Nodose Neurons Research Articles

Evidence for multiple bulbar and higher brain circuits processing sensory inputs from the respiratory system in humans.

Unpleasant respiratory sensations contribute to morbidity in pulmonary disease. In rodents, these sensations are processed by nodose and jugular vagal sensory neurons, two distinct cell populations that differentially project to the airways and brainstem. Whether similar differences exist in bronchopulmonary sensory pathways in humans is unknown. We use functional magnetic resonance imaging during inhalation of capsaicin and ATP, showing that airway nodose pathways project centrally to the nucleus of the solitary tract, whereas jugular pathways input into the trigeminal brainstem nuclei. We also show differences between the efficacy of nodose and jugular stimuli to evoke cough and activity in motor control regions of the brain. Our data suggest that humans have two distinct vagal sensory neural systems governing airway sensations and this may have implications for the development of new antitussive therapies. In rodents, nodose vagal sensory neurons preferentially innervate the distal airways and terminate centrally in the nucleus of the solitary tract. By contrast, jugular vagal sensory neurons preferentially innervate the proximal airways and terminate in the paratrigeminal nucleus in the dorsolateral medulla. This differential organization suggests distinct roles for nodose and jugular pathways in respiratory sensory processing. However, it is unknown whether bronchopulmonary afferent pathways are similarly arranged in humans. We set out to investigate this using high resolution brainstem and whole brain functional magnetic resonance imaging in healthy human participants when they were inhaling stimuli known to differentially activate nodose and jugular pathways. Inhalation of capsaicin or ATP evoked respiratory sensations described as an urge-to-cough, although ATP was significantly less effective compared to capsaicin at evoking the motor act of coughing. The nodose and jugular neuron stimulant capsaicin increased blood oxygen level-dependent (BOLD) signals extending across the dorsomedial and dorsolateral medulla, encompassing regions containing both the nucleus of the solitary tract and the paratrigeminal nucleus. By contrast, at perceptually comparable stimulus intensities, the nodose-selective stimulant ATP resulted in BOLD signal intensity changes that were confined to the area of the nucleus of the solitary tract. During whole brain imaging, capsaicin demonstrated a wider distributed network of activity compared to ATP, with significantly increased activity in regions involved with motor control functions. These data suggest that functional and neuroanatomical differences in bronchopulmonary nodose and jugular sensory pathway organization are conserved in humans and also that this has implications for understanding the neurobiological mechanisms underpinning cough.

Exaggerated potassium current reduction by oxytocin in visceral sensory neurons following chronic intermittent hypoxia

Oxytocin (OT) from the hypothalamus is increased in several cardiorespiratory nuclei and systemically in response to a variety of stimuli and stressors, including hypoxia. Within the nucleus tractus solitarii (nTS), the first integration site for cardiorespiratory reflexes, OT enhances synaptic transmission, action potential (AP) discharge, and cardiac baroreflex gain. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. The nTS receives sensory input from baroafferent neurons that originate in the nodose ganglia. Nodose neurons express the OT receptor (OTR) whose activation elevates intracellular calcium. However, the influence of OT on other ion channels, especially potassium channels important for neuronal activity during CIH, is less known. This study sought to determine the mechanism (s) by which OT modulates sensory afferent-nTS mediated reflexes normally and after CIH. Nodose ganglia neurons from male Sprague-Dawley rats were examined after 10d CIH (6% O2 every 3min) or their normoxic (21% O2) control. OTR mRNA and protein were identified in Norm and CIH ganglia and was similar between groups. To examine OTR function, APs and potassium currents (IK) were recorded in dissociated neurons. Compared to Norm, after CIH OT depolarized neurons and reduced current-induced AP discharge. After CIH OT also produced a greater reduction in IK that where tetraethylammonium-sensitive. These data demonstrate after CIH OT alters ionic currents in nodose ganglia cells to likely influence cardiorespiratory reflexes and overall function.

Acute activation of bronchopulmonary vagal nociceptors by type I interferons.

