Colonic Afferents Research Articles

945 CD24 Regulated the Properties of Self-Renewal, Drug-Resistant, and Tumorigenicity of Colorectal Cancer Stem Cell

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.

Differential roles of stretch-sensitive pelvic nerve afferents innervating mouse distal colon and rectum

Information about colorectal distension (i.e., colorectal dilation by increased intraluminal pressure) is primarily encoded by stretch-sensitive colorectal afferents in the pelvic nerve (PN). Despite anatomic differences between rectum and distal colon, little is known about the functional roles of colonic vs. rectal afferents in the PN pathway or the quantitative nature of mechanosensory encoding. We utilized an in vitro mouse colorectum-PN preparation to investigate pressure-encoding characteristics of colorectal afferents. The colorectum with PN attached was dissected, opened longitudinally, and pinned flat in a Sylgard-lined chamber. Action potentials of afferent fibers evoked by circumferential stretch (servo-controlled force actuator) were recorded from the PN. Stretch-sensitive fibers were categorized into the following four groups: colonic muscular, colonic muscular/mucosal, rectal muscular, and rectal muscular/mucosal. Seventy-nine stretch-sensitive PN afferents evenly distributed into the above four groups were studied. Rectal muscular afferents had significantly greater stretch-responses than the other three groups. Virtually all rectal afferents (98%) had low thresholds for response and encoded stimulus intensity into the noxious range without obvious saturation. Most colonic afferents (72%) also had low thresholds (<14 mmHg), but a significant proportion (28%) had high thresholds (>18 mmHg) for response. These high-threshold colonic afferents were sensitized to stretch by inflammatory soup; response threshold was significantly reduced (from 23 to 12 mmHg), and response magnitude significantly increased. These results suggest that the encoding of mechanosensory information differs between colonic and rectal stretch-sensitive PN afferents. Rectal afferents have a wide response range to stretch, whereas high-threshold colonic afferents likely contribute to visceral nociception.

Post‐inflammatory modification of colonic afferent mechanosensitivity

1. The present review discusses interactions between the immune and nervous systems in post-infectious irritable bowel syndrome (PI-IBS). 2. Visceral pain is the single symptom that most affects the quality of life of patients with irritable bowel syndrome (IBS), yet it is the least successfully managed. An underlying hypersensitivity of colonic afferents to mechanical stimuli has long been implicated in visceral pain in IBS, but little more is known of the physiological aetiology. 3. The PI-IBS patients are a cohort of IBS patients who attribute their symptoms to a preceding gastrointestinal infection by pathogens such as Campylobacter or Salmonella. Current evidence suggests that the immune system remains activated in these patients and contributes to their visceral hypersensitivity. This is characterized by a shift in the phenotype of circulating immune cells towards a Type 1 (Th1 predominating) state. Products from these immune cells sensitize colonic afferents to mechanical stimuli. 4. Rectal instillation of trinitrobenzene sulphonic acid induces a Th1-mediated inflammatory response, consistent with clinical observations in PI-IBS. The visceral hypersensitivity observed in this model is biphasic, with an initial onset characterized by visceral hypersensitivity correlating with histological damage followed by a delayed phase that occurs after histological recovery. Interestingly, this chronic visceral hypersensitivity is mediated by afferents in closest apposition to blood vessels, but furthest from the initial site of damage. 5. Both clinical and experimental evidence indicates that chronic dysregulation of the immune system induces visceral afferent hypersensitivity and, therefore, may be the central mechanism underlying PI-IBS.

