Nodose Neurons Research Articles

MrgprC11+ Jugular Neurons Control Airway Hyperresponsiveness in Allergic Airway Inflammation.

The lung is densely innervated by sensory nerves, the majority of which are derived from the vagal sensory neurons. Vagal ganglia consist of two different ganglia, termed nodose and jugular ganglia, with distinct embryonic origins, innervation patterns, and physiological functions in the periphery. Since nodose neurons constitute the majority of the vagal ganglia, our understanding of the function of jugular nerves in the lung is very limited. This study aims to investigate the role of MrgprC11+ jugular sensory neurons in a mouse allergic asthma model. Our previous study has shown that MrgprC11+ jugular neurons mediate cholinergic bronchoconstriction. In this study, we found that in addition to MrgprC11, several other Mrgpr family members including MrgprA3, MrgprB4, and MrgprD are also specifically expressed in the jugular sensory neurons. MrgprC11+ jugular neurons exhibit dense innervation in the respiratory tract including the larynx, trachea, proximal, and distal bronchus. We also found that receptors for IL-4 and oncostatin M, two critical cytokines promoting allergic airway inflammation, are mainly expressed in jugular sensory neurons. Both IL-4 and oncostatin M can sensitize the neuronal responses of MrgprC11+ jugular neurons. Moreover, ablation of MrgprC11+ neurons significantly inhibited airway hyperresponsiveness in the asthmatic lung, demonstrating the critical role of MrgprC11+ neurons in controlling airway constriction. Our results emphasize the critical role of jugular sensory neurons in respiratory diseases.

Nodose afferent fibers require PIEZO2 for esophageal mechanosensation

The esophagus receives dual innervation of extrinsic afferent fibers from both the dorsal root ganglia as well as the vagal ganglia. These are polymodal sensory afferents that are able to sense a variety of stimuli that include chemical and mechanical stimuli. Previous research has demonstrated that the esophagus is innervated by low threshold mechanoreceptors arising from the nodose ganglia, which provide critical peripheral feedback that is required for proper swallowing and peristaltic reflexes. We know that in the vagal system, PIEZO2 plays a critical role in lung inflation and baroreceptor function in the aorta and carotid sinus. Hence, we hypothesize that PIEZO2 is the mechanotransducer in these nodose esophageal low threshold mechanoreceptors. We used a two-pronged approach to elucidate PIEZO2’s role in esophageal mechanosensation: first, GCaMP6s imaging and then single fiber electrophysiology to understand the kinetics of nodose esophageal afferent fiber activation. Both of these approaches used our ex-vivo intact vagal esophageal preparation. Our proof-of-principle studies were conducted in PIRT-GCaMP6s mice, obtained through the crossbreeding of Pirt-Cre (Cre expressed in all sensory neurons) mice with Cre-dependent, ROSA26 based, GCaMP6s reporter (B6.129S6-Gt(ROSA)26Sor-tm96(CAG-CGaMP6s)Hze). Our preliminary data in Pirt-GCaMP6s mice showed a profound increase in the Ca2+ influx in a subset of nodose neurons in response to mechanical distention of the esophagus at 5, 10, and 30mmHg pressures. For our knockout group, we obtained nodose-specific knockout of PIEZO2 by crossing P2X2-Cre mice (P2X2 is expressed in all nodose neurons) with PIEZO2fl/fl mice (P2X2/PIEZO2 KO mice). Upon performing our GCaMP6s imaging experiments in this group, we saw that there was a significant reduction in the number of nodose neurons that were responsive to esophageal distention (58% down to 17%). Comparing the fluorescence intensities of all esophageal neurons between the control and KO groups, we observed a significant reduction at all three intraesophageal pressures (p<0.05, n=458 and 395 neurons in control and KO groups, respectively). In our single fiber electrophysiology experiments, our control and KO groups were PIEZO2fl/fl mice and P2X2/PIEZO2 KO respectively. We were able to successfully record APs from A-fiber mechanosensitive esophageal neurons. The number of APs recorded from KO group was reduced at all the three intraesophageal pressures when compared to the control PIEZO2fl/fl mice. Our findings support the hypothesis that PIEZO2 is the mechanotransduction protein involved in vagal nodose afferent fibers innervating the esophagus. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Selective KCNQ2/3 Potassium Channel Opener ICA069673 Inhibits Excitability in Mouse Vagal Sensory Neurons

