CGRPα within the Trpv1-Cre population contributes to visceral nociception.

The role of calcitonin gene-related peptide (CGRP) in visceral and somatic nociception is incompletely understood. CGRPα is highly expressed in sensory neurons of dorsal root ganglia and particularly in neurons that also express the transient receptor potential cation channel subfamily V member 1 (Trpv1). Therefore, we investigated changes in visceral and somatic nociception following deletion of CGRPα from the Trpv1-Cre population using the Cre/lox system. In control mice, acetic acid injection (0.6%, ip) caused significant immobility (time stationary), an established indicator of visceral pain. In CGRPα-mCherrylx/lx;Trpv1-Cre mice, the duration of immobility was significantly less than controls, and the distance CGRPα-mCherrylx/lx;Trpv1-Cre mice traveled over 20 min following acetic acid was significantly greater than controls. However, following acetic acid injection, there was no difference between genotypes in the writhing reflex, number of abdominal licks, or forepaw wipes of the cheek. CGRPα-mCherrylx/lx;Trpv1-Cre mice developed more pronounced inflammation-induced heat hypersensitivity above baseline values compared with controls. However, analyses of noxious acute heat or cold transmission revealed no difference between genotypes. Also, odor avoidance test, odor preference test, and buried food test for olfaction revealed no differences between genotypes. Our findings suggest that CGRPα-mediated transmission within the Trpv1-Cre population plays a significant role in visceral nociceptive pathways underlying voluntary movement. Monitoring changes in movement over time is a sensitive parameter to identify differences in visceral nociception, compared with writhing reflexes, abdominal licks, or forepaw wipes of the cheek that were unaffected by deletion of CGRPα- from Trpv1-Cre population and likely utilize different mechanisms. NEW & NOTEWORTHY The neuropeptide calcitonin gene-related peptide (CGRP) is highly colocalized with transient receptor potential cation channel subfamily V member 1 (TRPV1)-expressing primary afferent neurons, but the functional role of CGRPα specifically in these neurons is unknown in pain processing from visceral and somatic afferents. We used cre-lox recombination to conditionally delete CGRPα from TRPV1-expressing neurons in mice. We show that CGRPα from within TRPV1-cre population plays an important role in visceral nociception but less so in somatic nociception.

Electrophysiological and morphological features of myenteric neurons of human colon revealed by intracellular recording and dye fills.

BACKGROUND: Ex vivo intracellular recordings and dye fills, combined with immunohistochemistry, are a powerful way to analyze the enteric nervous system of laboratory animals. METHODS: Myenteric neurons were recorded in isolated specimens of human colon. A key determinant of successful recording was near-complete removal of circular muscle from the surface of ganglia. KEY RESULTS: Treatment with a collagenase/neutral protease mix before dissection significantly improved recording success and reduced damage to the plexus. Carboxyfluorescein in microelectrodes allowed recorded neurons to be routinely labeled, analyzed, and subjected to multi-layer immunohistochemistry. Carboxyfluorescein revealed morphological details that were not detected by immunohistochemical methods. Of 54 dye-filled myenteric neurons (n = 22), 45 were uni-axonal and eight were multi-axonal. There was a significant bias toward recordings from large neural somata. The close association between morphology and electrophysiology (long after-hyperpolarizations and fast EPSPs) seen in mice and guinea pigs did not hold for human myenteric neuron recordings. No slow EPSPs were recorded; however, disruption to the myenteric plexus during dissection may have led the proportion of cells receiving synaptic potentials to be underestimated. Neurons immunoreactive for nitric oxide synthase were more excitable than non-immunoreactive neurons. Distinctive grooves were observed on the serosal and/or mucosal faces of myenteric neurons in 3D reconstructions. These had varicose axons running through them and may represent a preferential site of synaptic inputs. CONCLUSIONS: Human enteric neurons share many features with laboratory animals, but the combinations of features in individual cells appear more variable.

Piezo2 channels expressed by colon-innervating TRPV1-lineage neurons mediate visceral mechanical hypersensitivity.