The Ion Channel TRPA1 Is Required for Normal Mechanosensation and Is Modulated by Algesic Stimuli

The transient receptor potential (TRP) channel family includes transducers of mechanical and chemical stimuli for visceral sensory neurons. TRP ankyrin 1 (TRPA1) is implicated in inflammatory pain; it interacts with G-protein-coupled receptors, but little is known about its role in the gastrointestinal (GI) tract. Sensory information from the GI tract is conducted via 5 afferent subtypes along 3 pathways. Nodose and dorsal root ganglia whose neurons innnervate 3 different regions of the GI tract were analyzed from wild-type and TRPA1(-/-) mice using quantitative reverse-transcription polymerase chain reaction, retrograde labeling, and in situ hybridization. Distal colon sections were analyzed by immunohistochemistry. In vitro electrophysiology and pharmacology studies were performed, and colorectal distension and visceromotor responses were measured. Colitis was induced by administration of trinitrobenzene sulphonic acid. TRPA1 is required for normal mechano- and chemosensory function in specific subsets of vagal, splanchnic, and pelvic afferents. The behavioral responses to noxious colonic distension were substantially reduced in TRPA1(-/-) mice. TRPA1 agonists caused mechanical hypersensitivity, which increased in mice with colitis. Colonic afferents were activated by bradykinin and capsaicin, which mimic effects of tissue damage; wild-type and TRPA1(-/-) mice had similar direct responses to these 2 stimuli. After activation by bradykinin, wild-type afferents had increased mechanosensitivity, whereas, after capsaicin exposure, mechanosensitivity was reduced: these changes were absent in TRPA1(-/-) mice. No interaction between protease-activated receptor-2 and TRPA1 was evident. These findings demonstrate a previously unrecognized role for TRPA1 in normal and inflamed mechanosensory function and nociception within the viscera.

Citrobacter rodentium colitis evokes post‐infectious hyperexcitability of mouse nociceptive colonic dorsal root ganglion neurons

To investigate the possible contribution of peripheral sensory mechanisms to abdominal pain following infectious colitis, we examined whether the Citrobacter rodentium mouse model of human E. coli infection caused hyperexcitability of nociceptive colonic dorsal root ganglion (DRG) neurons and whether these changes persisted following recovery from infection. Mice were gavaged with C. rodentium or distilled water. Perforated patch clamp recordings were obtained from acutely dissociated Fast Blue labelled colonic DRG neurons and afferent nerve recordings were obtained from colonic afferents during ramp colonic distensions. Recordings were obtained on day 10 (acute infection) and day 30 (infection resolved). Following gavage, colonic weights, myeloperoxidase (MPO) activity, stool cultures, and histological scoring established that infection caused colitis at day 10 which resolved by day 30 in most tissues. Electrophysiological recordings at day 10 demonstrated hyperexcitability of colonic DRG neurons (40% mean decrease in rheobase, P = 0.02; 50% mean increase in action potential discharge at twice rheobase, P = 0.02). At day 30, the increase in action potential discharge persisted (approximately 150% increase versus control; P = 0.04). In voltage clamp studies, transient outward (I(A)) and delayed rectifier (I(K)) currents were suppressed at day 10 and I(A) currents remained suppressed at day 30. Colonic afferent nerve recordings during colonic distension demonstrated enhanced firing at day 30 in infected animals. These studies demonstrate that acute infectious colitis evokes hyperexcitability of colonic DRG neurons which persists following resolution of the infection and that suppression of I(A) currents may play a role. Together, these findings suggest that peripheral pain mechanisms could contribute to post-infectious symptoms in conditions such as post-infectious irritable bowel syndrome.

S5.5 Localisation and function of transient receptor potential ion channel melastatin subtype 8 (TRPM8) in mouse colonic primary afferent nerve fibres

Also published as a book chapter: 6th Congress of the International Society for Autonomic Neuroscience (ISAN 2009), Sydney, Australia / J. Brock (ed.), pp.29

Role of TRPV1 in high-threshold rat colonic splanchnic afferents is revealed by inflammation

