium channel in DRG neurons from NCI rats. The expression of MT2 receptor at the protein level was markedly lower in NCI rats. 8MP, an agonist of MT2 receptor, enhanced the distention threshold in NCI rats. Application of 8MP reversed the enhanced hypersensitivity of DRG neurons from NCI rats. 8MP also reduced TTX-R sodium current density and modulated dynamics of TTX-R sodium current activation. These data suggest that sensitization of sodium channels of colon DRG neurons in NCI rats is most likely mediated by MT2 receptor, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

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

Cyclic analogues of α-conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain.

Patients with irritable bowel syndrome suffer from chronic visceral pain (CVP) and limited analgesic therapeutic options are currently available. We have shown that α-conotoxin Vc1.1 induced activation of GABAB receptors on the peripheral endings of colonic afferents and reduced nociceptive signalling from the viscera. However, the analgesic efficacy of more stable, cyclized versions of Vc1.1 on CVP remains to be determined. Using ex vivo colonic afferent preparations from mice, we determined the inhibitory actions of cyclized Vc1.1 (cVc1.1) and two cVc1.1 analogues on mouse colonic nociceptors in healthy and chronic visceral hypersensitivity (CVH) states. Using whole-cell patch clamp recordings, we also assessed the inhibitory actions of these peptides on the neuronal excitability of colonic innervating dorsal root ganglion neurons. In vivo, the analgesic efficacy of these analogues was assessed by determining the visceromotor response to colorectal distension in healthy and CVH mice. cVc1.1 and the cVc1.1 analogues, [C2H,C8F]cVc1.1 and [N9W]cVc1.1, all caused concentration-dependent inhibition of colonic nociceptors from healthy mice. Inhibition by these peptides was greater than those evoked by linear Vc1.1 and was substantially greater in colonic nociceptors from CVH mice. cVc1.1 also reduced excitability of colonic dorsal root ganglion neurons, with greater effect in CVH neurons. CVH mice treated with cVc1.1 intra-colonically displayed reduced pain responses to noxious colorectal distension compared with vehicle-treated CVH mice. Cyclic versions of Vc1.1 evoked significant anti-nociceptive actions in CVH states, suggesting that they could be novel candidates for treatment of CVP. This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors

Objectiveα-Conotoxin Vc1.1 is a small disulfide-bonded peptide from the venom of the marine cone snail Conus victoriae. Vc1.1 has antinociceptive actions in animal models of neuropathic pain, but its applicability...

Sensitization of Peripheral Sensory Nerves by Mediators From Colonic Biopsies of Diarrhea-Predominant Irritable Bowel Syndrome Patients: A Role for PAR2

This study examined whether mediators from biopsies of human irritable bowel syndrome (IBS) colons alter intrinsic excitability of colonic nociceptive dorsal root ganglion (DRG) neurons by a protease activated receptor 2 (PAR2)-mediated mechanism. Colonic mucosal biopsies from IBS patients with constipation (IBS-C) or diarrhea (IBS-D) and from healthy controls were incubated in medium, and supernatants were collected. Small-diameter mouse colonic DRG neurons were incubated in supernatants overnight and perforated patch current-clamp recordings obtained. Measurements of rheobase and action potential discharge at twice rheobase were compared between IBS and controls to assess differences in intrinsic excitability. Supernatants from IBS-D patients elicited a marked increase in neuronal excitability compared with controls. These changes were consistent among individual patients but the relative contribution of rheobase and action potential discharge varied. In contrast, no differences in neuronal excitability were seen with IBS-C patient supernatants. The increased excitability seen with IBS-D supernatant was not observed in PAR2 knockout mice. A cysteine protease inhibitor, which had no effect on the pronociceptive actions of a serine protease, inhibited the proexcitatory actions of IBS-D supernatant. Soluble mediators from colonic biopsies from IBS-D but not IBS-C patients sensitized colonic nociceptive DRG neurons, suggesting differences between these two groups. PAR2 signaling plays a role in this action and this protease signaling pathway could provide novel biomarkers and therapeutic targets for treatment.