Inflammatory and functional gastrointestinal disorders such as irritable bowel syndrome (IBS) and obstructive bowel disorder (OBD) underlie the most prevalent forms of visceral pain. Although visceral pain can be generally provoked by mechanical distension/stretch, the mechanisms that underlie visceral mechanosensitivity in colon-innervating visceral afferents remain elusive. Here, we show that virally mediated ablation of colon-innervating TRPV1-expressing nociceptors markedly reduces colorectal distention (CRD)-evoked visceromotor response (VMR) in mice. Selective ablation of the stretch-activated Piezo2 channels from TRPV1 lineage neurons substantially reduces mechanically evoked visceral afferent action potential firing and CRD-induced VMR under physiological conditions, as well as in mouse models of zymosan-induced IBS and partial colon obstruction (PCO). Collectively, our results demonstrate that mechanosensitive Piezo2 channels expressed by TRPV1-lineage nociceptors powerfully contribute to visceral mechanosensitivity and nociception under physiological conditions and visceral hypersensitivity under pathological conditions in mice, uncovering potential therapeutic targets for the treatment of visceral pain.

Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding.

BACKGROUND AND AIMS: Gut functions including motility, secretion, and blood flow are largely controlled by the enteric nervous system. Characterizing the different classes of enteric neurons in the human gut is an important step to understand how its circuitry is organized and how it is affected by disease. METHODS: Using multiplexed immunohistochemistry, 12 discriminating antisera were applied to distinguish different classes of myenteric neurons in the human colon (2596 neurons, 12 patients) according to their chemical coding. All antisera were applied to every neuron, in multiple layers, separated by elutions. RESULTS: A total of 164 combinations of immunohistochemical markers were present among the 2596 neurons, which could be divided into 20 classes, with statistical validation. Putative functions were ascribed for 4 classes of putative excitatory motor neurons (EMN1-4), 4 inhibitory motor neurons (IMN1-4), 3 ascending interneurons (AIN1-3), 6 descending interneurons (DIN1-6), 2 classes of multiaxonal sensory neurons (SN1-2), and a small, miscellaneous group (1.8% of total). Soma-dendritic morphology was analyzed, revealing 5 common shapes distributed differentially between the 20 classes. Distinctive baskets of axonal varicosities surrounded 45% of myenteric nerve cell bodies and were associated with close appositions, suggesting possible connectivity. Baskets of cholinergic terminals and several other types of baskets selectively targeted ascending interneurons and excitatory motor neurons but were significantly sparser around inhibitory motor neurons. CONCLUSIONS: Using a simple immunohistochemical method, human myenteric neurons were shown to comprise multiple classes based on chemical coding and morphology and dense clusters of axonal varicosities were selectively associated with some classes.

Characterization of viscerofugal neurons in human colon by retrograde tracing and multi-layer immunohistochemistry.

Background and Aims: Viscerofugal neurons (VFNs) have cell bodies in the myenteric plexus and axons that project to sympathetic prevertebral ganglia. In animals they activate sympathetic motility reflexes and may modulate glucose metabolism and feeding. We used rapid retrograde tracing from colonic nerves to identify VFNs in human colon for the first time, using ex vivo preparations with multi-layer immunohistochemistry. Methods: Colonic nerves were identified in isolated preparations of human colon and set up for axonal tracing with biotinamide. After fixation, labeled VFN cell bodies were subjected to multiplexed immunohistochemistry for 12 established nerve cell body markers. Results: Biotinamide tracing filled 903 viscerofugal nerve cell bodies (n = 23), most of which (85%) had axons projecting orally before entering colonic nerves. Morphologically, 97% of VFNs were uni-axonal. Of 215 VFNs studied in detail, 89% expressed ChAT, 13% NOS, 13% calbindin, 9% enkephalin, 7% substance P and 0 of 123 VFNs expressed CART. Few VFNs contained calretinin, VIP, 5HT, CGRP, or NPY. VFNs were often surrounded by dense baskets of axonal varicosities, probably reflecting patterns of connectivity; VAChT+ (cholinergic), SP+ and ENK+ varicosities were most abundant around them. Human VFNs were diverse; showing 27 combinations of immunohistochemical markers, 4 morphological types and a wide range of cell body sizes. However, 69% showed chemical coding, axonal projections, soma-dendritic morphology and connectivity similar to enteric excitatory motor neurons. Conclusion: Viscerofugal neurons are present in human colon and show very diverse combinations of features. High proportions express ChAT, consistent with cholinergic synaptic outputs onto postganglionic sympathetic neurons in prevertebral ganglia.

Sympathetic Pathways Target Cholinergic Neurons in the Human Colonic Myenteric Plexus.

