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Isolectin B4-binding (IB4+) dorsal root ganglion (DRG) neurons are distinct from peptidergic DRG neurons in their terminal location in the spinal cord and respective contributions to various classes and modalities of nociception. In DRG neurons innervating the mouse colon (c-DRG neurons), the reported proportion of IB4+ population is inconsistent across studies, and little is known regarding their role in colorectal mechanonociception. To address these issues, in C57BL/6J mice, we quantified IB4+ binding after labeling c-DRG neurons with Fast Blue and examined functional consequences of ablating these neurons by IB4-conjugated saporin. Sixty-one percent of Fast Blue-labeled neurons in the L6 DRG were IB4+, and 95% of these IB4+ c-DRG neurons were peptidergic. Intrathecal administration of IB4-conjugated saporin reduced the proportion of IB4+ c-DRG neurons to 37%, which was due to the loss of c-DRG neurons showing strong to medium IB4+ intensity; c-DRG neurons with weak IB4+ intensity were spared. However, this loss altered neither nociceptive behaviors to colorectal distension nor the relative proportions of stretch-sensitive colorectal afferent classes characterized by single-fiber recordings. These findings demonstrate that more than 1 half of viscerosensory L6 c-DRG neurons in C57BL/6J mouse are IB4+ and suggest, in contrast to the reported roles of IB4+/nonpeptidergic neurons in cutaneous mechanonociception, c-DRG neurons with strong-to-medium IB4+ intensity do not play a significant role in colorectal mechanonociception.

Stretch-sensitive afferents comprise ∼33% of the pelvic nerve innervation of mouse colorectum, which are activated by colorectal distension and encode visceral nociception. Stretch-sensitive colorectal afferent endings respond tonically to stepped or ramped colorectal stretch, whereas dissociated colorectal dorsal root ganglion neurons generally fail to spike repetitively upon stepped current stimulation. The present study investigated this difference in the neural encoding characteristics between the soma and afferent ending using pharmacological approaches in an in vitro mouse colon-nerve preparation and complementary computational simulations. Immunohistological staining and Western blots revealed the presence of voltage-gated sodium channel (NaV) 1.6 and NaV1.7 at sensory neuronal endings in mouse colorectal tissue. Responses of stretch-sensitive colorectal afferent endings were significantly reduced by targeting NaV1.6 using selective antagonists (μ-conotoxin GIIIa and μ-conotoxin PIIIa) or tetrodotoxin. In contrast, neither selective NaV1.8 (A803467) nor NaV1.7 (ProTX-II) antagonists attenuated afferent responses to stretch. Computational simulation of a colorectal afferent ending that incorporated independent Markov models for NaV1.6 and NaV1.7, respectively, recapitulated the experimental findings, suggesting a necessary role for NaV1.6 in encoding tonic spiking by stretch-sensitive afferents. In addition, computational simulation of a dorsal root ganglion soma showed that, by adding a NaV1.6 conductance, a single-spiking neuron was converted into a tonic spiking one. These results suggest a mechanism/channel to explain the difference in neural encoding characteristics between afferent somata and sensory endings, likely caused by differential expression of ion channels (e.g., NaV1.6) at different parts of the neuron., (Copyright © 2015 the American Physiological Society.)

Background: A low-level inflammation has been hypothesized to mediate visceral hypersensitivity in functional bowel disorders that persist after or even in the absence of gut inflammation. We aimed to test the efficacy of a steroidal anti-inflammatory treatment, and identify local inflammatory molecules mediating post- and non-inflammatory colorectal hypersensitivity using two mouse models., Methods: Visceromotor responses to colorectal distension were quantified as a measure of colorectal sensitivity. On day 1, mice received intracolonic saline (control), trinitrobenzenesulfonic acid (postinflammatory on day 15), or acidified hypertonic saline (non-inflammatory). Colorectal sensitivity before (day 10) and after (day 15) 4-day dexamethasone (Dex) treatment was compared, and colonic gene expression of inflammatory molecules was quantified., Key Results: Dexamethasone effectively inhibited gene expression of inflammatory molecules such as interleukin (IL)-1β and mast cell protease-1 in the colon, but did not attenuate colorectal hypersensitivity in either model. Gene expression of inflammatory molecules in the colon did not differ between control and the non-inflammatory model, but the postinflammatory model showed increased IL-10 and tight junction protein 2, and decreased IL-6, transforming growth factor (TGF)-β, a precursor of β-endorphin, occludin, and mucin 2. While no common molecule explained colorectal hypersensitivity in these models, hypersensitivity was positively correlated with TGF-β2 mRNA in control, and with IL-1β, inhibin βA, and prostaglandin E2 synthase in the Dex-treated postinflammatory model. In the non-inflammatory model, cyclooxygenase-2 mRNA was negatively correlated with colorectal sensitivity., Conclusions & Inferences: These results suggest that persistent functional colorectal hypersensitivity is mediated by condition-specific mediators whose gene expression in the colon is not inevitably sensitive to steroidal anti-inflammatory treatment., (© 2014 John Wiley & Sons Ltd.)