Type I interferon receptors are expressed by the majority of vagal C-fibre neurons innervating the respiratory tract Interferon alpha and beta acutely and directly activate vagal C-fibers in the airways. The interferon-induced activation of C-fibers occurs secondary to stimulation of type 1 interferon receptors Type 1 interferons may contribute to the symptoms as well as the spread of respiratory viral infections by causing coughing and other defensive reflexes associated with vagal C-fibre activation ABSTRACT: We evaluated the ability of type I interferons to acutely activate airway vagal afferent nerve terminals in mouse lungs. Using single cell RT-PCR of lung-specific vagal neurons we found that IFNAR1 and IFNAR2 were expressed in 70% of the TRPV1-positive neurons (a marker for vagal C-fibre neurons) and 44% of TRPV1-negative neurons. We employed an ex vivo vagal innervated mouse trachea-lung preparation to evaluate the effect of interferons in directly activating airway nerves. Utilizing 2-photon microscopy of the nodose ganglion neurons from Pirt-Cre;R26-GCaMP6s mice we found that applying IFNα or IFNβ to the lungs acutely activated the majority of vagal afferent nerve terminals. When the type 1 interferon receptor, IFNAR1, was blocked with a blocking antibody the response to IFNβ was largely inhibited. The type 2 interferon, IFNγ, also activated airway nerves and this was not inhibited by the IFNAR1 blocking antibody. The Janus kinase inhibitor GLPG0634 (1μm) virtually abolished the nerve activation caused by IFNβ. Consistent with the activation of vagal afferent C-fibers, infusing IFNβ into the mouse trachea led to defensive breathing reflexes including apneas and gasping. These reflexes were prevented by pretreatment with an IFN type-1 receptor blocking antibody. Finally, using whole cell patch-clamp electrophysiology of lung-specific neurons we found that IFNβ (1000Uml-1 ) directly depolarized the membrane potential of isolated nodose neurons, in some cases beyond to action potential threshold. This acute non-genomic activation of vagal sensory nerve terminals by interferons may contribute to the incessant coughing that is a hallmark of respiratory viral infections.

Chronic high fat diet impairs glucagon like peptide-1 sensitivity in vagal afferents

Dysfunction of the gut-brain axis is one of the potential contributors to the pathophysiology of obesity and is therefore a potential target for treatment. Vagal afferents innervating the gut play an important role in controlling energy homeostasis. There is an increasing evidence for the role of vagal afferents in mediating the anorexigenic effects of glucagon-like peptide-1 (GLP-1), an important satiety and incretin hormone. This study aimed to examine the effect of chronic high fat diet on GLP-1 sensitivity in vagal afferents. C57/BL6 mice were fed either a high-fat or low-fat diet for 6–8 weeks. To evaluate gastrointestinal afferent sensitivity and nodose neurons’ response to GLP-1, extracellular afferent recordings and patch clamp were performed, respectively. Exendin-4 (Ex-4) was used as an agonist of the GLP-1 receptor. C-Fos Expression was examined as an indication of afferent input to the nucleus tractus solitarius (NTS). Food intake was monitored in real-time before and after Ex-4 treatment to monitor the consequence of the high fat diet on the satiating effect of GLP-1. In high fat fed (HFF) mice, GLP-1 caused lower activation of intestinal afferent nerves, and failed to potentiate mechanosensitive nerve responses compared to low fat fed (LFF). GLP-1 increased excitability in LFF and this effect was reduced in HFF neurons. Consistent with these findings on vagal afferent nerves, GLP-1 receptor stimulation given systemically, had a reduced satiating effect in HFF compared to LFF mice, and neuronal activation in the NTS was also reduced. The present study demonstrated chronic high fat diet impaired vagal afferent responses to GLP-1, resulting in impaired satiety signaling. GLP-1 sensitivity may account for the impairment of satiety signaling in obesity and thus a therapeutic target for obesity treatment.

A Vagal-NTS Neural Pathway that Stimulates Feeding

A fundamental question of physiology is how gut-brain signaling stimulates appetite. While many studies have emphasized the importance of vagal afferents to the brain in inducing satiation, little is known about whether and how the vagal-mediated gut-brain pathway senses orexigenic signals and stimulates feeding. Here, we identified a previously uncharacterized population of fasting-activated catecholaminergic neurons in the nucleus of the solitary tract (NTS). After characterizing the anatomical complexity among NTS catecholaminergic neurons, we surprisingly found that activation of NTS epinephrine (ENTS) neurons co-expressing neuropeptide Y (NPY) stimulated feeding, whereas activation of NTS norepinephrine (NENTS) neurons suppressed feeding. Monosynaptic tracing/activation experiments then showed that these NTS neurons receive direct vagal afferents from nodose neurons. Moreover, activation of the vagal→NPY/ENTS neural circuit stimulated feeding. Our study reveals an orexigenic role of the vagal→NTS pathway in controlling feeding, thereby providing important insights about how gut-brain signaling regulates feeding behavior.

Intralaryngeal application of ATP evokes apneic response mainly via acting on P2X3/P2X2/3 receptors of the superior laryngeal nerve (SLN) in postnatal rats