Sensitization of sodium channels by cystathionine β-synthetase activation in colon sensory neurons in adult rats with neonatal maternal deprivation

BackgroundThe pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood and treatment remains difficult. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized and the expression of the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) was upregulated in a rat model of visceral hypersensitivity induced by neonatal maternal deprivation (NMD). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for CBS–H2S signaling in sensitization of sodium channels in a previously validated rat model of IBS. MethodsNeonatal male rats (postnatal days 2–15) were exposed to a 3hour period of daily maternal separation with temperature maintained at ~33°C. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. The expression of NaV1.8 was analyzed by Western blot and Immunofluorescence study. The endogenous H2S producing enzyme CBS antagonist was injected intraperitoneally. ResultsWe showed that CBS was colocalized with NaV1.8 in colon-specific DRG neurons pre-labeled with DiI. Pretreatment of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, significantly reduced expression of NaV1.8 in NMD rats. AOAA treatment also inhibited the TTX-R sodium current density, right-shifted the V1/2 of activation curve, and reversed hyperexcitability of colon-specific DRG neurons in NMD rats. Conversely, addition of NaHS, a donor of H2S, greatly enhanced TTX-R sodium current density, left shifted the activation curve and enhanced excitability of colon DRG neurons in age-matched healthy rats. Furthermore, application of H-89, an inhibitor of protein kinase A, markedly attenuated the potentiation of TTX-R sodium current density by NaHS. ConclusionThese data suggest that sensitization of sodium channels of colon DRG neurons in NMD rats is most likely mediated by CBS–H2S signaling, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

Neonatal colonic inflammation sensitizes voltage-gated Na+channels via upregulation of cystathionine β-synthetase expression in rat primary sensory neurons

The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood, and treatment remains difficult. We have previously reported that colon-specific dorsal root ganglion (DRG) neurons were hyperactive in a rat model of IBS induced by neonatal colonic inflammation (NCI). This study was designed to examine plasticity of voltage-gated Na(+) channel activities and roles for the endogenous hydrogen sulfide-producing enzyme cystathionine β-synthetase (CBS) in chronic visceral hyperalgesia. Abdominal withdrawal reflex (AWR) scores were recorded in response to graded colorectal distention in adult male rats as a measure of visceral hypersensitivity. Colon-specific DRG neurons were labeled with 1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate and acutely dissociated for measuring Na(+) channel currents. Western blot analysis was employed to detect changes in expressions of voltage-gated Na(+) (Na(V)) channel subtype 1.7, Na(V)1.8, and CBS. NCI significantly increased AWR scores when compared with age-matched controls. NCI also led to an ~2.5-fold increase in Na(+) current density in colon-specific DRG neurons. Furthermore, NCI dramatically enhanced expression of Na(V)1.7, Na(V)1.8, and CBS in colon-related DRGs. CBS was colocalized with Na(V)1.7 or -1.8 in colon-specific DRG neurons. Administration of O-(carboxymethyl)hydroxylamine hemihydrochloride (AOAA), an inhibitor for CBS, remarkably suppressed Na(+) current density and reduced expression of Na(V)1.7 and Na(V)1.8. More importantly, intraperitoneal or intrathecal application of AOAA attenuated AWR scores in NCI rats in a dose-dependent manner. These data suggest that NCI enhances Na(+) channel activity of colon DRG neurons, which is most likely mediated by upregulation of CBS expression, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

Role of Voltage Gated Ca2+ Channels in Rat Visceral Hypersensitivity Change Induced by 2, 4, 6-Trinitrobenzene Sulfonic Acid