Background: The sympathetic nervous system inhibits human colonic motility largely by effects on enteric neurons. Noradrenergic axons, which branch extensively in the myenteric plexus, are integral to this modulatory role, but whether they contact specific types of enteric neurons is unknown. The purpose of this study was to determine the association of noradrenergic varicosities with types of enteric neurons. Methods: Human colonic tissue from seven patients was fixed and dissected prior to multi-layer immunohistochemistry for human RNA binding proteins C and D (HuC/D) (pan-neuronal cell body labelling), tyrosine hydroxylase (TH, catecholaminergic labelling), Enkephalin (ENK), choline acetyltransferase (ChAT, cholinergic labelling) and/or nitric oxide synthase (NOS, nitrergic labelling) and imaged using confocal microscopy. TH-immunoreactive varicose nerve endings and myenteric cell bodies were reconstructed as three dimensional digital images. Data was exported to a purpose-built software package which quantified the density of varicosities close to the surface of each myenteric cell body. Results: TH-immunoreactive varicosities had a greater mean density within 1 µm of the surface of ChAT +/NOS- nerve cell bodies compared with ChAT-/NOS + cell bodies. Similarly, ENK-immunoreactive varicosities also had a greater mean density close to ChAT +/NOS- cell bodies compared with ChAT-/NOS + cells. Conclusion: A method for quantifying close associations between varicosities and nerve cell bodies was developed. Sympathetic axons in the myenteric plexus preferentially target cholinergic excitatory cells compared to nitrergic neurons (which are largely inhibitory). This connectivity is likely to be involved in inhibitory modulation of human colonic motility by the sympathetic nervous system.

Loss of visceral pain following colorectal distension in an endothelin-3 deficient mouse model of Hirschsprung's disease.

Endothelin peptides and their endogenous receptors play a major role in nociception in a variety of different organs. They also play an essential role in the development of the enteric nervous system. Mice with deletions of the endothelin-3 gene (lethal spotted mice, ls/ls) develop congenital aganglionosis. However, little is known about how nociception might be affected in the aganglionic rectum of mice deficient in endothelin-3. In this study we investigated changes in spinal afferent innervation and visceral pain transmission from the aganglionic rectum in ls/ls mice. Electromyogram recordings from anaesthetized ls/ls mice revealed a deficit in visceromotor responses arising from the aganglionic colorectum in response to noxious colorectal distension. Loss of visceromotor responses (VMRs) in ls/ls mice was selective, as no reduction in VMRs was detected after stimulation of the bladder or somatic organs. Calcitonin gene related peptide (CGRP) immunoreactivity, retrograde neuronal tracing and extracellular afferent recordings from the aganglionic rectum revealed decreased colorectal spinal innervation, combined with a reduction in mechanosensitivity of rectal afferents. The sensory defect in ls/ls mice is primarily associated with changes in low threshold wide dynamic range rectal afferents. In conclusion, disruption of endothelin 3 gene expression not only affects development and function of the enteric nervous system, but also specific classes of spinal rectal mechanoreceptors, which are required for visceral nociception from the colorectum.

Characterization of putative interneurons in the myenteric plexus of human colon.

BACKGROUND: The enteric nervous system contains multiple classes of neurons, distinguishable by morphology, immunohistochemical markers, and projections; however, specific combinations differ between species. Here, types of enteric neurons in human colon were characterized immunohistochemically, using retrograde tracing combined with multiple labeling immunohistochemistry, focussing on non-motor neurons. METHODS: The fluorescent carbocyanine tracer, DiI, was applied to the myenteric plexus in ex vivo preparations, filling neurons projecting within the plexus. Limits of projection lengths of motor neurons were established, allowing them to be excluded from the analysis. Long ascending and descending interneurons were then distinguished by labeling for discriminating immunohistochemical markers: calbindin, calretinin, enkephalin, 5-hydroxytryptamine, nitric oxide synthase, and substance P. These results were combined with a previous published study in which nitric oxide synthase and choline acetyltransferase immunoreactivities were established. KEY RESULTS: Long ascending neurons (with projections longer than 8 mm, which excludes more than 95% motor neurons) formed four types, in descending order of abundance, defined by immunoreactivity for: (a) ChAT+/ENK+, (b) ChAT+/ENK+/SP+, (c) ChAT+/Calb+, and (d) ChAT+/ENK+/Calb+. Long descending neurons, up to 70 mm long also formed at least four types, distinguished by immunoreactivity for (a) NOS + cells (without ChAT), (b) ChAT+/NOS+, (c) ChAT+/Calret+, and (d) ChAT+/5HT + cells (with or without NOS). CONCLUSIONS AND INFERENCES: Long interneurons, which do not innervate muscularis externa, are likely to coordinate neural activity over distances of many centimeters along the colon. Characterizing their neurochemical coding provides a basis for understanding their roles, investigating their connectivity, and building a comprehensive account of human colonic enteric neurons.