Background: Anterograde in vitro tracing of the pelvic nerve (PN) and visualization in the horizontal plane in whole mount preparations has been fundamental in the analysis of distribution of peripheral nerves innervating the colorectum. Here, we performed a similar analysis, but in cryostat sections of the mouse colorectum, allowing for a more direct visualization of nerve distribution in all tissue layers., Methods: Colorectum with attached PNs was dissected from adult male BalbC mice. Presence of active afferents was certified by single fiber recording of fine PN fibers. This was followed by 'bulk' (all fibers) anterograde tracing using biotinamide (BTA). Histo- and immunohistochemical techniques were used for visualization of BTA-positive nerves, and evaluation of co-localization with calcitonin gene-related peptide (CGRP), respectively. Tissue was analyzed using confocal microscopy on transverse or longitudinal colorectum sections., Key Results: Abundant BTA-positive nerves spanning all layers of the mouse colorectum and contacting myenteric plexus neurons, distributing within the muscle layer, penetrating deeper into the organ and contacting blood vessels, submucosal plexus neurons or even penetrating the mucosa, were regularly detected. Several traced axons co-localized CGRP, supporting their afferent nature. Finally, anterograde tracing of the PN also exposed abundant BTA-positive nerves in the major pelvic ganglion., Conclusions & Inferences: We present the patterns of innervation of extrinsic axons across layers in the mouse colorectum, including the labile mucosal layer. The proposed approach could also be useful in the analysis of associations between morphology and physiology of peripheral nerves targeting the different layers of the colorectum., (© 2014 John Wiley & Sons Ltd.)

Irritable bowel syndrome (IBS) is characterized by altered bowel habits, persistent pain and discomfort, and typically colorectal hypersensitivity. Linaclotide, a peripherally restricted 14 aa peptide approved for the treatment of IBS with constipation, relieves constipation and reduces IBS-associated pain in these patients presumably by activation of guanylate cyclase-C (GC-C), which stimulates production and release of cyclic guanosine monophosphate (cGMP) from intestinal epithelial cells. We investigated whether activation of GC-C by the endogenous agonist uroguanylin or the primary downstream effector of that activation, cGMP, directly modulates responses and sensitization of mechanosensitive colorectal primary afferents. The distal 2 cm of mouse colorectum with attached pelvic nerve was harvested and pinned flat mucosal side up for in vitro single-fiber recordings, and the encoding properties of mechanosensitive afferents (serosal, mucosal, muscular, and muscular-mucosal; M/M) to probing and circumferential stretch studied. Both cGMP (10-300 μM) and uroguanylin (1-1000 nM) applied directly to colorectal receptive endings significantly reduced responses of muscular and M/M afferents to stretch; serosal and mucosal afferents were not affected. Sensitized responses (i.e., increased responses to stretch) of muscular and M/M afferents were reversed by cGMP, returning responses to stretch to control. Blocking the transport of cGMP from colorectal epithelia by probenecid, a mechanism validated by studies in cultured intestinal T84 cells, abolished the inhibitory effect of uroguanylin on M/M afferents. These results suggest that GC-C agonists like linaclotide alleviate colorectal pain and hypersensitivity by dampening stretch-sensitive afferent mechanosensitivity and normalizing afferent sensitization.