BackgroundVisceral pain is common symptom involved in many gastrointestinal disorders such as inflammatory bowel disease. The underlying molecular mechanisms remain elusive. We investigated the molecular mechanisms and the role for voltage gated calcium channel (VGCC) in the pathogenesis in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced visceral inflammatory hypersensitivity.ResultsUsing Agilent cDNA arrays, we found 172 genes changed significantly in dorsal root ganglia (DRG) of TNBS treated rats. Among these changed genes, Cav1.2 and Cav2.3 were significantly up-regulated. Then the RT-PCR and Western blot further confirmed the up-regulation of Cav1.2 and Cav2.3. The whole cell patch clamp recording of acutely dissociated colonic specific DRG neurons showed that the peak IBa density was significantly increased in colonic neurons of TNBS treated rats compared with control rats (−127.82 ± 20.82 pA/pF Vs −91.67 ± 19.02 pA/pF, n = 9, *P < 0.05). To distinguish the different type of calcium currents with the corresponding selective channel blockers, we found that L-type (−38.56 ± 3.97 pA/pF Vs −25.75 ± 3.35 pA/pF, n = 9, * P < 0.05) and R-type (−13.31 ± 1.36 pA/pF Vs −8.60 ± 1.25 pA/pF, n = 9, * P < 0.05) calcium current density were significantly increased in colonic DRG neurons of TNBS treated rats compared with control rats. In addition, pharmacological blockade with L-type antagonist (nimodipine) and R-type antagonist (SNX-482) with intrathecal injection attenuates visceral pain in TNBS induced inflammatory visceral hypersensitivity.ConclusionCav1.2 and Cav2.3 in colonic primary sensory neurons play an important role in visceral inflammatory hyperalgesia, which maybe the potential therapeutic targets.

Neonatal maternal deprivation sensitizes voltage-gated sodium channel currents in colon-specific dorsal root ganglion neurons in rats

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by abdominal pain in association with altered bowel movements. The underlying mechanisms of visceral hypersensitivity remain elusive. This study was designed to examine the role for sodium channels in a rat model of chronic visceral hyperalgesia induced by neonatal maternal deprivation (NMD). Abdominal withdrawal reflex (AWR) scores were performed on adult male rats. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch-clamp configurations. The expression of Na(V)1.8 was analyzed by Western blot and quantitative real-time PCR. NMD significantly increased AWR scores, which lasted for ~6 wk in an association with hyperexcitability of colon DRG neurons. TTX-resistant but not TTX-sensitive sodium current density was greatly enhanced in colon DRG neurons in NMD rats. Compared with controls, activation curves showed a leftward shift in NMD rats whereas inactivation curves did not differ significantly. NMD markedly accelerated the activation time of peak current amplitude without any changes in inactivation time. Furthermore, NMD remarkably enhanced expression of Na(V)1.8 at protein levels but not at mRNA levels in colon-related DRGs. The expression of Na(V)1.9 was not altered after NMD. These data suggest that NMD enhances TTX-resistant sodium activity of colon DRG neurons, which is most likely mediated by a leftward shift of activation curve and by enhanced expression of Na(V)1.8 at protein levels, thus identifying a specific molecular mechanism underlying chronic visceral pain and sensitization in patients with IBS.

Release of endogenous opioids during a chronic IBD model suppresses the excitability of colonic DRG neurons

Endogenous opioids are implicated in pain-regulation in chronic inflammatory bowel disease (IBD). We sought to examine whether endogenous opioids suppress the excitability of colonic nociceptive dorsal root ganglia (DRG) neurons during chronic IBD, and if so, whether modulation of underlying voltage-gated K(+) currents was involved. The effects of chronic dextran sulfate sodium (DSS) colitis on afferent signaling in mice was studied using patch clamp recordings. Colonic DRG neurons were identified using Fast Blue retrograde labeling and recordings obtained from small DRG neurons (<40 pF). In current-clamp recordings, the rheobase of neurons was increased 47% (P < 0.01) and action potential discharge at twice rheobase decreased 23% (P < 0.05) following incubation in colonic supernatants from chronic DSS mice. β-endorphin increased 14-fold, and tissue opioid immunoreactivity and expression in CD4+ cells observed by flow cytometry increased in chronic DSS colons. Incubation of naïve neurons in the μ-opioid receptor agonist D-Ala(2), N- MePhe(4), Gly-ol (DAMGO) (10 nM) partially recapitulated the effects of supernatants from DSS mice on rheobase. Supernatant effects were blocked by the μ-opioid receptor antagonist naloxone. In voltage clamp, chronic DSS supernatants and DAMGO increased I(A) K(+) currents. The release of endogenous opioids during chronic inflammation in mice suppresses the excitability of nociceptive DRG neurons. Targeting immune cells may provide a novel means of modulating IBD pain.

