Sensory and motor innervation of the crural diaphragm by the vagus nerves.

BACKGROUND & AIMS: During gastroesophageal reflux, transient lower esophageal sphincter relaxation and crural diaphragm (CD) inhibition occur concomitantly. Modifying vagus nerve control of transient lower esophageal sphincter relaxation is a major focus of development of therapeutics for gastroesophageal reflux disease, but neural mechanisms that coordinate the CD are poorly understood. METHODS: Nerve tracing and immunolabeling were used to assess innervation of the diaphragm and lower esophageal sphincter in ferrets. Mechanosensory responses of vagal afferents in the CD and electromyography responses of the CD were recorded in novel in vitro preparations and in vivo. RESULTS: Retrograde tracing revealed a unique population of vagal CD sensory neurons in nodose ganglia and CD motor neurons in brainstem vagal nuclei. Anterograde tracing revealed specialized vagal endings in the CD and phrenoesophageal ligament-sites of vagal afferent mechanosensitivity recorded in vitro. Spontaneous electromyography activity persisted in the CD following bilateral phrenicotomy in vivo, while vagus nerve stimulation evoked electromyography responses in the CD in vitro and in vivo. CONCLUSIONS: We conclude that vagal sensory and motor neurons functionally innervate the CD and phrenoesophageal ligament. CD vagal afferents show mechanosensitivity to distortion of the gastroesophageal junction, while vagal motor neurons innervate both CD and distal esophagus and may represent a common substrate for motor control of the reflux barrier.

Nitric oxide as an endogenous peripheral modulator of visceral sensory neuronal function.

Nitric oxide (NO) plays important roles in CNS and smooth muscle function. Here we reveal an additional function in peripheral sensory transmission. We hypothesized that endogenous NO modulates the function of gastrointestinal vagal afferent endings. The nonselective NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride increased responses to tactile mechanical stimuli of mucosal afferent endings in two species, in some cases severalfold. This was mimicked by a neuronal NOS inhibitor but not an endothelial NOS inhibitor. NOS inhibitors did not affect the responsiveness of smooth muscle afferent endings, suggesting that the endogenous source of NO is exclusively accessible to mucosal receptors. The role of the NO-soluble guanylyl cyclase (sGC)-cGMP pathway was confirmed using the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one and the cGMP phosphodiesterase 5' inhibitor sildenafil. The first enhanced and the second inhibited mechanosensory function. Exogenous NO, from the donor S-nitroso-N-acetylpenicillamine, significantly reduced mechanosensitivity of both types of ending. Up to one-third of stomach-projecting afferent neurons in the nodose ganglia expressed neuronal NOS (nNOS). However, anterograde-traced vagal endings were nNOS negative, indicating NOS is not transported peripherally and there are alternative sources of NO for afferent modulation. A subpopulation of enteroendocrine cells in the gut mucosa were nNOS positive, which were found anatomically in close apposition with mucosal vagal afferent endings. These results indicate an inhibitory neuromodulatory role of epithelial NO, which targets a select population of vagal afferents. This interaction is likely to play a role in generation of symptoms and behaviors from the upper gastrointestinal system.

The ion channel TRPA1 is required for normal mechanosensation and is modulated by algesic stimuli.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These findings demonstrate a previously unrecognized role for TRPA1 in normal and inflamed mechanosensory function and nociception within the viscera.

Selective role for TRPV4 ion channels in visceral sensory pathways.

BACKGROUND & AIMS: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Anatomy and function of group III metabotropic glutamate receptors in gastric vagal pathways.