Previous studies in rat and mouse documented that a subpopulation of dorsal root ganglion (DRG) neurons innervating non-visceral tissues express tyrosine hydroxylase (TH). Here we studied whether or not mouse DRG neurons retrogradely traced with Fast Blue (FB) from colorectum or urinary bladder also express immunohistochemically detectable TH. The lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) were included in the analysis. Previously characterized antibodies against TH, norepinephrine transporter type 1 (NET-1) and calcitonin gene-related peptide (CGRP) were used. On average, ∼14% of colorectal and ∼17% of urinary bladder DRG neurons expressed TH and spanned virtually all neuronal sizes, although more often in the medium-sized to small ranges. Also, they were more abundant in lumbosacral than thoracolumbar DRGs, and often coexpressed CGRP. We also detected several TH-immunoreactive (IR) colorectal and urinary bladder neurons in the LSC and the MPG, more frequently in the former. No NET-1-IR neurons were detected in DRGs, whereas the majority of FB-labeled, TH-IR neurons in the LSC and MPG coexpressed this marker (as did most other TH-IR neurons not labeled from the target organs). TH-IR nerve fibers were detected in all layers of the colorectum and the urinary bladder, with some also reaching the basal mucosal cells. Most TH-IR fibers in these organs lacked CGRP. Taken together, we show: (1) that a previously undescribed population of colorectal and urinary bladder DRG neurons expresses TH, often CGRP but not NET-1, suggesting the absence of a noradrenergic phenotype; and (2) that TH-IR axons/terminals in the colon or urinary bladder, naturally expected to derive from autonomic sources, could also originate from sensory neurons., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Carbohydrate malabsorption such as in lactose intolerance or enteric infection causes symptoms that include abdominal pain. Because this digestive disorder increases intracolonic osmolarity and acidity by accumulation of undigested carbohydrates and fermented products, we tested whether these two factors (hypertonicity and acidity) would modulate colorectal afferents in association with colorectal nociception and hypersensitivity. In mouse colorectum-pelvic nerve preparations in vitro, afferent activities were monitored after application of acidic hypertonic saline (AHS; pH 6.0, 800 mosM). In other experiments, AHS was instilled intracolonically to mice and behavioral responses to colorectal distension (CRD) measured. Application of AHS in vitro excited 80% of serosal and 42% of mechanically-insensitive colorectal afferents (MIAs), sensitizing a proportion of MIAs to become mechanically sensitive and reversibly inhibiting stretch-sensitive afferents. Acute intracolonic AHS significantly increased expression of the neuronal activation marker pERK in colon sensory neurons and augmented noxious CRD-induced behavioral responses. After three consecutive daily intracolonic AHS treatments, mice were hypersensitive to CRD 4-15 days after the first treatment. In complementary single fiber recordings in vitro, the proportion of serosal class afferents increased at day 4; the proportion of MIAs decreased, and muscular class stretch-sensitive afferents were sensitized at days 11-15 in mice receiving AHS. These results indicate that luminal hypertonicity and acidity, two outcomes of carbohydrate malabsorption, can induce colorectal hypersensitivity to distension by altering the excitability and relative proportions of colorectal afferents, suggesting the potential involvement of these factors in the development of abdominal pain.

Inflammation of the distal bowel is often associated with abdominal pain and hypersensitivity, but whether and which colorectal afferents contribute to the hypersensitivity is unknown. Using a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, we investigated colorectal hypersensitivity following intracolonic TNBS and associated changes in colorectum and afferent functions. C57BL/6 mice were treated intracolonically with TNBS or saline. Visceromotor responses to colorectal distension (15-60 mmHg) were recorded over 8 wk in TNBS- and saline-treated (control) mice. In other mice treated with TNBS or saline, colorectal inflammation was assessed by myeloperoxidase assay and immunohistological staining. In vitro single-fiber recordings were conducted on both TNBS and saline-treated mice to assess colorectal afferent function. Mice exhibited significant colorectal hypersensitivity through day 14 after TNBS treatment that resolved by day 28 with no resensitization through day 56. TNBS induced a neutrophil- and macrophage-based colorectal inflammation as well as loss of nerve fibers, all of which resolved by days 14-28. Single-fiber recordings revealed a net increase in afferent drive from stretch-sensitive colorectal afferents at day 14 post-TNBS and reduced proportions of mechanically insensitive afferents (MIAs) at days 14-28. Intracolonic TNBS-induced colorectal inflammation was associated with the development and recovery of hypersensitivity in mice, which correlated with a transient increase and recovery of sensitization of stretch-sensitive colorectal afferents and MIAs. These results indicate that the development and maintenance of colorectal hypersensitivity following inflammation are mediated by peripheral drive from stretch-sensitive colorectal afferents and a potential contribution from MIAs.