ATP is reportedly capable of stimulating bronchopulmonary C‐ and/or Aδ‐fibers to evoke apnea, bronchoconstriction, and bradycardia via acting on P2X (P2X2/3) receptors. Stimulation of the SLN evokes apnea, bradycardia, and hypertension, especially in early life. Although P2X3/P2X2/3 receptors are present in the nodose and jugular (N/J) ganglion neurons, it is undetermined whether ATP applied onto the larynx mucosa can elicit the cardiorespiratory responses via activation of P2X3/P2X2/3 receptors of the SLN. To address this issue, the cardiorespiratory responses to intralaryngeal perfusion of ATP (a non‐selective P2‐receptor agonist, 5–10 mg/ml) or α,β‐methylene ATP (a selective P2X‐receptor agonist, 2.5–5 mg/ml) were recorded in anesthetized rat pups (P14‐P18) under the following conditions: 1) before and after bilateral SLN section and 2) immediately after 5 min intralaryngeal pretreatment with saline (Ctrl) or saline containing A‐317491 (a P2X3/P2X2/3 receptor antagonist, 2.5–5 mg/ml). The immunoreactivity (IR) of P2X3 receptor was then identified in N/J ganglion neurons retrogradely traced by DiI that was previously injected into the SLN 10 days ago. Whole‐cell patch clamp recording was used to determine the ATP‐ or α,β‐methylene ATP‐induced currents of N/J neurons in primary culture, particularly those labeled by DiI. We found that intralaryngeal perfusion of ATP and α,β‐methylene ATP induced similar apnea (latency < 1.0 s), hypertension, and bradycardia, which were eliminated after bilateral section of the SLN. A‐317491 but not saline pretreatment attenuated the apneic response to ATP and α,β‐methylene ATP by 63% and 76% respectively and failed to affect the evoked hypertension and bradycardia. P2X3 receptor‐IR was identified in ~35% of both N/J ganglion neurons. 12% of these neurons were labeled by DiI, ~60% of which expressed P2X3 receptor‐IR. Both ATP‐ and α,β‐methylene ATP‐induced currents were recorded in DiI‐labeled neurons. The former was reduced by 75% and the latter abolished by A‐317491. We conclude that ATP and α,β‐methylene ATP applied onto the laryngeal mucosa elicit an apnea predominately through activation of P2X3/P2X2/3 receptors of the SLN.

Gastric Distension‐induced Nodose Ganglionic Cell Responses Using a High‐throughput Multi‐electrode Array in the Ferret

The nodose ganglion (NG) contains cell bodies of gastrointestinal afferents, which play critical roles in metabolic and feeding disorders and gastrointestinal and inflammatory diseases. To further understand the population coding of nodose ganglionic signals, we used a 32‐chan multi‐electrode array (MEA; 4×8, 400 μm pitch; Blackrock Microsystems) to record single‐unit responses with 3 main objectives: (1) determine the stability of multiple single‐unit responses during several hours of in vivo electrophysiological recording; (2) measure the activation patterns of these neurons to prototypical stimulation, such as gastric distension; and (3) investigate the phenotypes of nodose neurons using mechanical probing and vagus nerve electrical stimulation to determine conduction velocities. Acute experiments were performed in anesthetized ferrets using inhalational isoflurane (~2.5%) anesthesia. MEAs were inserted in the nodose ganglion using a pneumatic inserter, and a balloon‐catheter was advanced into the stomach through a small incision on the lateral edge of the gastric fundus. Two volumes of saline, 10 and 20 ml, were infused into the balloon to provide distension. Nerve cuff electrodes were placed on the abdominal vagus trunk to determine conduction velocity. Ganglionic single‐unit activity was successfully recorded from the NG for up to 5.3 h. In our preliminary analysis using spike sorting on each channel (MKsort, https://github.com/ripple‐neuro/mksort; Matlab), we were able to identify a total of 233 units in 6 animals (16 to 55 units per animal). Gastric balloon distension produced >20% increase in neuronal activity in an average of 48% of units to 10 ml inflation and 56% of units to 20 ml inflation. These results demonstrate the feasibility to record multiple single‐unit responses from the ferret NG to provide insight into gastric‐related population coding. Future effort could focus on delineating the physiological roles of these gastric neurons for the design of neuromodulation devices, which could be used to treat diseases involving vagal sensory input such as obesity and the chronic nausea associated with gastroparesis.Support or Funding InformationThis research was supported by funding from the NIH SPARC Common Fund Program (award #U18TR002205).

SPARC: The Structure and Location of Vagal Nociceptive Nerve Fibers in the Mouse Trachea

