Colonic Nerves Research Articles

The Severity of Neural Invasion Is Associated with Shortened Survival in Colon Cancer

Neural invasion (NI) is a histopathologic feature of colon cancer that receives little consideration. Therefore, we conducted a morphologic and functional characterization of NI in colon cancer. NI was investigated in 673 patients with colon cancer. Localization and severity of NI was determined and related to patient's prognosis and survival. The neuro-affinity of colon cancer cells (HT29, HCT-116, SW620, and DLD-1) was compared with pancreatic cancer (T3M4 and SU86.86) and rectal cancer cells (CMT-93) in the in vitro three-dimensional (3D)-neural-migration assay and analyzed via live-cell imaging. Immunoreactivity of the neuroplasticity marker GAP-43, and the neurotrophic-chemoattractant factors Artemin and nerve growth factor (NGF), was quantified in colon cancer and pancreatic cancer nerves. Dorsal root ganglia of newborn rats were exposed to supernatants of colon cancer, rectal cancer, and pancreatic cancer cells and neurite density was determined. NI was detected in 210 of 673 patients (31.2%). Although increasing NI severity scores were associated with a significantly poorer survival, presence of NI was not an independent prognostic factor in colon cancer. In the 3D migration assay, colon cancer and rectal cancer cells showed much less neurite-targeted migration when compared with pancreatic cancer cells. Supernatants of pancreatic cancer and rectal cancer cells induced a much higher neurite density than those of colon cancer cells. Accordingly, NGF, Artemin, and GAP-43 were much more pronounced in nerves in pancreatic cancer than in colon cancer. NI is not an independent prognostic factor in colon cancer. The lack of a considerable biologic affinity between colon cancer cells and neurons, the low expression profile of colonic nerves for chemoattractant molecules, and the absence of a major neuroplasticity in colon cancer may explain the low prevalence and impact of NI in colon cancer.

P21 Endogenous H2S produced by CSE selectively facilitates acetylcholine release from synaptic terminals of central origin in mouse superior mesenteric ganglion

Abdominal sympathetic prevertebral ganglia in mammals play a major physiological role in regulating motility, absorption and secretion in the gastrointestinal tract and its accessory organs. Hydrogen sulfide (H2S) has emerged as an important messenger molecule in the central nervous system and enteric nervous system. We designed experiments to test the hypotheses that prevertebral ganglia have the enzymatic machinery to generate H2S, that H2S is continuously generated and released in the prevertebral ganglia and that H2S modulates fast cholinergic nicotinic excitatory synaptic input (F-EPSP). Methods The superior mesenteric ganglion (SMG) and attached splanchnic and colonic nerve trunks were dissected from adult SJL/J mice. The use of mouse tissue was approved by the Institutional Animal Care and Use Committee. Sharp glass microelectrodes were used for intracellular recordings and bi-polar stimulating electrodes were used to evoke F-EPSPs. Immunostaining was performed with antibodies for cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) and with vesicular acetylcholine transporter to identify preganglionic cholinergic terminals. Since the amount of tissue in mouse prevertebral ganglia was less than the amount required for the assay method (gas chromatography), we used guinea pig prevertebral ganglia to measure endogenous H2S generation. Results Immunoreactivity of CSE was found in neurons and glia cells. There was no colocalization of CSE with immunoreactivity of vesicular acetylcholine transporter. Immunoreactivity of CBS was only found in some glia cells. There was no colocalization between immunoreactivity of CBS and immunoreactivity of vesicular acetylcholine transporter. Endogenous H2S was generated and released from homogenized guinea pig sympathetic prevertebral ganglia at a rate of 2.52 ± 0.52 pmol/min/mg tissue weight, suggesting that H2S is generated in intact prevertebral ganglia. To determine whether the F-EPSPs are potentiated by endogenously released H2S in mouse SMG, we tested the effect of inhibiting H2S break-down using stigmatellin, a specific sulfide quinone reductase inhibitor. Stigmatellin (1 μM) significantly (p dl -propargylglycine (PAG) (1 μM) on F-EPSPs evoked by splanchnic and colonic nerve stimulation in the presence of PAG. The amplitude and area of F-EPSPs evoked by splanchnic nerve stimulation were significantly (p 0.05) effect on F-EPSPs evoked by colonic nerve stimulation (30.2 ± 6.1 mV and 808 ± 235 ms · mV with PAG vs. 26.9 ± 7.6 mV and 855 ± 241 ms · mV before PAG; n = 5). Conclusions H2S is enzymatically generated and released in the mouse prevertebral ganglia where it potentiates fast cholinergic synaptic input in a pathway specific manner.