Identification of medium/high-threshold extrinsic mechanosensitive afferent nerves to the gastrointestinal tract.

BACKGROUND & AIMS: Large distentions reliably evoke sensation from the noninflamed, nonischemic bowel, but the specialized afferent axonal structures responsible have not been morphologically identified. We investigated whether their transduction sites are located on major blood vessels close to and within the gut wall. METHODS: In vitro extracellular recordings were made from mesenteric nerve trunks in guinea pig ileum, combined with rapid axonal dye filling and immunohistochemical analysis of nerve trunks. RESULTS: Recordings revealed sensory fibers with focal mechanosensitive sites in the mesenteries that could be activated by von Frey hairs and by stretch. Dye filling revealed varicose branching sensory axons on mesenteric blood vessels but no other anatomically specialized structures in mesenteric membranes or the serosa. Large-amplitude stretch and von Frey hairs also activated sensory endings within the gut wall itself but only if the submucosa was present; mechanotransduction sites in the serosa or outer muscle layers were sparse. Mechanosensitive sites in submucosa were exclusively associated with submucosal blood vessels. Submucosal endings had significantly higher thresholds to stretch than specialized low-threshold mechanoreceptors characterized previously in the rectum (P < .05) and were therefore classified as medium/high-threshold mechanoreceptors. Capsaicin (0.3-1 micromol/L) activated most mechanosensitive mesenteric (68%) and submucosal (85%) afferent endings. Similar intramural mechanosensitive afferent endings on blood vessels also exist in the colon and bladder. CONCLUSIONS: Varicose branching axons of sensory neurons on intramural blood vessels, previously shown to mediate sensory vasodilation, are transduction sites for medium/high-threshold, stretch-sensitive mechanoreceptors, encoding large distentions in hollow viscera.

Characterization of projections of longitudinal muscle motor neurons in human colon.

BACKGROUND: The enteric nervous system contains inhibitory and excitatory motor neurons which modulate smooth muscle contractility. Cell bodies of longitudinal muscle motor neurons have not been identified in human intestine. METHODS: We used retrograde tracing ex vivo with DiI, with multiple labeling immunohistochemistry, to characterize motor neurons innervating tenial and inter-tenial longitudinal muscle of human colon. KEY RESULTS: The most abundant immunohistochemical markers in the tertiary plexus were vesicular acetylcholine transporter, nitric oxide synthase (NOS), and vasoactive intestinal polypeptide (VIP). Of retrogradely traced motor neurons innervating inter-tenial longitudinal muscle, 95% were located within 6mm oral or anal to the DiI application site. Excitatory motor neuron cell bodies, immunoreactive for choline acetyltransferase (ChAT), were clustered aborally, whereas NOS-immunoreactive cell bodies were distributed either side of the DiI application site. Motor neurons had small cell bodies, averaging 438 + 18µm2 in cross-sectional area, similar for ChAT- and NOS-immunoreactive subtypes. Motor neurons innervating the tenia had slightly longer axial projections, with 95% located within 9mm. ChAT-immunoreactive excitatory motor neurons to tenia were clustered aborally, whereas NOS-immunoreactive inhibitory motor neurons had both ascending and descending projections. VIP immunoreactivity was rarely present without NOS immunoreactivity in motor neurons. CONCLUSIONS AND INFERENCES: Tenial and inter-tenial motor neurons innervating the longitudinal muscle have short projections. Inhibitory motor neurons have less polarized projections than cholinergic excitatory motor neurons. Longitudinal and circular muscle layers are innervated by distinct local populations of excitatory and inhibitory motor neurons. A population of human enteric neurons that contribute significantly to colonic motility has been characterized.

Comparison of extrinsic efferent innervation of guinea pig distal colon and rectum.