The vanilloid-1 receptor TRPV1 is known to play a role in extrinsic gastrointestinal afferent function. We investigated the role of TRPV1 in mechanosensitivity in afferents from normal and inflamed tissue. Colonic mechanosensitivity was determined in an in vitro rat colon preparation by recording from attached splanchnic nerves. Recordings were made from serosal/mesenteric afferents responding only at high thresholds to graded mechanical stimulation with von Frey probes. Colonic inflammation was induced by adding 5% dextran sulphate sodium (DSS) to the drinking water for 5 days, and was confirmed by histopathology. The selective TRPV1 antagonist, SB-750364 (10 −8 to 10 −6 M), was tested on mechanosensory stimulus response functions of afferents from normal and inflamed preparations ( N = 7 each). Mechanosensory responses had thresholds of 1–2 g, and maximal responses were observed at 12 g. The stimulus response function was not affected by DSS-induced colitis. SB-750364 had no effect on stimulus response functions in normal preparations, but reduced (up to 60%) in a concentration-dependent manner those in inflammation (2-way ANOVA, p < 0.05). Moreover, in inflamed tissue, spontaneous afferent activity showed a dose-dependent trend toward reduction with SB-750364. We conclude that mechanosensitivity of high-threshold serosal colonic splanchnic afferents to graded stimuli is unaffected during DSS colitis. However, there is a positive influence of TRPV1 in mechanosensitivity in inflammation, suggesting up-regulation of excitatory TRPV1-mediated mechanisms.

W1729 Volume Dependent Recruitment of Adaptive Esophageal Peristaltic and Upper Esophageal Sphincter Contractile Reflexes Is Preserved in Sleep State in Healthy Premature Infants

Background: Serotonin (5-HT) plays a major role in gastrointestinal (GI) functions. Of several subtypes of 5-HT receptors, 5-HT3 and 5-HT4 are involved in GI motility and pain. 5-HT4 receptor agonist tegaserod (TEG) has been shown to modulate visceral pain in both humans and animals. However, its underlying mechanism of action remains unknown. Aim: The objective of the present study is to examine the site of action of this drug in colorectal distension (CRD)-induced visceral pain in naive and post-inflamed sensitized rats. Methods: In each of 47 male Sprague-Dawley rats under fully anesthetized state, a bipolar electrode was implanted surgically into the external oblique muscle of the abdomen to measure the visceromotor response (VMR) represented as electrical activity (EMG) of the muscle to graded CRD (10-80mmHg). VMR was recorded in fully awake rats 72 hours following the surgery. To induce colonic inflammation, 0.5ml of tri-nitrobenzene sulfonic acid (TNBS) in 50% ethanol was infused into the colon under the influence of anesthesia and rats were tested 7days after TNBS injection. Drugs were injected either intraperitoneally (ip), intravenously (iv) or subcutaneously (sc). Electrophysiological recordings were made from the S1 sacral dorsal root receiving pelvic nerve innervating the colon. Responses of pelvic afferents to CRD were recorded before and after TEG injection. Results: Rats exhibited linear increasing VMR to graded CRD. TNBS-treated rats exhibited significantly (p<0.05 vs naive) greater VMR suggesting the sensitization following inflammation. High doses of TEG (5 and 10mg/ kg, ip) resulted in severe CNS effect including agitation, motor impairment and sedation (n=4). CNS effects were delayed in atropine (5mg/kg, ip) pretreated rats (n=4). Lower doses (0.5 and 1mg/kg, ip) of TEG did not produce any visible CNS effect. TEG (1mg/kg) significantly attenuated VMR in both naive and post-inflamed rats. Atropine failed to block the effect of TEG, suggesting that visceral analgesia was not mediated via muscariniccholinergic system. Selective 5-HT4 antagonist GR113808 (5mg/kg, sc) significantly reversed the effect of TEG. Similarly, opioid receptor antagonist naloxone (5mg/kg, iv) significantly reversed the effect of TEG. In electrophysiology experiments, TEG (1mg/kg, ip) did not attenuate the mechanotransduction of colonic pelvic nerve afferents in either naive or postinflamed rats. Conclusion: Results suggest that TEG produces visceral analgesia via the activation of central 5-HT4 receptors. Since opioid antagonist naloxone blocks the effect of TEG, it suggests that the central opioidergic system is linked to 5-HT4 mediated visceral analgesia.