Afferent input contributes significantly to the pain and colorectal hypersensitivity that characterize irritable bowel syndrome. In the present study, we investigated the contributions of mechanically sensitive and mechanically insensitive afferents (MIAs; or silent afferents) to colorectal hypersensitivity. The visceromotor response to colorectal distension (CRD; 15-60 mmHg) was recorded in mice before and for weeks after intracolonic treatment with zymosan or saline. After CRD tests, the distal colorectum with the pelvic nerve attached was removed for single-fiber electrophysiological recordings. Colorectal afferent endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. Intracolonic zymosan produced persistent colorectal hypersensitivity (>24 days) associated with brief colorectal inflammation. Pelvic nerve muscular-mucosal but not muscular mechanosensitive afferents recorded from mice with colorectal hypersensitivity exhibited persistent sensitization. In addition, the proportion of MIAs (relative to control) was significantly reduced from 27% to 13%, whereas the proportion of serosal afferents was significantly increased from 34% to 53%, suggesting that MIAs acquired mechanosensitivity. PGP9.5 immunostaining revealed no significant loss of colorectal nerve fiber density, suggesting that the reduction in MIAs is not due to peripheral fiber loss after intracolonic zymosan. These results indicate that colorectal MIAs and sensitized muscular-mucosal afferents that respond to stretch contribute significantly to the afferent input that sustains hypersensitivity to CRD, suggesting that targeted management of colorectal afferent input could significantly reduce patients' complaints of pain and hypersensitivity.

Vesicular glutamate transporters (VGLUTs) have been extensively studied in various neuronal systems, but their expression in visceral sensory and autonomic neurons remains to be analyzed in detail. Here we studied VGLUTs type 1 and 2 (VGLUT(1) and VGLUT(2) , respectively) in neurons innervating the mouse colorectum. Lumbosacral and thoracolumbar dorsal root ganglion (DRG), lumbar sympathetic chain (LSC), and major pelvic ganglion (MPG) neurons innervating the colorectum of BALB/C mice were retrogradely traced with Fast Blue, dissected, and processed for immunohistochemistry. Tissue from additional naïve mice was included. Previously characterized antibodies against VGLUT(1) , VGLUT(2) , and calcitonin gene-related peptide (CGRP) were used. Riboprobe in situ hybridization, using probes against VGLUT(1) and VGLUT(2) , was also performed. Most colorectal DRG neurons expressed VGLUT(2) and often colocalized with CGRP. A smaller percentage of neurons expressed VGLUT(1) . VGLUT(2) -immunoreactive (IR) neurons in the MPG were rare. Abundant VGLUT(2) -IR nerves were detected in all layers of the colorectum; VGLUT(1) -IR nerves were sparse. A subpopulation of myenteric plexus neurons expressed VGLUT2 protein and mRNA, but VGLUT1 mRNA was undetectable. In conclusion, we show 1) that most colorectal DRG neurons express VGLUT(2) , and to a lesser extent, VGLUT(1) ; 2) abundance of VGLUT2-IR fibers innervating colorectum; and 3) a subpopulation of myenteric plexus neurons expressing VGLUT(2). Altogether, our data suggests a role for VGLUT(2) in colorectal glutamatergic neurotransmission, potentially influencing colorectal sensitivity and motility., (Copyright © 2011 Wiley-Liss, Inc.)

The bladder and distal colon are innervated by lumbar splanchnic (LSN) and pelvic nerves (PN) whose axons arise from dorsal root ganglia (DRG) neurons at thoracolumbar (TL) and lumbosacral (LS) spinal levels, respectively. In an attempt to understand the molecular basis of differences between LSN and PN mechanosensitive afferents, we analyzed the gene expression of two potentially counteracting ion channel groups involved in mechanosensation, transient receptor potential channels (TRPV1 and TRPA1) and mechanosensitive two pore-domain K(+) (K(2P)) channels (TREK-1, TREK-2 and TRAAK), in TL and LS DRG neurons innervating mouse bladder or distal colon. The proportion of TRPV1-expressing cells (41∼61%) did not differ between TL and LS neurons innervating bladder or colon. TRPA1 was seldom detected in bladder LS neurons whereas it was expressed in 64∼66% of bladder TL, colon TL and colon LS neurons. Coexpression of TRPV1 and TRPA1 was frequent. TREK-1-expressing cells were more prevalent in LS than TL ganglia in both bladder- and colon-DRG neurons. All three K(2P) channels were detected more frequently in TRPV1-positive neurons in TL ganglia. More than half of TL neurons expressing only TRPA1 were devoid of any of the three K(2P) channels, whereas all TL neurons expressing both TRPA1 and TRPV1 expressed at least one of the K(2P) channels. These results reveal clear differences between LSN and PN sensory pathways in TRPA1 and TREK-1 gene expression and in the gene expression of K(2P) channels in TRPV1-expressing neurons. This study further documents heterogeneity of visceral afferents based on combinations of the five channels examined., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