The extrinsic efferent innervation of the distal colon and rectum of the guinea pig was compared, by using retrograde tracing combined with immunohistochemistry. Application of the carbocyanine tracer DiI to the rectum filled significantly greater numbers of extrinsic neurons than similar injections into the distal colon. Approximately three-fourths of all filled neurons from either location were either sympathetic or parasympathetic; the rest were spinal sensory neurons. Nerve cell bodies in sympathetic prevertebral ganglia labelled from the two regions were similar in number. Both regions were innervated by sympathetic neurons in paravertebral ganglia; however, the rectum received much more input from this source than the colon. The rectum received significantly more input from pelvic ganglia than the colon. The rectum also received direct innervation from two groups of neurons in the spinal cord. Neurons located in the spinal parasympathetic nucleus in segment S2 and S3 were labelled by DiI injected into the rectal wall. Similar numbers of neurons, located in intermediolateral cell column and dorsal commissural nucleus of lumbar segments, also projected directly to rectum, but not colon. The great majority (>80%) of retrogradely labelled nerve cell bodies in sympathetic ganglia were immunoreactive for tyrosine hydroxylase. In pelvic ganglia, retrogradely labelled neurons contained choline acetyltransferase and/or nitric oxide synthase or tyrosine hydroxylase. Although the rectum and colon in this species are continuous and macroscopically indistinguishable, they have significantly different patterns of extrinsic efferent innervation, presumably reflecting their different functions.

Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon.

Extrinsic nerves to the gut influence the absorption of water and electrolytes and expulsion of waste contents, largely via regulation of enteric neural circuits; they also contribute to control of blood flow. The distal colon is innervated by extrinsic sympathetic and parasympathetic efferent and spinal afferent neurons, via axons in colonic nerve trunks. In the present study, biotinamide tracing of colonic nerves was combined with immunohistochemical labeling for markers of sympathetic, parasympathetic, and spinal afferent neurons to quantify their relative contribution to the extrinsic innervation. Calcitonin gene-related peptide, vesicular acetylcholine transporter, and tyrosine hydroxylase, which selectively label spinal afferent, parasympathetic, and sympathetic axons, respectively, were detected immunohistochemically in 1 ± 0.5% (n = 7), 15 ± 4.7% (n = 6), and 24 ± 4% (n = 7) of biotinamide-labeled extrinsic axons in myenteric ganglia. Immunoreactivity for vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, and vesicular glutamate transporters 1 and 2 accounted for a combined maximum of 14% of biotinamide-labeled axons in myenteric ganglia. Thus, a maximum of 53% of biotinamide-labeled extrinsic axons in myenteric ganglia were labeled by antisera to one of these eight markers. Viscerofugal neurons were also labeled by biotinamide. They had distinct morphologies and spatial distributions that correlated closely with their immunoreactivity for nitric oxide synthase and choline acetyltransferase. As reported for the rectum, nearly half of all extrinsic nerve fibers to the distal colon lack the key immunohistochemical markers commonly used for their identification. Their abundance may therefore have been significantly underestimated in previous immunohistochemical studies.

4-aminopyridine- and dendrotoxin-sensitive potassium channels influence excitability of vagal mechano-sensitive endings in guinea-pig oesophagus.

1. Distension-sensitive vagal afferent fibres from the guinea-pig oesophagus were recorded extracellularly in vitro. Most recorded units were spontaneously active firing at 3.2+/-0.3 Hz (n=41, N=41) and had low thresholds (less than 1 mm) to circumferential stretch. Dynamic and adapted phases of stretch-evoked firing, as well as a silent period were linearly dependent on the amplitude of stretch. 2. High K+ (7-12 mM) Krebs solution dose-dependently increased both spontaneous and stretch-evoked firing and reduced the duration of the silent period. 3. Charybdotoxin (ChTX, 100 nM) slightly increased spontaneous and stretch-evoked firing and decreased the silent period, while neither iberiotoxin (100 nM) nor apamin (0.5 microM) had significant effects. omega-Conotoxin GVIA (0.5 microM) did not significantly affect firing of vagal mechanoreceptors. 4. In the majority of single units, 4-aminopyridine (4-AP) concentration-dependently (EC(50) approximately 28 microM) increased spontaneous firing, strongly reduced the silent period but did not affect stretch (3 mm)-induced firing. Firing evoked by 1-2 mm was increased by 4-AP. 5. Alpha-dendrotoxin (DnTX, 300 nM) and DnTX K (30 nM) slightly increased spontaneous and stretch-evoked firing. There was no additive effect on spontaneous firing when ChTX and DnTX K were applied simultaneously. 6. Barium (100 microM) increased stretch-induced firing, probably due to an increase in intramural tension. Glibenclamide (10 microM) had no effect on spontaneous or stretch-induced firing. 7. The results indicate that voltage-gated 4-AP- and dendrotoxin-sensitive K+ channels are the main type of K+ channels that influence excitability of vagal mechano-sensitive endings of the guinea-pig oesophagus. They were involved in control of spontaneous firing and in stretch-induced firing evoked by moderate stretch, but none of the K+ channels appeared to be involved in adaptation to maintained stretch by their slowly adapting vagal mechanoreceptors.