Post-inflammatory colonic afferent sensitisation: different subtypes, different pathways and different time courses

Objective:Intestinal infection evokes hypersensitivity in a subgroup of patients with irritable bowel syndrome (IBS) long after healing of the initial injury. Trinitrobenzene sulfonic acid (TNBS)-induced colitis in rodents likewise results...

TRPV1 Expression Defines Functionally Distinct Pelvic Colon Afferents

Changes in primary sensory neurons are likely to contribute to the emergence of chronic visceral pain. An important step in understanding visceral pain is the development of comprehensive phenotypes that combines functional and anatomical properties for these neurons. We developed a novel ex vivo physiology preparation in mice that allows intracellular recording from colon sensory neurons during colon distension, in the presence and absence of pharmacologic agents. This preparation also allows recovery of functionally characterized afferents for histochemical analysis. Recordings obtained from L6 dorsal root ganglion cells in C57BL/6 mice identified two distinct populations of distension-responsive colon afferents: high-firing frequency (HF) and low-firing frequency (LF) cells. Fluid distension of the colon elicited rapid firing (>20 Hz) in HF cells, whereas LF cells seldom fired >5 Hz. Distension response thresholds were significantly lower in HF cells (LF, 17.5 +/- 1.1 cmH(2)O; HF, 2.6 +/- 1.0 cmH(2)O). Responses of most LF afferents to colon distension were sensitized by luminal application of capsaicin (1 microm; 8 of 9 LF cells), mustard oil (100 microm; 10 of 12 LF cells), and low pH (pH 4.0; 5 of 6 LF cells). In contrast, few HF afferents were sensitized by capsaicin (3 of 9), mustard oil (2 of 7), or low pH (1 of 6) application. Few HF afferents (4 of 23) expressed the capsaicin receptor, TRPV1. In contrast, 87% (25 of 29) of LF afferents expressed TRPV1. TRPV1 has been shown to be required for development of inflammatory hyperalgesia. These results suggest a unique functional role of TRPV1-positive colon afferents that could be exploited to design specific therapies for visceral hypersensitivity.

Intraspinal sprouting of unmyelinated pelvic afferents after complete spinal cord injury is correlated with autonomic dysreflexia induced by visceral pain

Autonomic dysreflexia is a potentially life-threatening hypertensive syndrome following high thoracic (T) spinal cord injury (SCI). It is commonly triggered by noxious pelvic stimuli below the injury site that correlates with increased sprouting of primary afferent C-fibers into the lumbosacral (L/S) spinal cord. We have recently demonstrated that injury-induced plasticity of (L/S) propriospinal neurons, which relay pelvic visceral sensations to thoracolumbar sympathetic preganglionic neurons, is also correlated with the development of this syndrome. To determine the phenotype of pelvic afferent fiber sprouts after SCI, cholera toxin subunit beta (CTb) was injected into the distal colon 2 weeks post–T4 transection/sham to label colonic visceral afferents. After 1 week of transport, the (L/S) spinal cords were cryosectioned and immunohistochemically stained for CTb, the nociceptive-specific marker calcitonin gene-related peptide (CGRP), and the myelinated fiber marker RT97. Quantitative analysis showed that the density of CGRP+ afferent fibers was significantly increased in the L/S dorsal horns of T4-transected versus sham rats, whereas RT97+ afferent fiber density showed no change. Importantly, CTb-labeled pelvic afferent fibers were co-localized with CGRP+ fibers, but not with RT97+ fibers. These results suggest that the sprouting of unmyelinated nociceptive pelvic afferents following high thoracic SCI, but not myelinated fibers, contributes to hypertensive autonomic dysreflexia induced by pelvic visceral pain.