Identification and mechanosensitivity of viscerofugal neurons

Enteric viscerofugal neurons are interneurons with cell bodies in the gut wall; they project to prevertebral ganglia where they provide excitatory synaptic drive to sympathetic neurons which control intestinal motility and secretion. Here, we studied the mechanosensitivity and firing of single, identified viscerofugal neurons in guinea-pig distal colon. Flat sheet preparations of gut were set up in vitro and conventional extracellular recordings made from colonic nerve trunks. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (1mM), was locally pressure ejected onto individual myenteric ganglia. In a few ganglia, DMPP promptly evoked firing in colonic nerves. Biotinamide filling of colonic nerves revealed that DMPP-responsive sites corresponded to viscerofugal nerve cell bodies. This provides a robust means to positively identify viscerofugal neuron firing. Of 15 single units identified in this way, none responded to locally-applied capsaicin (1μM). Probing with von Frey hairs at DMPP-responsive sites reliably evoked firing in all identified viscerofugal neurons (18/18 units tested; 0.8–5mN). Circumferential stretch of the preparation increased firing in all 14/14 units (1–5g, p<0.05). Both stretch and von Frey hair responses persisted in Ca2+-free solution (6mMMg2+, 1mM EDTA), indicating that viscerofugal neurons are directly mechanosensitive. To investigate their adequate stimulus, circular muscle tension and length were independently modulated (BAY K8644, 1μM and 10μM, respectively). Increases in intramural tension without changes in length did not affect firing. However, contraction-evoked shortening, under constant load, significantly decreased firing (p<0.001). In conclusion, viscerofugal neuron action potentials contribute to recordings from colonic nerve trunks, in vitro. They provide a significant primary afferent output from the colon, encoding circumferential length, largely independent of muscle tension. All viscerofugal neurons are directly mechanosensitive, although they have been reported to receive synaptic inputs. In short, viscerofugal neurons combine interneuronal function with length-sensitive mechanosensitivity.

Viscerofugal neurons recorded from guinea‐pig colonic nerves after organ culture

Enteric viscerofugal neurons provide cholinergic synaptic inputs to prevertebral sympathetic neurons, forming reflex circuits that control motility and secretion. Extracellular recordings of identified viscerofugal neurons have not been reported. Preparations of guinea pig distal colon were maintained in organotypic culture for 4-6 days (n = 12), before biotinamide tracing, immunohistochemistry, or extracellular electrophysiological recordings from colonic nerves. After 4-6 days in organ culture, calcitonin gene-related peptide and tyrosine hydroxylase immunoreactivity in enteric ganglia was depleted, and capsaicin-induced firing (0.4 μmol L(-1) ) was not detected, indicating that extrinsic sympathetic and sensory axons degenerate in organ culture. Neuroanatomical tracing of colonic nerves revealed that viscerofugal neurons persist and increase as a proportion of surviving axons. Extracellular recordings of colonic nerves revealed ongoing action potentials. Interestingly, synchronous bursts of action potentials were seen in 10 of 12 preparations; bursts were abolished by hexamethonium, which also reduced firing rate (400 μmol L(-1) , P < 0.01, n = 7). DMPP (1,1-dimethyl-4-phenylpiperazinium; 10(-4) mol L(-1) ) evoked prolonged action potential discharge. Increased firing preceded both spontaneous and stretch-evoked contractions (χ(2) = 11.8, df = 1, P < 0.001). Firing was also modestly increased during distensions that did not evoke reflex contractions. All single units (11/11) responded to von Frey hairs (100-300 mg) in hexamethonium or Ca(2+) -free solution. Action potentials recorded from colonic nerves in organ cultured preparations originated from viscerofugal neurons. They receive nicotinic input, which coordinates ongoing burst firing. Large bursts preceded spontaneous and reflex-evoked contractions, suggesting their synaptic inputs may arise from enteric circuitry that also drives motility. Viscerofugal neurons were directly mechanosensitive to focal compression by von Frey hairs.