Neurochemical coding compared between varicose axons and cell bodies of myenteric neurons in the guinea-pig ileum.

The discrete functional classes of enteric neurons in the mammalian gastrointestinal tract have been successfully distinguished on the basis of the unique combination of molecules and enzymes in their cell bodies ("chemical coding"). Whether the same chemical coding exists in varicose axons of different functional classes has not been systematically tested. In this study, we quantified the coexistence of markers that define classes of nerve cell bodies in the myenteric plexus of the guinea-pig ileum, in varicose axons of the same neurons. Profound differences between the combinations of immunohistochemical markers in myenteric nerve cell bodies and in their varicosities were identified. These discrepancies were particularly notable for classes of neurons that had previously been classified as cholinergic, based on immunoreactivity for choline acetyltransferase (ChAT) in their cell bodies. To detect cholinergic varicose axons of enteric neurons in this study, we used antiserum against the vesicular acetylcholine transporter (VAChT). ChAT-immunoreactivity has been reported to be consistently co-localized with 5-hydroxytryptamine (5-HT) in interneuronal cell bodies, yet only 29±5% (n=4) of 5-HT-immunoreactive varicosities contained vesicular acetylcholine transporter (VAChT). Somatostatin coexists with ChAT-immunoreactivity in a class of descending interneuron but only 21±1% (n=4) of somatostatin-immunoreactive varicosities were VAChT-immunoreactive. Comparable discrepancies were also noted for non-cholinergic markers. The results suggest that chemical coding of cell bodies does not necessarily reflect chemical coding of varicose axon terminals and that the assumption that nerve cell bodies that contain ChAT are functionally cholinergic may be questionable.

Mechanotransduction by intraganglionic laminar endings of vagal tension receptors in the guinea-pig oesophagus.

Vagal mechanoreceptors to the guinea-pig oesophagus, recorded extracellularly, in vitro, fired spontaneously at 3.3 +/- 0.2 Hz, (n = 75, from 57 animals), and had low thresholds to circumferential stretch. In this study, we have investigated whether mechanotransduction by intraganglionic laminar endings (IGLEs) directly relies on mechano-gated ion channels, or whether it is due to chemical activation by neurotransmitters (glutamate or ATP) released from other cells during mechanical distortion. Rapid distortion of focal transduction sites (IGLEs) evoked action potentials with a latency of < 10 ms. Antagonists to ionotropic (AP5, memantine and 6,7-dinitroquinoxaline-2,3-dione (DNQX)) and metabotropic glutamate receptors (N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) and (RS)-a-methyl-4-phosphono-phenylglycine (MPPG)) did not affect mechano-transduction. Glutamate, NMDA and the selective mGluR group II and III agonists, (2R, 4R)-APDC and L-AP4, had no effect on spontaneous or stretch-induced firing. The P2X purinoreceptor agonist, alpha,beta-methylene ATP, caused concentration-dependent excitation of vagal mechanoreceptors (EC50 = 22.2 microM) which was blocked by the non-selective P2 antagonist PPADS (30 microM). On its own, PPADS affected neither stretch-induced firing nor spontaneous firing. Neither Ca(2+)-free solution (1 mM EDTA, 3.6 mM Mg(2+)) solution nor Cd(2+) (100 microM) blocked stretch-induced firing. Thus chemical transmission is not involved in activation of vagal mechanoreceptors. The blocker of stretch-activated channels, Gd(3+) (300 microM), did not inhibit stretch-induced firing. However, benzamil (100 microM) significantly inhibited spontaneous and distension-evoked firing in a stretch-dependent manner; proportionally greater inhibition was seen with larger stretches. The results suggest that IGLEs of vagal tension receptors directly transduce mechanical stimuli probably via benzamil-sensitive, Gd3+-insensitive, stretch-activated ion channels, and that chemical transmission is not involved in transduction.