Vagal afferent innervation of the airways and lungs comprises of afferent fibers from vagal nodose neurons (developmentally derived from epibranchial placodes) and vagal jugular neurons (derived from neural crest). Here we evaluated the structure and the location in tracheal wall of afferent nerve fibers innervating the trachea focusing on the nociceptive TRPV1‐positive fibers (putative C‐fibers). We used genetic neuronal tracing in several mutant mice, including two new strains we generated. The wholemount preparations of the trachea were immunostained for fluorescent proteins and imaged by spinning‐disk confocal microscopy. In the TRPV1‐Flp/Tac1‐Cre/RC:FLTG mice (N=3) in which GFP is selectively expressed in cells positive for both TRPV1 and Tac1 (in the vagal system the marker of TRPV1‐positive fibers derived from neural crest) we observed dense innervation of the trachea. In the anterolateral tracheal wall the TRPV1+Tac1+ fibers were located beneath the epithelium in the mucosa above tracheal ligaments (which connect tracheal rings) running transversally (i.e. in parallel with tracheal rings). These fibers ramified richly and repetitively. In the dorsal (membranous) tracheal wall the TRPV1+Tac1+ fibers were also located beneath the epithelium in the mucosa above tracheal smooth muscle, running craniocaudally and often branching. These fibers occasionally penetrated through the tracheal smooth muscle. Some TRPV1+Tac1+ fibers were also observed in the epithelium in the sublaryngeal region of the trachea. In the TRPV1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP or AAV9‐FLEX‐tdTomato virus vector into vagal ganglia (N=4), in which only the vagal TRPV1‐positive fibers express GFP or tdTomato, the vagal TRPV1+ fibers had the same pattern as TRPV1+/Tac1+ fibers, but with expected lower density and patchy distribution. Similarly, in the Tac1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP vector into vagal ganglia (N=3) in which only the vagal Tac1‐positive fibers express GFP had the same pattern. In contrast, in the TRPV1‐Flp/P2X2‐Cre/RC:FLTG mice (N=3) in which GFP is selectively expressed in cells positive for both TRPV1 and P2X2 (the marker of vagal nociceptors derived from placodes) the TRPV1+/P2X2+ fibers were almost absent in the trachea. However, in the P2X2‐Cre mice unilaterally injected with AAV9‐FLEXEGFP vector into vagal ganglia (N=2) in which GFP is selectively expressed in vagal P2X2− positive fibers, the P2X2+ fibers had distinct distribution from TRPV1+Tac1+ and often located in the tracheal smooth muscle and tracheal epithelium. Our data indicate that vagal afferent nociceptive (TRPV1+) nerve fibers (putative C‐fibers) innervating the mouse trachea originate almost exclusively from jugular ganglion neurons. In contrast, vagal nodose ganglia project only TRPV1‐fibers into the trachea. Our results are in agreement with electrophysiological studies in other species.Support or Funding InformationNIH SPARC

Mapping of Sensory Nerve Subsets within the Vagal Ganglia and the Brainstem Using Reporter Mice for Pirt, TRPV1, 5-HT3, and Tac1 Expression.

Vagal afferent sensory nerves, originating in jugular and nodose ganglia, are composed of functionally distinct subsets whose activation evokes distinct thoracic and abdominal reflex responses. We used Cre-expressing mouse strains to identify specific vagal afferent populations and map their central projections within the brainstem. We show that Pirt is expressed in virtually all vagal afferents; whereas, 5-HT3 is expressed only in nodose neurons, with little expression in jugular neurons. Transient receptor potential vanilloid 1 (TRPV1), the capsaicin receptor, is expressed in a subset of small nodose and jugular neurons. Tac1, the gene for tachykinins, is expressed predominantly in jugular neurons, some of which also express TRPV1. Vagal fibers project centrally to the nucleus tractus solitarius (nTS), paratrigeminal complex, area postrema, and to a limited extent the dorsal motor nucleus of the vagus. nTS subnuclei preferentially receive projections by specific afferent subsets, with TRPV1+ fibers terminating in medial and dorsal regions predominantly caudal of obex, whereas TRPV1− fibers terminate in ventral and lateral regions throughout the rostral–caudal aspect of the medulla. Many vagal Tac1+ afferents (mostly derived from the jugular ganglion) terminate in the nTS. The paratrigeminal complex was the target of multiple vagal afferent subsets. Importantly, lung-specific TRPV1+ and Tac1+ afferent terminations were restricted to the caudal medial nTS, with no innervation of other medulla regions. In summary, this study identifies the specific medulla regions innervated by vagal afferent subsets. The distinct terminations provide a neuroanatomic substrate for the diverse range of reflexes initiated by vagal afferent activation.

Abdominal vagotomy reveals majority of small intestinal mucosal afferents labeled in nav 1.8cre-rosa26tdTomato mice are vagal in origin.

Vagal afferents innervating the small intestinal mucosa regulate feeding, gastrointestinal (GI) digestive, and immune functions. Their anatomical-functional characterization has been impeded by the inability to selectively label and manipulate them. Nav 1.8-Cre-tdTomato mice label 80% of nodose and dorsal root ganglia neurons. Here, the origin of these neuron's terminals and their distribution in the small intestinal mucosa were examined by quantitatively comparing tdTomato-labeled innervation in nonoperated (control), subdiaphragmatic vagotomy (VAGX), and sham-operated mice. Control mice exhibited a large proximal-to-distal decrease and a moderate mesentery-to-antimesentery decrease in villus innervation. VAGX reduced this innervation to a greater degree proximally (91-93%) than distally (65-72%), resulting in flat proximal-distal distributions. Therefore, estimates of vagal villus afferent distributions (control minus VAGX) paralleled control distributions, but were slightly reduced in magnitude. Compared with villus afferents, crypt innervation exhibited a muted proximal-to-distal decrease in control mice and a smaller loss after VAGX (45-48%). Sham-operated mice exhibited similar distributions of villus and crypt afferents as control mice, suggesting surgery did not contribute to the effects of VAGX. Most crypt and villus afferent terminals along the entire proximal-distal small intestinal axis had similar morphology to those previously reported in the proximal duodenum, but the density of terminal branches varied. Our findings suggest the majority of small intestinal mucosal innervation labeled in Nav 1.8-Cre-tdTomato mice is vagal in origin. Therefore, these mice will be valuable for studying vagal mucosal afferent morphology, interactions with other GI elements, plasticity, and function.