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...

OC-119 Mechanistic randomised control trial of mesalazine in symptomatic diverticular disease

IntroductionPainful symptomatic diverticular disease (SDD) is a common but poorly understood condition. Approximately 20% of patients with diverticulosis complain of pain, but there are currently no effective treatments. We have...

489 Endogenous H2S Produced in Prevertebral Sympathetic Ganglia Predominantly by Cse in Neurons and Glia Cells Modulates Central Cholinergic Synaptic Input

Hydrogen sulfide(H2S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia. Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H2S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs). In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H2S on central sympathetic input. Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice. Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals. Microelectrodes were used for intracellular recordings. Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR. CBS-IR was found only in glia cells. To determine whether the F-EPSPs could be potentiated by endogenously released H2S, we tested the effect of inhibiting H2S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs. Stigmatellin(1μM) significantly(P<0.05) increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms●mV with stigmatellin vs. 18.4±5.3mV and 338±108ms●mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation. In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05) decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms●mV with PAG vs. 27.0±4.1mV and 1230±414ms●mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation. Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mV and 651±455ms●mV with AOAA vs. 20.2±10.9mV and 438±318ms●mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms●mV with AOAA vs. 28.9±8.1mV and 1084±402ms●mV before AOAA, n=6, P<0.05). Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA. Conclusion: We conclude that endogenously produced H2S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner. H2S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input. (Funded by NIH DK17238)

490 Studies of the Psychophysiological Markers and the Brain Processing of Nausea in Healthy Humans Using a Novel Virtual Reality Video

Hydrogen sulfide(H2S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia. Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H2S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs). In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H2S on central sympathetic input. Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice. Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals. Microelectrodes were used for intracellular recordings. Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR. CBS-IR was found only in glia cells. To determine whether the F-EPSPs could be potentiated by endogenously released H2S, we tested the effect of inhibiting H2S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs. Stigmatellin(1μM) significantly(P<0.05) increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms●mV with stigmatellin vs. 18.4±5.3mV and 338±108ms●mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation. In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05) decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms●mV with PAG vs. 27.0±4.1mV and 1230±414ms●mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation. Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mV and 651±455ms●mV with AOAA vs. 20.2±10.9mV and 438±318ms●mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms●mV with AOAA vs. 28.9±8.1mV and 1084±402ms●mV before AOAA, n=6, P<0.05). Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA. Conclusion: We conclude that endogenously produced H2S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner. H2S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input. (Funded by NIH DK17238)

Mo1860 Identification of Kappa Opioid Receptors on Colonic Sensory Nerves and Their Up-Regulation in a Mouse Model of Chronic Visceral Hypersensitivity