TRPV1 fans the flames of visceral pain

TRPV1 fans the flames of visceral pain

Oral Cyclic Guanosine Monophosphate (cGMP) Desensitizes Colonic Afferents in an Animal Model of Experimental Colitis

Purpose: Irritable bowel syndrome (IBS) is characterized by lower abdominal pain and/or discomfort in association with alterations in intestinal motility. Visceral afferent sensitization is thought to play a key role. Linaclotide, a novel, orally-administered agonist of guanylate cyclase-C receptors (GC-C) markedly increases intracellular and extracellular cGMP levels. Furthermore, linaclotide decreases visceral pain in animal models and reduced pain in an IBS-constipation study. To better understand these analgesic effects, we examined whether cGMP decreases pelvic afferent activity. The aim of this study was to explore the antinociceptive effects of cGMP in an animal model of colonic afferent sensitization. Methods: Colonic sensitization was produced in female Sprague-Dawley rats by intra-rectal administration of trinitrobenzesulfonic acid (TNBS). Responses of colonic afferents to colonic distension (CD), capsaicin (CP, 0.1–5.0 μg), and Substance P (SP, 0.1–10 μg) were tested before and 30 min after intra-duodenal administration of cGMP (30 mg/kg) either 1 hour (N = 8) or 8–10 days post-TNBS (N = 8) and were compared to intra-rectal saline controls (N = 12). Afferent activity was recorded in fibers sensitive to CD and CP from the fine bundles of the right pelvic nerve. Resting activity represented maximal activity recorded 1 min before each intervention. Changes in afferent activity calculated as number of impulses per minute were expressed as percent change from the resting firing rate. Results: In intra-rectal saline controls, cGMP slightly increased baseline afferent firing rates but had no effect on the afferent response to CD or CP. The increase in afferent firing rates produced by SP, however, was significantly attenuated by cGMP (148 ± 34% vs. 989 ± 391%, P < 0.05). One hour after colonic irritation, basal afferent activity was increased and not affected by cGMP; their responses to CD, CP (493 ± 233% vs. 1846 ± 569%, P < 0.05), and SP (267 ± 65% vs. 964 ± 294%, P < 0.05), however, were all significantly attenuated. Afferent responses to CD (>30 mmHg) and cGMP measured 8–10 days after TNBS were similar to those recorded acutely. Afferent responses to SP were significantly reduced by cGMP 8–10 days after TNBS (255 ± 63% vs. 455 ± 58%, P < 0.05), while responses trended towards normalization in response to CP. Conclusion: Oral cGMP reduced the response of sensitized pelvic afferent neurons in rats to different stimuli, particularly those chemically responsive to SP. These findings suggest that the production and secretion of cGMP elicited by oral linaclotide may explain the activity of linaclotide on visceral pain. Additional studies are needed to further characterize the role of cGMP in attenuating afferent responses to noxious stimuli.

Distinct chemical classes of medium-sized transient receptor potential channel vanilloid 1-immunoreactive dorsal root ganglion neurons innervate the adult mouse jejunum and colon

Physiological studies suggest visceral spinal afferents are generally small diameter, unmyelinated C-fibers or myelinated Aδ-fibers, but little is known about the size and chemical phenotypes of visceral sensory neurons supplying the small intestine. This study examines the size and expression patterns of transient receptor potential vanilloid 1 (TRPV1), calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (NOS) and isolectin B 4-binding (IB 4) in dorsal root ganglion (DRG) neurons projecting to the gastrointestinal tract. The spinal afferent innervation of mouse jejunum and distal colon was investigated with retrograde neuronal tracing and multi-label immunohistochemistry. Expression of histochemical markers and soma sizes of retrogradely labeled DRG profiles were determined with confocal microscopy. Most (>75%) jejunal and colonic afferent neurons were medium- and large-sized cells. The majority (82%) of jejunal afferents expressed TRPV1, but few bound IB 4. All retrogradely labeled jejunal afferents expressing NOS-immunoreactivity (64%) also expressed TRPV1 and CGRP and most expressed SP. Most labeled colonic afferents expressed TRPV1 (62%) and half expressed NOS. Taken together these data demonstrate that the spinal afferent supply of the jejunum and colon is largely from medium and large sensory neurons, suggesting most intestinal afferent axons are A fibers. The various chemically-defined subpopulations of afferents may play multiple roles in sensory innervation of the jejunum apart from nociceptive transduction. Additionally, we have identified a unique chemical code, TRPV1/NOS/CGRP/SP, that distinguishes many spinal afferent terminals from those of enteric neurons.