Role of TRP channels in Gq-coupled protease-activated receptor 1-mediated activation of mouse nodose pulmonary C-fibers.

We evaluated the mechanisms underlying protease-activated receptor 1 (PAR1)-mediated activation of nodose C-fibers in mouse lungs. The PAR1-induced action potential discharge at the terminals was strongly inhibited in phospholipase C-β3 (PLCβ3)-deficient animals. At the level of the cell soma, PAR1 activation led to an increase in cytosolic calcium that was largely inhibited by transient receptor potential (TRP) A1 antagonism. Patch-clamp recordings, however, revealed that neither TRPA1 nor TRPV1 or any other ruthenium red-sensitive ion channels are required for the PAR1-mediated inward current or membrane depolarization in isolated nodose neurons. Consistent with these findings, PAR1-mediated action potential discharge in mouse lung nodose C-fiber terminals was unaltered in Trpa1/Trpv1 double-knockout animals and Trpc3/Trpc6 double-knockout animals. The activation of the C-fibers was also not inhibited by ruthenium red at concentrations that blocked TRPV1- and TRPA1-dependent responses. The biophysical data show that PAR1/Gq-mediated activation of nodose C-fibers may involve multiple ion channels downstream from PLCβ3 activation. TRPA1 is an ion channel that participates in PAR1/Gq-mediated elevation in intracellular calcium. There is little evidence, however, that TRPA1, TRPV1, TRPC3, TRPC6, or other ruthenium red-sensitive TRP channels are required for PAR1/Gq-PLCβ3-mediated membrane depolarization and action potential discharge in bronchopulmonary nodose C-fibers in the mouse.

Transcriptional Profiling of Individual Airway Projecting Vagal Sensory Neurons.

Bronchopulmonary sensory neurons are derived from the vagal sensory ganglia and are essential for monitoring the physical and chemical environment of the airways and lungs. Subtypes are heterogenous in their responsiveness to stimuli, phenotype, and developmental origin, but they collectively serve to regulate normal respiratory and pulmonary processes and elicit a diverse range of defensive physiological responses that protect against noxious stimuli. In this study, we aimed to investigate the transcriptional features of vagal bronchopulmonary sensory neurons using single-cell RNA sequencing (scRNA-seq) to provide a deeper insight into their molecular profiles. Retrogradely labeled vagal sensory neurons projecting to the airways and lungs were hierarchically clustered into five types reflecting their developmental lineage (neural crest versus placodal) and putative function (nociceptors versus mechanoreceptors). The purinergic receptor subunit P2rx2 is known to display restricted expression in placodal-derived nodose neurons, and we demonstrate that the gene profiles defining cells high and low in expression of P2rx2 include G protein coupled receptors and ion channels, indicative of preferential expression in nodose or jugular neurons. Our results provide valuable insight into the transcriptional characteristics of bronchopulmonary sensory neurons and provide rational targets for future physiological investigations.

Vagal Transient Receptor Potential Ankyrin 1 Mediates Stress-exacerbated Visceral Mechanonociception After Antral Cold Exposure.

Background/AimsAbdominal pain can be evoked or exacerbated after gastrointestinal cold stimulation in some patients with diarrhea-predominant irritable bowel syndrome (IBS-D), indicating a low temperature-induced sensitization of visceral perception. We investigated the role of vagal transient receptor potential ankyrin 1 (TRPA1, a cold-sensing ion channel) in cold-aggravated visceral mechanonociception in a stress-induced IBS animal model.MethodsTRPA1 expression was examined in antral biopsies of healthy controls and IBS-D patients. Abdominal symptoms were assessed before and after warm or cold water intake. The visceromotor response (VMR) to colorectal distention (CRD) following intra-antral infusion of cold saline was measured in animals undergoing sham or chronic water avoidance stress. TRPA1 expression, extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation, and neuronal calcium influx in vagal afferents were assessed.ResultsCompared to healthy controls, IBS-D patients displayed elevated antral TRPA1 expression, which was associated with symptom scores after cold (4°C) water intake. Intra-antral infusion of cold saline increased VMR to CRD in naive rats, an effect dependent on vagal afferents. In stressed rats, this effect was greatly enhanced. Functional blockade and gene deletion of TRPA1 abolished the cold effect on visceral nociception. TRPA1 expression in vagal (but not spinal) afferents increased after stress. Moreover, the cold-induced, TRPA1-dependent ERK1/2 activation and calcium influx in nodose neurons were more robust in stressed rats.ConclusionsStress-exaggerated visceral mechanonociception after antral cold exposure may involve up-regulation of TRPA1 expression and function on vagal afferents. Our findings reveal a novel mechanism for abnormal gastrointestinal cold sensing in IBS.