Background: Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract. This relapsing condition is often associated with debilitating abdominal pain, which in some cases can persist throughout quiescent stages of the disease. The regulatory mechanisms involved in central sensitization and peripheral neuroplasticity in the context of IBD-related visceral pain remains elusive. A growing body of evidence suggests a role for the transient receptor potential vanilloid 1 (TRPV1) in visceral hypersensitivity. TRPV1 is expressed in sensory dorsal root ganglion (DRG) neurons and is directly involved in nociception. Previous studies have demonstrated that TRPV1-deficient mice experience reduced thermal hyperalgesia during inflammation, thus suggesting a role for TRPV1 in inflammatory pain. The objective of the present study was therefore to investigate the role of TRPV1 in visceral pain in the dextran sulfate sodium (DSS) murine model of colitis. Methods: Mice were given DSS in drinking water for 7 days, after which spontaneous visceral pain was assessed using the non-invasive, animal behavior recognition system LABORAS. Control and DSS-treated mice were then sacrificed and colon-innervating DRGs collected for quantitative PCR, immunostaining, and neuronal dissociation. In Vitro studies were performed with human neuroblastoma NG108 cells. Results: DSS-treated mice showed reduced mobility and frequency of grooming and climbing, which likely reflect spontaneous visceral pain. Remarkably, while controls cells showed very few processes, dissociated DRG neurons isolated from DSS-treated mice displayed extensive neurite outgrowth after 24 h in culture. Immunocytochemistry later revealed TRPV1 expression along the entire length of these axons. These results are supported by In Vitro data demonstrating that exposure to a mixture of proinflammatory cytokines (bradykinin, histamine, and serotonin) induces neurite outgrowth and TRPV1 membrane trafficking in TRPV1-transfected neuronal NG108 cells. Finally, PCR analysis demonstrated that the neuronal morphological changes observed in DSS-treated animals were associated with a significant decrease in the expression of the small Rho GTPase RhoA and Rho kinase, both of which are known to modulate neurite outgrowth. Conclusion: These results demonstrate that DSS-colitis induces changes in neuronal morphology and modulation of TRPV1 trafficking, which may play a role in visceral hypersensitivity in the context of gastrointestinal inflammatory disorders. Current experiments using pharmacological inhibitors of the RhoA/Rho kinase signaling axis and TRPV1deficient animals will provide important insights into the exact role of TRPV1 in IBD-related visceral hypersensitivity and perhaps identify new targets for the treatment of chronic pain in these patients.

A Case of Triple Cancer of the Pancreas, Colon, and Peripheral Nerve Sheath in Type 2 Diabetes found by Progressive Body Weight Loss and Deteriorating Glycemic Control

A Case of Triple Cancer of the Pancreas, Colon, and Peripheral Nerve Sheath in Type 2 Diabetes found by Progressive Body Weight Loss and Deteriorating Glycemic Control

Relaxations mediated by adrenergic and non-adrenergic nerves in human isolated taenia coli.

In human taenia coli electrical field stimulation after cholinergic blockade excited mainly non-adrenergic inhibitory nerves. Adrenergic relaxations during stimulation were demonstrated only with short electrical pulses at high frequencies or with ganglion stimulants. Reduction by tetrodotoxin of relaxations to indirectly acting sympathomimetics supports the histochemical finding that colonic adrenergic nerves are mainly preganglionic, and possibly synapse with non-adrenergic inhibitory nerves. When the latter undergo direct maximal stimulation, responses to adrenergic nerve excitation are masked.

The influence of ileitis on the neurochemistry of the caudal mesenteric ganglion in the pig

Some literature data suggest that there is a regulatory neuronal circuit between the small and the large bowel. To verify this hypothesis the present study investigated: (i) the distribution, chemical coding and routing of caudal mesenteric ganglion (CaMG) neurons participating in an intestinointestinal reflex pathway involving ileal descending neurons and viscerofugal colonic neurons and (ii) possible changes in the neuroarchitecture of this pathway evoked by chemically induced ileitis in juvenile pigs (n=16). Combined retrograde tract tracing and transections of the intermesenteric or caudal colonic nerves were applied. In addition, double immunostainings was used to investigate the chemical coding of retrogradely labeled CaMG neurons and intraganglionic nerve terminals apposed to them, under normal and inflammatory conditions. The majority of the ileum-projecting neurons were found in the caudal part of CaMG. Disruption of particular nerve pathways resulted in diminished number of retrogradely labeled neurons, ipsilateral to the side of manipulation. In normal pigs, ileum-projecting CaMG neurons stained for tyrosine hydroxylase, dopamine-β-hydroxylase, neuropeptide Y (NPY), somatostatin and galanin (GAL). The number and chemical coding of the neurons in the inflamed animals were similar to those observed in the normal pigs. However, in the inflamed pigs, the number of NPY-, GAL- or substance P-positive nerve terminals supplying retrogradely labeled neurons was increased. The present results suggest that inflammatory processes of the porcine ileum are able to induce changes in the intraganglionic architecture of a sympathetic ganglion located at discrete distance from the affected bowel segment.