The excitability of vagal sensory neurons is abnormally enhanced in inflammatory visceral diseases, contributing to the unproductive and diffcult-to-treat neuronally based symptoms such as non-cardiac chest pain and excessive coughing. Identification of targets and modulators capable of regulating the excitability of vagal sensory neurons may lead to novel therapeutic options. The KCNQ/KV7/M-channel is increasingly recognized as an intrinsic regulator of excitability in neurons, and several neuronal KCNQ2/3-selective M-channel openers have been developed. This study aimed to characterize the effects of one of these KCNQ2/3-selective activators, ICA-069673, on the M-current (IM) in mouse nodose neurons, and to determine its effects on excitability and action potential firings using perforated patch-clamp technique. The results showed that ICA-069673 enhanced IM density, accelerated the activation and delayed the deactivation of M-channels in a concentration-dependent manner. The EC50 of ICA-069673 in enhancing IM determined at -30 mV is 0.52 μM. ICA-069673 at 10 μM shifted the voltage-dependent activation of IM in hyperpolarizing direction by -25.5 ± 4.1 mV (p<0.001) and increased the maximal conductance from 8.1 ± 4.0 to 12.6±7.4 nS/pF (p=0.031). In a total of 48 nodose neurons studied on voltage-clamp mode, ICA-069673 enhanced IM in 41 neurons (~85%). Consistent with its effects on IM, ICA-069673 induced a marked hyperpolarization of resting potential and reduced the input resistance. The hyperpolarizing effect was more pronounced in partially depolarized neurons. Moreover, ICA-069673 caused a 3-fold increase in the minimal amount of depolarizing current needed to evoke an action potential, and significantly limited the action potential firings in response to sustained suprathreshold stimulations. These results indicate that opening KCNQ2/3-mediated M-channels is suffcient to suppress the excitability and enhance spike accommodation in vagal sensory neurons, and provide evidence supporting further investigation into the effcacy of ICA-069673 in normalizing the aberrant vagal visceral nociception. The study is supported by NIH Grant R35HL155671. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Selective KCNQ2/3 Potassium Channel Opener ICA-069673 Inhibits Excitability in Mouse Vagal Sensory Neurons.

Heightened excitability of vagal sensory neurons in inflammatory visceral diseases contributes to unproductive and difficult-to-treat neuronally based symptoms such as visceral pain and dysfunction. Identification of targets and modulators capable of regulating the excitability of vagal sensory neurons may lead to novel therapeutic options. KCNQ1-KCNQ5 genes encode KV7.1-7.5 potassium channel α-subunits. Homotetrameric or heterotetrameric KV7.2-7.5 channels can generate the so-called M-current (IM) known to decrease the excitability of neurons including visceral sensory neurons. This study aimed to address the hypothesis that KV7.2/7.3 channels are key regulators of vagal sensory neuron excitability by evaluating the effects of KCNQ2/3-selective activator, ICA-069673, on IM in mouse nodose neurons and determining its effects on excitability and action potential firings using patch clamp technique. The results showed that ICA-069673 enhanced IM density, accelerated the activation, and delayed the deactivation of M-channels in a concentration-dependent manner. ICA-069673 negatively shifted the voltage-dependent activation of IM and increased the maximal conductance. Consistent with its effects on IM, ICA-069673 induced a marked hyperpolarization of resting potential and reduced the input resistance. The hyperpolarizing effect was more pronounced in partially depolarized neurons. Moreover, ICA-069673 caused a 3-fold increase in the minimal amount of depolarizing current needed to evoke an action potential, and significantly limited the action potential firings in response to sustained suprathreshold stimulations. ICA-069673 had no effect on membrane currents when Kcnq2 and Kcnq3 were deleted. These results indicate that opening KCNQ2/3-mediated M-channels is sufficient to suppress the excitability and enhance spike accommodation in vagal visceral sensory neurons. SIGNIFICANCE STATEMENT: This study supports the hypothesis that selectively activating KCNQ2/3-mediated M-channels is sufficient to suppress the excitability and action potential firings in vagal sensory neurons. These results provide evidence in support of further investigations into the treatment of various visceral disorders that involve nociceptor hyperexcitability with selective KCNQ2/3 M-channel openers.

Gut mucosal cells transfer α-synuclein to the vagus nerve.

Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson's disease. Although past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut-to-brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of intestinal mucosa express α-synuclein. Here, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from epithelial cells in organoids to cocultured nodose neurons devoid of α-synuclein. In mice expressing human α-synuclein, but no mouse α-synuclein, α-synuclein fibril-templating activity emerged in α-synuclein-seeded fibril aggregation assays in intestine, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfered to the vagus nerve and dorsal motor nucleus. Subdiaphragmatic vagotomy prior to induction of α-synuclein expression in intestinal epithelial cells effectively protected the hindbrain from emergence of α-synuclein fibril-templating activity. Overall, these findings highlight a potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.

Kv2 channels contribute to neuronal activity within the vagal afferent-nTS reflex arc.