Phenotypic distinctions between the nodose and jugular TRPV1-positive vagal sensory neurons in the cynomolgus monkey.

Vagal capsaicin-sensitive afferent C-fibers play an important role in the maintenance of visceral homeostasis and contribute to symptoms in visceral diseases. Based on their developmental origin two functionally distinct types of vagal C-fibers are recognized: those with neurons in the vagal nodose ganglia (derived from epibranchial placodes) and in the vagal jugular ganglia (from neural crest). Studies in nonprimate species demonstrated that the vagal nodose and jugular C-fibers differ in activation profile, neurotrophic regulation, and expression of neurotransmitters. We hypothesized that the expression of selected markers related to key phenotypic properties of vagal C-fibers in the cynomolgus monkey is similar to that reported in nonprimate species. We performed single-cell RT-PCR on nodose and jugular putative C-fiber (TRPV1-positive) neurons isolated from the cynomolgus monkey. We found that the expression of purinergic P2X receptors that underlie selective responsiveness of nodose C-fiber terminals to ATP was conserved in that P2X2 and P2X3 subunits were expressed in nodose, but only P2X3 subunit was expressed in jugular TRPV1-positive neurons. Also conserved was the preferential expression of neurotrophic receptor TrkB in the nodose and preprotachykinin-A in the jugular TRPV1-positive neurons. Several key distinctions in gene expression between nodose and jugular TRPV1-positive (C-fiber) neurons that have been noted in mice, rats, and guinea pigs, are conserved in the cynomolgus monkey. Our results support the translatability of distinct vagal C-fiber phenotypes to primates.

Effects of ginger constituent 6-shogaol on gastroesophageal vagal afferent C-fibers.

Ginger has been used as an herbal medicine worldwide to relieve nausea/vomiting and gastrointestinal discomfort, but the cellular and molecular mechanisms of its neuronal action remain unclear. The present study aimed to determine the effects of ginger constituent 6-shogaol on gastroesophageal vagal nodose C-fibers. Extracellular single-unit recording and two-photon nodose neuron imaging were performed, respectively, in ex vivo gastroesophageal-vagal preparations from wild type and Pirt-GCaMP6 transgenic mice. The action potential discharge or calcium influx evoked by mechanical distension and chemical perfusions applied to the gastroesophageal vagal afferent nerve endings were recorded, respectively, at their intact neuronal cell soma in vagal nodose ganglia. The effects of 6-shogaol on nodose C-fiber neurons were then compared and determined. Gastroesophageal application of 6-shogaol-elicited intensive calcium influxes in nodose neurons and evoked robust action potential discharges in most studied nodose C-fibers. Such activation effects were followed by a desensitized response to the second application of 6-shogaol. However, action potential discharges evoked by esophageal mechanical distension, after 6-shogaol perfusion, did not significantly change. Pretreatment with TRPA1 selective blocker HC-030031 inhibited 6-shogaol-induced action potential discharges in gastric and esophageal nodose C-fiber neurons, suggesting that TRPA1 played a role in mediating 6-shogaol-induced activation response. This study provides evidence that ginger constituent 6-shogaol directly activates vagal afferent C-fiber peripheral gastrointestinal endings. This activation leads to desensitization to subsequent application of 6-shogaol but not subsequent esophageal mechanical distension. Further investigation is required to establish a possible contribution in its anti-emetic effects.

Expression and Function of Oxytocin Signaling on Nodose Sensory Neuron Potassium Currents after Chronic Intermittent Hypoxia (CIH)