Metabotropic glutamate receptors (mGluR) are classified into groups I (excitatory), II and III (inhibitory) mGluR. Activation of peripheral group III mGluR (mGluR4, mGluR6, mGluR7, mGluR8), particularly mGluR8, inhibits vagal afferent mechanosensitivity in vitro which translates into reduced triggering of transient lower oesophageal sphincter relaxations and gastroesophageal reflux in vivo. However, the expression and function of group III mGluR in central gastrointestinal vagal reflex pathways is not known. Here we assessed the expression of group III mGluR in identified gastric vagal afferents in the nodose ganglion (NG) and in the dorsal medulla. We also determined the central action of the mGluR8a agonist S-3,4-DCPG (DCPG) on nucleus tractus solitarius (NTS) neurons with gastric mechanosensory input in vivo. Labelling for mGluR4 and mGluR8 was abundant in gastric vagal afferents in the NG, at their termination site in the NTS (subnucleus gelatinosus) and in gastric vagal motorneurons, while labelling for mGluR6 and mGluR7 was weaker in these regions. DCPG (0.1 nmol or 0.001-10 nmol i.c.v.) inhibited or markedly attenuated responses of 8/10 NTS neurons excited by isobaric gastric distension with no effect on blood pressure or respiration; 2 NTS neurons were unaffected. The effects of DCPG were significantly reversed by the group III mGluR antagonist MAP4 (10 nmol, i.c.v.). In contrast, 4/4 NTS neurons inhibited by gastric distension were unaffected by DCPG. We conclude that group III mGluR are expressed in peripheral and central vagal pathways, and that mGluR8 within the NTS selectively reduce excitatory transmission along gastric vagal pathways.

Peripheral versus central modulation of gastric vagal pathways by metabotropic glutamate receptor 5.

Metabotropic glutamate receptors (mGluR) are classified into group I, II, and III mGluR. Group I (mGluR1, mGluR5) are excitatory, whereas group II and III are inhibitory. mGluR5 antagonism potently reduces triggering of transient lower esophageal sphincter relaxations and gastroesophageal reflux. Transient lower esophageal sphincter relaxations are mediated via a vagal pathway and initiated by distension of the proximal stomach. Here, we determined the site of action of mGluR5 in gastric vagal pathways by investigating peripheral responses of ferret gastroesophageal vagal afferents to graded mechanical stimuli in vitro and central responses of nucleus tractus solitarius (NTS) neurons with gastric input in vivo in the presence or absence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). mGluR5 were also identified immunohistochemically in the nodose ganglia and NTS after extrinsic vagal inputs had been traced from the proximal stomach. Gastroesophageal vagal afferents were classified as mucosal, tension, or tension-mucosal (TM) receptors. MPEP (1-10 microM) inhibited responses to circumferential tension of tension and TM receptors. Responses to mucosal stroking of mucosal and TM receptors were unaffected. MPEP (0.001-10 nmol icv) had no major effect on the majority of NTS neurons excited by gastric distension or on NTS neurons inhibited by distension. mGluR5 labeling was abundant in gastric vagal afferent neurons and sparse in fibers within NTS vagal subnuclei. We conclude that mGluR5 play a prominent role at gastroesophageal vagal afferent endings but a minor role in central gastric vagal pathways. Peripheral mGluR5 may prove a suitable target for reducing mechanosensory input from the periphery, for therapeutic benefit.

Phenotypic characterization of taste cells of the mouse small intestine.

Nutrient-evoked gastrointestinal reflexes are likely initiated by specialized epithelial cells located in the small intestine that detect luminal stimuli and release mediators that activate vagal endings. The G-protein alpha-gustducin, a key signal molecule in lingual taste detection, has been identified in mouse small intestine, where it may also subserve nutrient detection; however, the phenotype of alpha-gustducin cells is unknown. Immunohistochemistry was performed throughout the mouse small intestine for alpha-gustducin, enteroendocrine cell markers 5-HT and glucagon-like peptide-1 (GLP-1), and brush cell markers neuronal nitric oxide synthase and Ulex europaeus agglutinin-1 (UEA-1) lectin binding, singly, and in combination. alpha-Gustducin was expressed in solitary epithelial cells of the mid to upper villus, which were distributed in a regional manner with most occurring within the midjejunum. Here, 27% of alpha-gustducin cells colabeled for 5-HT and 15% for GLP-1; 57% of alpha-gustducin cells colabeled UEA-1, with no triple labeling. alpha-Gustducin cells that colabeled for 5-HT or GLP-1 were of distinct morphology and exhibited a different alpha-gustducin immunolabeling pattern to those colabeled with UEA-1. Neuronal nitric oxide synthase was absent from intestinal epithelium despite strong labeling in the myenteric plexus. We conclude that subsets of enteroendocrine cells in the midjejunum and brush cells (more generally distributed) are equipped to utilize alpha-gustducin signaling in mice. Intestinal taste modalities may be signaled by these enteroendocrine cells via the release of 5-HT, GLP-1, or coexpressed mediators or by brush cells via a nonnitrergic mediator in distinct regions of the intestine.