Diversity in the functional expression of ion channels contributes to the unique patterns of activity generated in visceral sensory A-type myelinated neurons versus C-type unmyelinated neurons in response to their natural stimuli. In the present study, Kv2 channels were identified as underlying a previously uncharacterized delayed rectifying potassium current expressed in both A- and C-type nodose ganglion neurons. Kv2.1 and 2.2 appear confined to the soma and initial segment of these sensory neurons; however, neither was identified in their central presynaptic terminals projecting onto relay neurons in the nucleus of the solitary tract (nTS). Kv2.1 and Kv2.2 were also not detected in the peripheral axons and sensory terminals in the aortic arch. Functionally, in nodose neuron somas, Kv2 currents exhibited frequency-dependent current inactivation and contributed to action potential repolarization in C-type neurons but not A-type neurons. Within the nTS, the block of Kv2 currents does not influence afferent presynaptic calcium influx or glutamate release in response to afferent activation, supporting our immunohistochemical observations. On the other hand, Kv2 channels contribute to membrane hyperpolarization and limit action potential discharge rate in second-order neurons. Together, these data demonstrate that Kv2 channels influence neuronal discharge within the vagal afferent-nTS circuit and indicate they may play a significant role in viscerosensory reflex function.NEW & NOTEWORTHY We demonstrate the expression and function of the voltage-gated delayed rectifier potassium channel Kv2 in vagal nodose neurons. Within sensory neurons, Kv2 channels limit the width of the broader C-type but not narrow A-type action potential. Within the nucleus of the solitary tract (nTS), the location of the vagal terminal field, Kv2 does not influence glutamate release. However, Kv2 limits the action potential discharge of nTS relay neurons. These data suggest a critical role for Kv2 in the vagal-nTS reflex arc.

Mechanical activation of vagal afferents involves opposing cation and TREK1 currents and NO regulation.

Vagal afferents convey signals of mechanical stimulation in the gut to the brain, which is essential for the regulation of food intake. However, ion channels sensing mechanical stimuli are not fully understood. This study aimed to examine the ionic currents activated by mechanical stimulation and a possible neuro-modulatory role of nitric oxide on vagal afferents. Nodose neuronal currents and potentials, and intestinal afferent firing by mechanical stimulation were measured by whole-cell patch clamp, and in vitro afferent recording, respectively. Osmotically activated cation and two-pore domain K+ currents were identified in nodose neurons. The membrane potential displayed a biphasic change under hypotonic stimulation. Cation channel-mediated depolarization was followed by a hyperpolarization mediated by K+ channels. The latter was inhibited by l-methionine (TREK1 channel inhibitor) and l-NNA (nitric oxide synthase inhibitor). Correspondingly, mechanical stimulation activated opposing cation and TREK1 currents. NOS inhibition decreased TREK1 currents and potentiated jejunal afferent nerve firing induced by mechanical stimuli. This study suggested a novel activation mechanism of ion channels underlying adaptation under mechanical distension in vagal afferent neurons. The guts' ability to perceive mechanical stimuli is vital in determining how it responds to food intake. The mechanosensation through ion channels could initiate and control gut function.

Genetic inhibition of KV7/M-channels increases the excitability of bronchopulmonary vagal nociceptive nerves

The KV7/M-channel, encoded by Kcnq2-5 genes, generates a “braking” potassium current (IM) and contributes to the regulation of neuronal excitability. We have previously reported that the vagal sensory nerves terminated in the mouse lungs expressed mainly Kcnq3 and Kcnq2, as well as the functional M-channels. Opening the M-channel with retigabine almost abolished the agonist-evoked action potential (AP) discharges in bronchopulmonary nociceptive C-fibers, and suppressed the irritant gases-induced, C-fiber-mediated coughing in awake freely moving mice. Inhibition of IM using XE991 slightly but consistently depolarized the nodose neurons. The use of XE991 to evaluate whether IM plays a role in regulating the activation of bronchopulmonary vagal sensory nerves was, however, limited due to its off-target effects on the fast Na+ current in these neurons. This study aimed to address this key issue using the genetic approach. We generated the sensory neuron-specific Kcnq2 and Kcnq3 double-KO mice (Kcnq2/3-KO) by crossing the Kcnq2/3fl/fl mice with the Pirt-driven Cre (Pirt-Cre) mice. Single-neuron RT-PCR assay showed that none of the 60 tested Kcnq2/3-KO nodose neurons (30 Trpv1-positive and 30 Trpv1-negative) expressed Kcnq2 or Kcnq3. Eliminating Kcnq2 and Kcnq3 did not increase Kcnq4 or Kcnq5 expression. IM, defined as the retigabine-enhanced and XE991-inhibited tail currents (elicited by repolarizing steps from -30 to -60 mV), was recorded in all 22 control neurons using perforated patch clamp technique, but only in 2 out of 14 Kcnq2/3-KO nodose neurons (probably due to Kcnq5) with a much lower IM density (0.4 and 2.1 pA/pF vs. 8.5±2.9 pA/pF in controls). These results confirmed an effective elimination of the dominant KV7 genes in the nodose neurons of our Kcnq2/3-KO mice. Genetic inhibition of Kcnq2/3 did not significantly alter the resting potential (-68.0±1.9, n=12 vs. -69.1±1.4 mV, n=13; p=0.644) or the minimal amount of currents to evoke a single AP (rheobase: 41.2±6.1 pA, n=12 vs. 48.1±6.2 pA, n=13; p=0.442) in capsaicin-sensitive nodose neurons. However, the nociceptive nodose neurons isolated from Kcnq2/3-KO mice fired more APs and at a higher frequency in response to a series of either step (50-500 pA, 600 ms) or ramp (50-800 pA, 1 second) depolarization compared to their littermate controls (P<0.001, two-way ANOVA). Extracellular recordings in ex-vivo vagally innervated trachea-lungs preparations also revealed that Kcnq2/3 deficiency led to enhanced AP discharges at the nerve endings of the bronchopulmonary C-fibers in response to the agonist of Gq-coupled protease-activated receptor 1 (3, 10 and 30 μM) applied to the mouse lungs via trachea (number of AP: 393±45, n=8 vs. 183±43, n=9; p=0.001 by two-way ANOVA). These results indicate that the M-channel may serve as an intrinsic inhibitory mechanism in the bronchopulmonary vagal nociceptive neurons by limiting the excessive repetitive AP firings. Johns Hopkins Blaustein Pain Research Funds Grant 80048814, and NIH R01 HL137807/R35 HL155671 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of NaV 1.9 in inflammatory mediator-induced activation of mouse airway vagal C-fibres.