Sensory neuro-immune interactions differ between Irritable Bowel Syndrome subtypes

ObjectiveThe gut is a major site of contact between immune and sensory systems and evidence suggests that patients with irritable bowel syndrome (IBS) have immune dysfunction. Here we show how...

637 Activation of Mu-Opioid Receptors Decreases Excitability of Colonic Dorsal Root Ganglion Neurons by Enhancing Voltage Gated Potassium Channels in a Chronic Mouse Model of Inflammatory Bowel Disease

637 Activation of Mu-Opioid Receptors Decreases Excitability of Colonic Dorsal Root Ganglion Neurons by Enhancing Voltage Gated Potassium Channels in a Chronic Mouse Model of Inflammatory Bowel Disease

Brain–Gut Interactions Increase Peripheral Nociceptive Signaling in Mice With Postinfectious Irritable Bowel Syndrome

To investigate the peripheral sensory effects of repeated stress in patients with postinfectious irritable bowel syndrome (IBS), we tested whether stress following self-limiting bacterial colitis increases colonic dorsal root ganglia (DRG) nociceptive signaling. C57BL/6 mice were infected with Citrobacter rodentium. Stress was induced using a 9-day water avoidance paradigm (days 21-30 after infection). Colonic DRG neuronal excitability was measured using perforated patch clamp techniques, in vitro multi-unit afferent recordings, and measurements of visceromotor reflexes. Combined stress and prior infection increased corticosterone and epinephrine levels, compared with infected animals, but did not alter the resolution of colonic inflammation. These changes were associated with increased neuronal excitability and parallel changes in multi-unit afferent recordings and visceromotor reflex thresholds. Protease activity was increased at day 30 following infection with C rodentium. Protease inhibitors markedly reduced the effects of colonic supernatants on neuronal excitability from C rodentium but not stressed animals. Colonic DRG neurons expressed messenger RNAs for the β(2) adrenergic and glucocorticoid receptors; incubation with stress mediators recapitulated the effects on neuronal excitability observed with chronic stress alone. PAR2 activation with concentrations of the activating peptide SLIGRL that had no effect on neuronal excitability in controls caused marked increases in excitability when applied to neurons from chronically stressed animals. Stress, combined with prior acute colitis, results in exaggerated peripheral nociceptive signaling. Proteases and stress mediators can signal directly to colonic DRG neurons; further analysis of these pathways could provide new targets for treatment of patients with postinfectious IBS.

Characterization of the Cysteinyl Leukotriene 2 Receptor in Novel Expression Sites of the Gastrointestinal Tract

Cysteinyl leukotrienes (cysLTs: LTC₄, LTD₄, and LTE₄) are pro-inflammatory lipid molecules synthesized from arachidonic acid. They exert their actions on at least two cysLT receptors (CysLT₁R and CysLT₂R). Endothelial expression and activation of these receptors is linked to vasoactive responses and to the promotion of vascular permeability. Here we track the expression pattern of CysLT₂R in a loss-of-function murine model (CysLT₂R-LacZ) to neurons of the myenteric and submucosal plexus in the small intestine, colonic myenteric plexus, dorsal root ganglia, and nodose ganglion. Cysteinyl leukotriene (LTC₄/D₄) stimulation of colonic submucosal venules elicited a greater permeability response in wild-type mice. In a dextran sulfate sodium-induced colon inflammation model, the disease activity index and colonic edema (measured by wet:dry weights and submucosal thickness) were significantly reduced in knockout (KO) mice compared to controls. Tumor necrosis factor-α levels in colon tissue were significantly lower in KO mice; however, myeloperoxidase activity was similar in both the KO and wild-type groups. Finally, patch-clamp recordings of basal neuronal activity of colonic-projecting nociceptive neurons from dorsal root ganglia (T9-13) revealed significantly higher excitability in KO neurons compared to wild type. These results suggest that a lack of neuronal expression of CysLT₂R in the murine colonic myenteric plexus attenuates colitis disease progression via a reduction in inflammation-associated tissue edema and increases neuronal sensitivity to nociceptive stimuli.

Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Epigenetic Regulation of DNA Methyltransferase I (Dnmt1) in Dorsal Root Ganglion (DRG) Neurons Innervating the Colon in the Rat

Introduction: Increasing evidence suggests that chronic stress alters behavior and modifies epigenetic regulation of genes in the central nervous system. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), is an important epigenetic mechanism of transcriptional control of gene expression. We reported previously that chronic psychological stress induced visceral hyperalgesia and differential alterations in the expression of several genes in DRG neurons innervating the colon in the rat. A potential role for epigenetic regulation in peripheral sensory pathways has not been investigated. Objectives: We examined the hypothesis that DNMTs play an important role in the regulation of chronic stress-induced visceral hyperalgesia. Methods: Male rats were exposed to 1-hour water avoidance (WA) stress daily for 10 consecutive days as a chronic stress paradigm. SiRNA for DNMT1 was administrated in situ to L6-S2 DRGs every other day during the stress procedure. The visceromotor response (VMR) to colorectal distension was measured. Retrograde labeling with cholera toxin B (CTB)-FITCwas employed to identify colonDRG neurons. Immunofluorescence andWestern blot analysis were used to assess protein expression. In Vitro studies were performed in isolated control DRGs in the presence or absence of corticosterone (CORT; 10 μM) and RU-486 (corticoid receptor antagonist, 500 nM). Results: WA stress rats demonstrated significant increases in the level of DNMT1 and DNMT3b but not DNMT3a in L6-S2 DRGs compared with the controls. Enzyme activity assessment showed a 42% increase in DNMT1 activity in L6-S2 DRGs in stressed rats. Immunofluorescence studies revealed a significant increase in DNMT1 in small-sized, C-fiber neurons in WA stressed rats (52.3±2.2%) compared with the control (31.7±1.6%). Retrograde labeling demonstrated that 72.0±2.1% of the CTB-FITC labeled colonic DRG neurons were positive for DNMT1 in stressed rats compared to 40.0±6.5% in controls (P<0.05; n=4). The VMR in WA stressed rats was increased 68% and 92% above control responses at pressures of 40 and 60 mm Hg, respectively. Treatment of stressed rats with siRNA for DNMT1 prevented the VMR enhancement and changes in DNMT1 proteins levels in L6-S2 DRGs. In addition, treatment of control L6-S2 DRGs In Vitro with CORT (10 μM) increased DNMT1 expression level that was prevented by RU-486 (500 nM) (P<0.05). Conclusions: These data support the novel and provocative interpretation that: 1. Chronic stress induces epigenetic regulation of genes in primary nociceptive neurons; 2. DNA methyltransferase 1 (DNMT1) plays an important role in modulation of chronic stress-induced visceral hyperalgesia; and 3. DNA methyltransferases represent a potential target for treatment of functional GI disorders associated with visceral hyperalgesia.

Enhanced Excitability and Down-Regulated Voltage-Gated Potassium Channels in Colonic DRG Neurons from Neonatal Maternal Separation Rats

Irritable bowel syndrome (IBS), characterized mainly by abdominal pain, is a functional bowel disorder. The present study aimed to examine changes in the excitability and the activity of the voltage-gated K+ channel in dorsal root ganglia (DRG) neurons innervating the colon of rats subjected to neonatal maternal separation (NMS). Colonic DRG neurons from NMS rats as identified by FAST DiI™ labeling showed an increased cell size compared with those from nonhandled (NH) rats. Whole cell current-clamp recordings showed that colonic DRG neurons from NMS rats displayed: 1) depolarized resting membrane potential; 2) increased input resistance; 3) a dramatic reduction in rheobase; and 4) a significant increase in the number of action potentials evoked at twice rheobase. Whole cell voltage-clamp recordings revealed that neurons from both groups exhibited transient A-type (IA) and delayed rectifier (IK) K+ currents. Compared with NH rat neurons, the averaged density of IK was significantly reduced in NMS rat neurons. Furthermore, the Kv1.2 expression was significantly decreased in NMS rat colonic DRG neurons. These results suggest that NMS increases the excitability of colonic DRG neurons mainly by suppressing the IK current, which is likely accounted for by the downregulation of the Kv1.2 expression and somal hypertrophy. PerspectiveThis study demonstrates the alteration of delayed rectifier K current and Kv1.2 expression in DRG neurons from IBS model rats, representing a molecular mechanism underlying visceral pain and sensitization in IBS, suggesting the potential of Kv1.2 as a therapeutic target for the treatment of IBS.