Modulation of gastro-oesophageal vagal afferents by galanin in mouse and ferret.

The neuropeptide galanin is found in the central and peripheral nervous systems. It may have excitatory or inhibitory actions via three subtypes of G-protein-coupled receptor, and it modulates the mechanosensitivity of somatic sensory fibres. We aimed to determine if galanin also modulates vagal afferent mechanosensitivity, and to localize endogenous sources. The responses of ferret and mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated in vitro. The effects of galanin and/or the galanin receptor antagonist galantide on these responses were quantified. Immunohistochemistry for galanin was performed in ferret and mouse proximal stomach and nodose ganglion. In ferrets, retrograde labelling of gastric afferents to the nodose ganglion was combined with immunohistochemistry. When exposed to galanin (1-10 nM), 18/31 ferret and 12/15 mouse gastro-oesophageal afferents (tension, mucosal and tension/mucosal receptors) showed inhibition of mechanosensitivity. Four of 31 ferret afferents showed potentiation of mechanosensitivity, and 9/31 were unaffected (2/15 and 1/15 in mouse, respectively). Galanin effects were reversed after washout or by galantide (10-30 nM). Galantide given alone increased mechanosensitivity. Galanin immunoreactivity was found in nodose neurones, including those innervating the stomach in ferret. Enteric neurones were also galanin immunoreactive, as were endings associated with myenteric ganglia and smooth muscle. We conclude that galanin potently modulates mechanosensitivity of gastro-oesophageal vagal afferents with either facilitatory or inhibitory actions on individual afferent fibres. Both intrinsic and extrinsic (vagal) neurones contain galanin and are therefore potential sources of endogenous galanin.

Metabotropic glutamate receptors inhibit mechanosensitivity in vagal sensory neurons.

BACKGROUND AND AIMS: Inhibitory G-protein-coupled receptors have demonstrated potential in treatment of gastroesophageal reflux disease (GERD) through actions on vagal afferent signaling. Metabotropic glutamate receptors (mGluR) belong to this receptor family and have great pharmacologic and molecular diversity, with 8 subtypes. We investigated mGluR in the vagal system of humans and other species. METHODS: Expression of mGluR1-8 in human, dog, ferret, and rodent nodose ganglia was investigated by reverse-transcription polymerase chain reaction. mGluR1-8 immunohistochemistry was performed in combination with retrograde tracing of vagal afferents from ferret proximal stomach to nodose ganglia. Transport of mGluR peripherally was investigated by vagal ligation, followed by immunohistochemistry. Glutamate receptor pharmacology of ferret and rodent gastroesophageal vagal afferents was investigated by testing single fiber responses to graded mechanical stimuli during drug application to their peripheral endings. RESULTS: Messenger RNA for several mGluR was detected in the nodose ganglia of all species. Retrograde tracing indicated that ferret gastric vagal afferents express mGluR protein. Accumulation of immunoreactivity proximal to a ligature showed that mGluR were transported peripherally in the vagus nerves. Glutamate (1-30 mumol/L with kynurenate 0.1 mmol/L) concentration dependently inhibited vagal afferent mechanosensitivity. This was mimicked by selective group II and III mGluR agonists but not by a group I agonist. Conversely, a group III mGluR antagonist increased mechanosensitivity to intense stimuli. CONCLUSIONS: Both exogenous and endogenous glutamate inhibits mechanosensitivity of vagal afferents. Group II (mGluR2 and 3) and group III mGluR (mGluR4, 6, 7, 8) are novel targets for inhibition of vagal signaling with therapeutic potential in, for example, GERD.