T1437 TPRV1 Is Selectively Expressed in a Functionally Distinct Subset of Colonic Afferents

T1437 TPRV1 Is Selectively Expressed in a Functionally Distinct Subset of Colonic Afferents

T1426 Delayed Post-Inflammatory Hypersensitivity in Sub-Populations of Colonic Afferents

T1426 Delayed Post-Inflammatory Hypersensitivity in Sub-Populations of Colonic Afferents

Transient receptor potential vanilloid 4 mediates protease activated receptor 2-induced sensitization of colonic afferent nerves and visceral hyperalgesia

Protease-activated receptor (PAR(2)) is expressed by nociceptive neurons and activated during inflammation by proteases from mast cells, the intestinal lumen, and the circulation. Agonists of PAR(2) cause hyperexcitability of intestinal sensory neurons and hyperalgesia to distensive stimuli by unknown mechanisms. We evaluated the role of the transient receptor potential vanilloid 4 (TRPV4) in PAR(2)-induced mechanical hyperalgesia of the mouse colon. Colonic sensory neurons, identified by retrograde tracing, expressed immunoreactive TRPV4, PAR(2), and calcitonin gene-related peptide and are thus implicated in nociception. To assess nociception, visceromotor responses (VMR) to colorectal distension (CRD) were measured by electromyography of abdominal muscles. In TRPV4(+/+) mice, intraluminal PAR(2) activating peptide (PAR(2)-AP) exacerbated VMR to graded CRD from 6-24 h, indicative of mechanical hyperalgesia. PAR(2)-induced hyperalgesia was not observed in TRPV4(-/-) mice. PAR(2)-AP evoked discharge of action potentials from colonic afferent neurons in TRPV4(+/+) mice, but not from TRPV4(-/-) mice. The TRPV4 agonists 5',6'-epoxyeicosatrienoic acid and 4alpha-phorbol 12,13-didecanoate stimulated discharge of action potentials in colonic afferent fibers and enhanced current responses recorded from retrogradely labeled colonic dorsal root ganglia neurons, confirming expression of functional TRPV4. PAR(2)-AP enhanced these responses, indicating sensitization of TRPV4. Thus TRPV4 is expressed by primary spinal afferent neurons innervating the colon. Activation of PAR(2) increases currents in these neurons, evokes discharge of action potentials from colonic afferent fibers, and induces mechanical hyperalgesia. These responses require the presence of functional TRPV4. Therefore, TRPV4 is required for PAR(2)-induced mechanical hyperalgesia and excitation of colonic afferent neurons.

Selective Role for TRPV4 Ion Channels in Visceral Sensory Pathways

Although there are many candidates as molecular mechanotransducers, so far there has been no evidence for molecular specialization of visceral afferents. Here, we show that colonic afferents express a specific molecular transducer that underlies their specialized mechanosensory function: the transient receptor potential channel, vanilloid 4 (TRPV4). We found TRPV4 mRNA is highly enriched in colonic sensory neurons compared with other visceral and somatic sensory neurons. TRPV4 protein was found in colonic nerve fibers from patients with inflammatory bowel disease, and it colocalized in a subset of fibers with the sensory neuropeptide CGRP in mice. We characterized the responses of 8 subtypes of vagal, splanchnic, and pelvic mechanoreceptors. Mechanosensory responses of colonic serosal and mesenteric afferents were enhanced by a TRPV4 agonist and dramatically reduced by targeted deletion of TRPV4 or by a TRP antagonist. Other subtypes of vagal and pelvic afferents, by contrast, were unaffected by these interventions. The behavioral responses to noxious colonic distention were also substantially reduced in mice lacking TRPV4. These data indicate that TRPV4 contributes to mechanically evoked visceral pain, with relevance to human disease. In view of its distribution in favor of specific populations of visceral afferents, we propose that TRPV4 may present a selective novel target for the reduction of visceral pain, which is an important opportunity in the absence of current treatments.