Neonatal colon insult alters growth factor expression and TRPA1 responses in adult mice

Inflammation or pain during neonatal development can result in long-term structural and functional alterations of nociceptive pathways, ultimately altering pain perception in adulthood. We have developed a mouse model of neonatal colon irritation (NCI) to investigate the plasticity of pain processing within the viscerosensory system. Mouse pups received an intracolonic administration of 2% mustard oil (MO) on postnatal days 8 and 10. Distal colons were processed at subsequent timepoints for myeloperoxidase (MPO) activity and growth factor expression. Adult mice were assessed for visceral hypersensitivity by measuring the visceromotor response during colorectal distension. Dorsal root ganglion (DRG) neurons from adult mice were retrogradely labeled from the distal colon and calcium imaging was used to measure transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) responses to acute application of capsaicin and MO, respectively. Despite the absence of inflammation (as indicated by MPO activity), neonatal exposure to intracolonic MO transiently maintained a higher expression level of growth factor messenger RNA (mRNA). Adult NCI mice displayed significant visceral hypersensitivity, as well as increased sensitivity to mechanical stimulation of the hindpaw, compared to control mice. The percentage of TRPA1-expressing colon afferents was significantly increased in NCI mice, however they displayed no increase in the percentage of TRPV1-immunopositive or capsaicin-sensitive colon DRG neurons. These results suggest that early neonatal colon injury results in a long-lasting visceral hypersensitivity, possibly driven by an early increase in growth factor expression and maintained by permanent changes in TRPA1 function.

α7-nAChR-mediated suppression of hyperexcitability of colonic dorsal root ganglia neurons in experimental colitis

Controlled clinical trials of nicotine transdermal patch for treatment of ulcerative colitis have been shown to improve histological and global clinical scores of colitis. Here we report that nicotine (1 microM) suppresses in vitro hyperexcitability of colonic dorsal root ganglia (DRG) (L(1)-L(2)) neurons in the dextran sodium sulfate (DSS)-induced mouse model of acute colonic inflammation. Nicotine gradually reduced regenerative multiple-spike action potentials in colitis mice to a single action potential. Nicotine's effect on hyperexcitability of inflamed neurons was blocked in the presence of an alpha(7)-nicotinic acetylcholine receptor (nAChR) antagonist, methyllicaconitine, while choline, the alpha(7)-nAChR agonist, induced a similar effect to that of nicotine. Consistent with these findings, nicotine failed to suppress hyperexcitability in colonic DRG neurons from DSS-treated alpha(7) knockout mice. Furthermore, colonic DRG neurons from DSS-treated alpha(7) knockout mice were characterized by lower rheobase (10 +/- 5 vs. 77 +/- 13 pA, respectively) and current threshold (28 +/- 4 vs. 103 +/- 8 pA, respectively) levels than DSS-treated C57BL/J6 mice. An interesting observation of this study is that 8 of 12 colonic DRG (L(1)-L(2)) neurons from control alpha(7) knockout mice exhibited multiple-spike action potential firing while no wild-type neurons did. Overall, our findings suggest that nicotine at low 1 microM concentration suppresses in vitro hyperexcitability of inflamed colonic DRG neurons in a mouse model of acute colonic inflammation via activation of alpha(7)-nAChRs.