Hypothalamic‐derived oxytocin (OT) increases systemically and in the dorsal vagal complex in response to a variety of stimuli and stressors, such as hypoxia. Within the nucleus tractus solitarii (nTS), OT modifies baroreflex sensitivity suggestive of an influence of OT on presynaptic afferents originating in the nodose ganglia or postsynaptic nTS neurons. nTS slice studies demonstrated an excitatory influence of OT on afferent terminals, and nodose neurons express the OT receptor (OTR) whose activation elevates somal calcium. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. During CIH, however, enhancing OT in the dorsal vagal complex reduces blood pressure. To determine the mechanism(s) by which OT and OTR modulates sensory afferent‐nTS activity and mediated reflexes normally and after CIH we used immunohistochemistry, immunoblot, and electrophysiology on nodose ganglia tissue. Nodose ganglia of 5–6 week old male and female Sprague‐Dawley rats were studied after 10 days of Normoxia (Norm, 21% O2) or CIH (alternating 21% & 6% O2, 8 hr/day). Immunohistochemistry of cryostat‐sectioned (10 μm) nodose ganglia demonstrated OTRs in a subset of Norm somas and fibers. Immunoblot demonstrated a 0.52 fold reduction in OTR protein after CIH vs. Norm (n=4 each). OT plasma levels were reduced in CIH (n=12) when compared to Norm (n=10). To determine the functional effects of these changes, nodose neurons were dissociated, and 2–5 hours after isolation outward potassium currents (IK) were evoked via depolarizing voltages (10 mV steps, −70 to +60 mV, 140 ms) from a holding potential of −60 mV. The IK‐voltage relationship was measured in the presence of vehicle or 600 nM OT with or without the application of the OTR antagonist L‐368,899 (1 μM). OT‐sensitive IK was also characterized by the application of the K+ channel blockers Tetraethylammonium (TEA, 10 mM) or 4 ‐Aminopyridine (4‐AP, 5 mM), prior to and during the application of vehicle or 600 nM OT. In male nodose neurons, vehicle application after Norm and CIH did not alter IK, confirming no time dependent changes in IK. By contrast, OT decreased IK after Norm (n=7) and CIH (n=9), with the attenuation more prominent in CIH. The application of the OTR antagonist or TEA prior to and during 600 nM OT blocked the reduction of IK in CIH (n=5–7) and Norm (n=4–5). Conversely, 4‐AP did not block the OT‐reduction in IK after CIH (n=4) and Norm (n=6). To determine if there was a sex‐dependent influence of OT on IK, female nodose neurons were examined. OT induced a similar reduction of IK as males (n=5). Taken together, these data demonstrate OTR in nodose ganglia neurons and its activation via OT induces decreases IK that are consistent with increasing excitability in CIH. These data may also shed light on the influence of OT blood pressure control.Support or Funding InformationRO1 HL128454 & HL098602This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Potassium Channel Subtypes that Regulate Airway Vagal Sensory Nerve Excitability

Vagal sensory afferent nerves innervating the respiratory tracts play a defensive and homeostatic role in cardiopulmonary physiology. The majority of vagal afferent nerves in the airways are nociceptive in nature. Exaggerated activation of airway nociceptive nerve fibers by inflammatory mediators and other potentially noxious stimuli can lead to chronic cough, dyspnea, and aberrant reflex responses such as airway constriction and excessive mucus secretion. Most investigations on airway vagal nociceptive nerves have focused on the mediators and ion channels that underlie their activation while very little research attention has been given to the inhibitory K+ channels that operate at resting and/or subthreshold membrane potentials. These channels serve as brakes against excessive activation. In this study, we investigate the role of KCNQ/KV7/M‐channels, KCNA/KV1 channels and KCNK/two‐pore domain K+ (K2P) channels in regulating the intrinsic excitability of mouse nodose neurons and airway C‐fibers. Ultra‐low input RNA sequencing experiments showed that both lung‐specific and general population of isolated mouse nodose neurons express members of KV7, KV1, as well as TWIK, TASK and TREK subfamilies of K2P channels. Patch clamp recordings revealed an XE991‐sensitive KCNQ/KV7/M‐channel current (IM), α‐dendrotoxin (DTX)‐sensitive KV1‐mediated D‐current (ID) and an extracellular acidification‐inhibited TASK current in the majority of nodose neurons examined. IM activates around −65 mV with V1/2 for activation being at −38±3 mV. ID isolated with 10 nM α‐DTX has two components, and the low‐threshold component activates between −70 and −60 mV. At a holding potential of −60 mV, lowering the pH of the bath solution from 7.3 to 5.5 abolished the outward TASK current. The resting potential of mouse nodose neurons determined by perforated patch clamp technique averaged at −61±0.5 mV (n=65). Bath application of 10 μM M‐channel inhibitor XE991 or 30 nM ID channel blocker α‐DTX, or bath acidification led to a depolarization of 2 to 15 mV. Extracellular recordings of nodose neuron activities in the isolated and perfused vagus‐innervated mouse lung preparation revealed that the M‐channel opener retigabine applied locally to the lungs abolished the α,β‐meATP‐evoked action potential firing in pulmonary C‐fiber terminals. In the same kind of vagus‐lung preparations, application of 100 nM α‐DTX to the mouse lungs elicited action potential firing in about 20% of nodose fibers in the lungs as detected by two‐photon microscopy using mice expressing the Ca2+ indicator GCaMP6 under the Pirt promoter. In conclusion, our results indicate that KV7/M‐channels, KV1/ID channels and K2P/TASK channels contribute to maintaining the negative resting membrane potential in mouse nodose neurons and serve as a brake on their activation. Down regulation of these channels in visceral inflammatory diseases may contribute to sensory hyperexcitability. Alternatively, approaches enhancing the function of these K+ channels may be beneficial in the management of inflammatory diseases associated with vagal afferent hyperexcitability.(This study was conducted in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education).Support or Funding InformationR01 HL122228; R01 HL137807‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Airway Vagal Afferent C‐fibers by Type I and Type II Interferons