Identification of the Visceral Pain Pathway Activated by Noxious Colorectal Distension in Mice

In patients with irritable bowel syndrome, visceral pain is evoked more readily following distension of the colorectum. However, the identity of extrinsic afferent nerve pathway that detects and transmits visceral pain from the colorectum to the spinal cord is unclear. In this study, we identified which extrinsic nerve pathway(s) underlies nociception from the colorectum to the spinal cord of rodents. Electromyogram recordings were made from the transverse oblique abdominal muscles in anesthetized wild type (C57BL/6) mice and acute noxious intraluminal distension stimuli (100–120 mmHg) were applied to the terminal 15 mm of colorectum to activate visceromotor responses (VMRs). Lesioning the lumbar colonic nerves in vivo had no detectable effect on the VMRs evoked by colorectal distension. Also, lesions applied to the right or left hypogastric nerves failed to reduce VMRs. However, lesions applied to both left and right branches of the rectal nerves abolished VMRs, regardless of whether the lumbar colonic or hypogastric nerves were severed. Electrical stimulation applied to either the lumbar colonic or hypogastric nerves in vivo, failed to elicit a VMR. In contrast, electrical stimulation (2–5 Hz, 0.4 ms, 60 V) applied to the rectum reliably elicited VMRs, which were abolished by selective lesioning of the rectal nerves. DiI retrograde labeling from the colorectum (injection sites 9–15 mm from the anus, measured in unstretched preparations) labeled sensory neurons primarily in dorsal root ganglia (DRG) of the lumbosacral region of the spinal cord (L6-S1). In contrast, injection of DiI into the mid to proximal colon (injection sites 30–75 mm from the anus, measured in unstretched preparations) labeled sensory neurons in DRG primarily of the lower thoracic level (T6-L2) of the spinal cord. The visceral pain pathway activated by acute noxious distension of the terminal 15 mm of mouse colorectum is transmitted predominantly, if not solely, through rectal/pelvic afferent nerve fibers to the spinal cord. The sensory neurons of this spinal afferent pathway lie primarily in the lumbosacral region of the spinal cord, between L6 and S1.

Analysis of Autonomic Nerve Preservation and Pouch Reconstruction Influencing Fragmentation of Defecation after Sphincter-Preserving Surgery for Rectal Cancer

Our questionnaire survey on defecation disorders after rectal cancer surgery revealed that 66.7% of postoperative patients were most annoyed with fragmentation of defecation. Therefore, we performed a change-over-time analysis on the relationship of fragmentation and factors including location of rectal cancer, surgical technique, anastomosis method, pouch reconstruction, extent of lymph node dissection, and degree of pelvic and colonic nerve preservation surrounding the superior mesenteric artery. The fragmentation decreased over time at the postoperative time points of 6 months, 2 and 5 years. A statistical analysis of factors influencing fragmentation revealed that location of cancer, reconstruction technique, anastomosis method and degree of pelvic nerve preservation were significant factors for the entire patient population and that colonic nerve preservation was a significant factor 5 years after surgery. Analysis of patients with lower rectal cancer only showed that in addition to surgical technique and anastomosis method, pouch reconstruction was effective and autonomic nerve preservation was effective 5 years after surgery. As a result, when the anastomotic site was closer to the anus, the frequency of fragmentation increased; we concluded that pouch reconstruction was an effective surgical technique and colonic nerve preservation was effective in the longer term.

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.

S1799 Specialized Spinal Afferent Neurons to Detect Mucosal Shear in Guinea-Pig Colon

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

S1800 Characterization of a Reproducible Gastric Pain Model Using Oral Capsaicin Titration: A Placebo-Controlled, Double-Blind Study

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

S1803 Early Life Esophageal Acid Exposure Accentuates the Expression of Nr1 and PSD-95 in ACC Neurons in Response to Acid Rechallenge in Adulthood

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

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