Bacterial cell products signal to mouse colonic nociceptive dorsal root ganglia neurons

This study examined whether bacterial cell products that might gain access to the intestinal interstitium could activate mouse colonic nociceptive dorsal root ganglion (DRG) neurons using molecular and electrophysiological recording techniques. Colonic projecting neurons were identified by using the retrograde tracer fast blue and Toll-like receptor (TLR) 1, 2, 3, 4, 5, 6, 9, adapter proteins Md-1 and Md-2, and MYD88 mRNA expression was observed in laser-captured fast blue-labeled neurons. Ultrapure LPS 1 microg/ml phosphorylated p65 NF-kappaB subunits increased transcript for TNF-alpha and IL-1beta and stimulated secretion of TNF-alpha from acutely dissociated DRG neurons. In current-clamp recordings from colonic DRG neurons, chronic incubation (24 h) of ultrapure LPS significantly increased neuronal excitability. In acute studies, 3-min superfusion of standard-grade LPS (3-30 microg/ml) reduced the rheobase by up to 40% and doubled action potential discharge rate. The LPS effects were not significantly different in TLR4 knockout mice compared with wild-type mice. In contrast to standard-grade LPS, acute application of ultrapure LPS did not increase neuronal excitability in whole cell recordings or afferent nerve recordings from colonic mesenteric nerves. However, acute application of bacterial lysate (Escherichia coli NLM28) increased action potential discharge over 60% compared with control medium. Moreover, lysate also activated afferent discharge from colonic mesenteric nerves, and this was significantly increased in chronic dextran sulfate sodium salt mice. These data demonstrate that bacterial cell products can directly activate colonic DRG neurons leading to production of inflammatory cytokines by neurons and increased excitability. Standard-grade LPS may also have actions independent of TLR signaling.

943 Adenosine-Induced Activation of the Esophageal Nociceptors

isms of any sensitizing or direct action. Methods: Afferent mechanoand chemosensitivity was assessed in mouse colo-rectum In Vitro. High-threshold nociceptive afferents were characterised by responses to mechanical stimuli (3). Cytokine receptor abundance was assessed using PCR of Dorsal Root Ganglia (DRG) (L6-S1) and specifically in laser-captured colonic DRG neurons. Healthy and PIVH (TNBS 130μg/mL with 28 day recovery) mice were compared (3). Results: In healthy mice TNF-α (100ng/mL) induced potent mechanical hypersensitivity (P<0.0001) that was blocked by ruthenium red and selective TRPA1 antagonism (HC-030031), but not by selective antagonism of TRPV1 (capsazepine). TNF-α had no direct chemosensory effects. A switch in effect occurred in PIVH whereby TNF-α inhibited mechanical hypersensitivity. This inhibition was blocked by iberiotoxin, a selective potassium channel (BKCa) antagonist. TNF1 or 2 receptor mRNA abundance in DRG was unaltered. In healthy mice IL-1β (20ng/mL) induced modest mechanical hypersensitivity but caused a direct chemosensory effect in 4/7 afferent recordings, that was inhibited by tetrodotoxin (TTX). Similar responses occurred in PIVH, and IL-1R1 mRNA abundance was unaltered. IL-2 (100ng/mL) had no effect in healthy mice (n=7), but induced a profound TTX-sensitive chemosensory effect in PIVH (7/10 afferents), that was greater than observed for IL-1β, and a significant (P<0.05) increase in IL-2 receptor mRNA. Data are shown for pelvic serosal afferents, but were broadly similar in splanchnic serosal afferents. Discussion: IL-1β and IL2 induce direct chemosensory effects in colonic afferents via sodium channels without alteringmechanosensory function. In contrast, TNF-α exclusively induced mechanical hypersensitivity, predominately via TRPA1. In PIVH, IL-1β effects are unchanged, IL-2 effects are increased, and TNF-α effects are in the opposite direction whereby TNF-α now causes inhibition via BKCa channels. Therefore cytokines interact with visceral afferents via different mechanisms, which undergo markedly different changes in PIVH. 1: Leibregts Gastro 2007. 2: Hughes Gut 2009. 3: Hughes Gut 2009.