Pelvic Nerve Input Mediates Descending Modulation of Homovisceral Processing in the Thoracolumbar Spinal Cord of the Rat

Colonic afferents project to the lumbosacral and thoracolumbar spinal cord via the pelvic and hypogastric/lumbar colonic nerves, respectively. Both spinal regions process inflammatory colonic stimuli. The role of thoracolumbar segments in processing acute colorectal pain is questionable, however, because the lumbosacral spinal cord appears sufficient to process reflex responses to acute pain. Here, we show that activity in pelvic nerve colonic afferents actively modulates thoracolumbar dorsal horn neuron processing of the same colonic stimulus through a supraspinal loop: homovisceral descending modulation. Dorsal horn neurons were recorded in the rat thoracolumbar spinal cord after acute or chronic pelvic neurectomy and cervical cold block. Acute pelvic neurectomy or lidocaine inhibition of lumbosacral dorsal roots facilitated the excitatory response of thoracolumbar dorsal horn neurons to colorectal distention (CRD) and decreased the percentage of neurons inhibited by CRD, suggesting colonic input over the pelvic nerve inhibits thoracolumbar processing of the same stimulus. Ectopic activity developed in the proximal pelvic nerve after chronic neurectomy reactivating the inhibitory circuit, inhibiting thoracolumbar neurons. Cervical cold block alleviated the inhibition in intact or chronic neurectomized rats. However, the facilitated response after acute pelvic neurectomy was inhibited by cervical cold block, exposing an underlying descending facilitation. Inhibiting pelvic nerve input after cervical cold block had minimal effect. These data demonstrate that input over the pelvic nerve modulates the response of thoracolumbar spinal neurons to CRD by a supraspinal loop and that increasing thoracolumbar processing increases visceral hyperalgesia.

Acid sensing ion channels 2 and 3 are required for inhibition of visceral nociceptors by benzamil

The Deg/ENaC family of ion channels, including ASIC1, 2 and 3, are candidate mechanotransducers in visceral and somatic sensory neurons, although each channel may play a different role in different sensory pathways. Here we determined which distinct populations of visceral sensory neurons are sensitive to the non-selective Deg/ENaC blocker benzamil, and which ASIC channels are targets for benzamil by studying its actions in knockout mice. Single afferent fiber recordings were made in vitro from mouse high threshold colonic thoracolumbar splanchnic afferents and low threshold gastroesophageal vagal afferents. mRNA expression of ASIC subtypes was compared between colonic and gastroesophageal afferents by quantitative RT-PCR of transcripts following laser capture microdissection of retrogradely labeled cell bodies. Mechanosensitivity of colonic afferents was potently reduced by benzamil (10−6–3×10−4M), whereas gastroesophageal afferents were marginally inhibited. Inhibition of colonic afferent mechanosensitivity by benzamil was markedly diminished in ASIC2−/− and ASIC3−/− mice, but unchanged in ASIC1a−/−. Therefore ASIC2 and 3 are targets for benzamil to inhibit colonic afferent mechanosensitivity. Conversely, gastroesophageal afferents are less sensitive to benzamil, and its action depends less on ASIC expression. mRNA for ASIC3 showed higher and ASIC1a showed lower relative expression in colonic afferents from thoracolumbar dorsal root ganglia than in gastric afferents from nodose (vagal) ganglia. These data indicate that ASICs on colonic afferents present distinct pharmacological targets for visceral pain.