TREK-1, TREK-2 and TRAAK are mechanosensitive two-pore domain K(+) (K(2P)) channels thought to be involved in the attenuation of mechanotransduction. Because colon inflammation is associated with colon mechanohypersensitivity, we hypothesized that the role of these channels in colon sensory (dorsal root ganglion, DRG) neurons would be reduced by colon inflammation. Accordingly, we studied the functional expression of mechanosensitive K(2P) channels in colon sensory neurons in both thoracolumbar (TL) and lumbosacral (LS) DRG that represent the splanchnic and pelvic nerve innervations of the colon, respectively. In colon DRG neurons identified by retrograde tracer previously injected into the colon wall, 62% of TL neurons and 83% of LS neurons expressed at least one of three K(2P) channel mRNAs; the proportion of neurons expressing the TREK-1 gene was greater in LS than in TL DRG. In electrophysiological studies, single-channel activities of TREK-1a, TREK-1b, TREK-2, and TRAAK-like channels were detected in cultured colon DRG neuronal membranes. After trinitrobenzene sulfonic acid-induced colon inflammation, we observed significant decreases in the amount of TREK-1 mRNA, in the response of TREK-2-like channels to membrane stretch, and in the whole cell outward current during osmotic stretch in LS colon DRG neurons. These findings document that the majority of DRG neurons innervating the mouse colon express mechanosensitive K(2P) channels and suggest that a decrease in their expression and activities contributes to the increased colon mechanosensitivity that develops in inflammatory bowel conditions.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by pain and hypersensitivity in the relative absence of colon inflammation or structural changes. To assess the role of P2X receptors expressed in colorectal dorsal root ganglion (c-DRG) neurons and colon hypersensitivity, we studied excitability and purinergic signaling of retrogradely labeled mouse thoracolumbar (TL) and lumbosacral (LS) c-DRG neurons after intracolonic treatment with saline or zymosan (which reproduces 2 major features of IBS-persistent colorectal hypersensitivity without inflammation) using patch-clamp, immunohistochemical, and RT-PCR techniques. Although whole cell capacitances did not differ between LS and TL c-DRG neurons and were not changed after zymosan treatment, membrane excitability was increased in LS and TL c-DRG neurons from zymosan-treated mice. Purinergic agonist adenosine-5'-triphosphate (ATP) and α,β-methylene ATP [α,β-meATP] produced inward currents in TL c-DRG neurons were predominantly P2X(3)-like fast (∼70% of responsive neurons); P2X(2/3)-like slow currents were more common in LS c-DRG neurons (∼35% of responsive neurons). Transient currents were not produced by either agonist in c-DRG neurons from P2X(3)(-/-) mice. Neither total whole cell Kv current density nor the sustained or transient Kv components was changed in c-DRG neurons after zymosan treatment. The number of cells expressing P2X(3) protein and its mRNA and the kinetic properties of ATP- and α,β-meATP-evoked currents in c-DRG neurons were not changed by zymosan treatment. However, the EC(50) of α,β-meATP for the fast current decreased significantly in TL c-DRG neurons. These findings suggest that colorectal hypersensitivity produced by intracolonic zymosan increases excitability and enhances purinergic signaling in c-DRG neurons.

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4 % acetic acid in the rat. Animals were sacrificed at 4 h and 48 h postacetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nomega-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acidtreated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.

Aim: Irritable bowel syndrome (IBS) is a functional bowel disorder. Its major symptom is bowel dysmotility, yet the mechanism of the symptom is poorly understood. Since the neurokinin-1 receptor (NK1R)-mediated signaling in the gut is important in the control of normal bowel motor function, we aimed to investigate whether the NK1R-mediated bowel motor function was altered in IBS, using a rat IBS model that was previously reported to show colonic dysmotility in response to restraint stress., Methods: IBS symptoms were produced in male Sprague-Dawley rats by inducing colitis with acetic acid. Rats were left to recover from colitis for 6 d, and used for experiments 7 d post-induction of colitis. Motor activities of distal colon were recorded in vitro., Results: The contractile sensitivity of isolated colon to a NK1R agonist (Sar9,Met(O2)11)-substance P (1-30 nmol/L) was higher in IBS rats than that in normal rats. After the enteric neurotransmission was blocked by tetrodotoxin (TTX, 1 micromol/L), the contractile sensitivity to the NK1R agonist was increased in normal colon but not in IBS rat colon. The NK1R agonist-induced contraction was not different between the two groups when the agonist was challenged to the TTX-treated colon or the isolated colonic myocytes. A nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L) augmented the NK1R agonist-induced contraction only in normal rat colon., Conclusion: These results suggest that the NK1R-meidated colonic motor response is increased in IBS rats, due to the decrease in the nitrergic inhibitory neural component.