During respiratory viral infections and inflammatory airway diseases, both type I and type II interferons are elevated in the respiratory tract. Interferons are known for their ability to orchestrate anti‐viral defenses and other immunological responses in the airways. Our objective was to address the hypothesis that interferons may also play an important heretofore under‐appreciated role in recruiting the nocifensive nervous system during viral infection and inflammatory disease. We previously noted in an RNAseq evaluation that receptors for both type I and type II interferons are richly expressed by capsaicin‐sensitive vagal nodose and jugular C‐fiber neurons (PLoS One. 2017 Oct 5;12). In this study we show that both type I and II interferons (a, b, and g)directly and rapidly stimulate vagal C‐fiber terminals in mouse lungs to evoke action potential discharge.MethodsWe used single cell RT‐PCR specifically of nodose neurons retrogradely labeled from the mouse lungs to detect the presence of type I and II interferon receptors in airway afferent C‐fibers. To study action potential discharge from C‐fiber terminals we isolated the lungs, vagus nerve, along with vagal sensory ganglia from Pirt‐cre;GCaMP6smice. The nodose ganglion was positioned beneath a 20X, 2‐Photon microscope objective that assessed the elevations in calcium that were caused by action potentials conducted from the terminals in the lungs to cell bodies in the nodose ganglion. We noted that this system is sensitive enough to detect a single conducted action potential. This allowed us to simultaneously monitor the action potentials arriving at hundreds of vagal ganglion neurons in response to interferons infused specifically into the trachea/lung compartment.ResultsThe interferon receptors (ifnar1, ifnar2, ifngr1 and ifngr2) were expressed in 60–70% of airway‐specific TRPV1‐positive nodose neurons. In our 2 Photon imaging of the nodose ganglion we found that within minutes of perfusing the trachea/lungs with type I or II interferons action potentials were discharged from more than 70% of capsaicin‐sensitive C‐fibers (approximately 4000 neurons evaluated in 4 experiments). Vehicle perfusion was without effect. Among the interferons studies the relative responsiveness of vagal C‐fibers was b> g> aConclusionsThis study supports the hypothesis that type I and II interferons, in addition to orchestrating the immune responses by regulating chemokine and cytokine gene expression, can also more acutely recruit the nervous system by activating visceral sensory nociceptors. If dysregulated this may contribute to the nocifensive reflexes and sensations including bronchospasm, mucus secretion, urge to cough, and dyspnea, that can accompany airway inflammatory diseases and viral infections in asthmatic subjects.Support or Funding InformationNIH RO1HL137807, and David Marsh Symposium Research Award (Johns Hopkins)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

KCNQ/M-channels regulate mouse vagal bronchopulmonary C-fiber excitability and cough sensitivity.

Increased airway vagal sensory C-fiber activity contributes to the symptoms of inflammatory airway diseases. The KCNQ/Kv7/M-channel is a well-known determinant of neuronal excitability, yet whether it regulates the activity of vagal bronchopulmonary C-fibers and airway reflex sensitivity remains unknown. Here we addressed this issue using single-cell RT-PCR, patch clamp technique, extracellular recording of single vagal nerve fibers innervating the mouse lungs, and telemetric recording of cough in free-moving mice. Single-cell mRNA analysis and biophysical properties of M-current (IM) suggest that KCNQ3/Kv7.3 is the major M-channel subunit in mouse nodose neurons. The M-channel opener retigabine negatively shifted the voltage-dependent activation of IM, leading to membrane hyperpolarization, increased rheobase, and suppression of both evoked and spontaneous action potential (AP) firing in nodose neurons in an M-channel inhibitor XE991-sensitive manner. Retigabine also markedly suppressed the α,β-methylene ATP-induced AP firing in nodose C-fiber terminals innervating the mouse lungs, and coughing evoked by irritant gases in awake mice. In conclusion, KCNQ/M-channels play a role in regulating the excitability of vagal airway C-fibers at both the cell soma and nerve terminals. Drugs that open M-channels in airway sensory afferents may relieve the sufferings associated with pulmonary inflammatory diseases such as chronic coughing.

TMEM16B determines cholecystokinin sensitivity of intestinal vagal afferents of nodose neurons.

The satiety effects and metabolic actions of cholecystokinin (CCK) have been recognized as potential therapeutic targets in obesity for decades. We identified a potentially novel Ca2+-activated chloride (Cl-) current (CaCC) that is induced by CCK in intestinal vagal afferents of nodose neurons. The CaCC subunit Anoctamin 2 (Ano2/TMEM16B) is the dominant contributor to this current. Its expression is reduced, as is CCK current activity in obese mice on a high-fat diet (HFD). Reduced expression of TMEM16B in the heterozygote KO of the channel in sensory neurons results in an obese phenotype with a loss of CCK sensitivity in intestinal nodose neurons, a loss of CCK-induced satiety, and metabolic changes, including decreased energy expenditure. The effect on energy expenditure is further supported by evidence in rats showing that CCK enhances sympathetic nerve activity and thermogenesis in brown adipose tissue, and these effects are abrogated by a HFD and vagotomy. Our findings reveal that Ano2/TMEM16B is a Ca2+-activated chloride channel in vagal afferents of nodose neurons and a major determinant of CCK-induced satiety, body weight control, and energy expenditure, making it a potential therapeutic target in obesity.