The influence of NaV 1.9 on inflammatory mediator-induced activation of airway vagal nodose C-fibres was evaluated by comparing responses in wild-type versus NaV 1.9-/- mice. A single-cell RT-PCR analysis indicated that virtually all nodose C-fibre neurons expressed NaV 1.9 (SCN11A) mRNA. Using extracellular electrophysiological recordings in an isolated vagally innervated mouse trachea-lung preparation, it was noted that mediators acting via G protein-coupled receptors (PAR2), or ionotropic receptors (P2×3) were 70-85% less effective in evoking action potential discharge in the absence of NaV 1.9. However, there was no difference in action potential discharge between wild-type and NaV 1.9-/- when the stimulus was a rapid punctate mechanical stimulus. An analysis of the passive and active properties of isolated nodose neurons revealed no difference between neurons from wild-type and NaV 1.9-/- mice, with the exception of a modest difference in the duration of the afterhyperpolarization. There was also no difference in the amount of current required to evoke action potentials (rheobase) or the action potential voltage threshold. The inward current evoked by the chemical mediator by a P2×3 agonist was the same in wild-type versus NaV 1.9-/- neurons. However, the current was sufficient to evoke action potential only in the wild-type neurons. The data support the speculation that NaV 1.9 could be an attractive therapeutic target for inflammatory airway disease by selectively inhibiting inflammatory mediator-associated vagal C-fibre activation. KEY POINTS: Inflammatory mediators were much less effective in activating the terminals of vagal airway C-fibres in mice lacking NaV 1.9. The active and passive properties of nodose neurons were the same between wild-type neurons and NaV 1.9-/- neurons. Nerves lacking NaV 1.9 responded, normally, with action potential discharge to rapid punctate mechanical stimulation of the terminals or the rapid stimulation of the cell bodies with inward current injections. NaV 1.9 channels could be an attractive target to selectively inhibit vagal nociceptive C-fibre activation evoked by inflammatory mediators without blocking the nerves' responses to the potentially hazardous stimuli associated with aspiration.

Diabetes mellitus differently affects electrical membrane properties of vagal afferent neurons of rats.

To study whether diabetes mellitus (DM) would cause electrophysiological alterations in nodose ganglion (NG) neurons, we used patch clamp and intracellular recording for voltage and current clamp configuration, respectively, on cell bodies of NG from rats with DM. Intracellular microelectrodes recording, according to the waveform of the first derivative of the action potential, revealed three neuronal groups (A0 , Ainf , and Cinf ), which were differently affected. Diabetes only depolarized the resting potential of A0 (from -55 to -44 mV) and Cinf (from -49 to -45 mV) somas. In Ainf neurons, diabetes increased action potential and the after-hyperpolarization durations (from 1.9 and 18 to 2.3 and 32 ms, respectively) and reduced dV/dtdesc (from -63 to -52 V s-1 ). Diabetes reduced the action potential amplitude while increasing the after-hyperpolarization amplitude of Cinf neurons (from 83 and -14 mV to 75 and -16 mV, respectively). Using whole cell patch clamp recording, we observed that diabetes produced an increase in peak amplitude of sodium current density (from -68 to -176 pA pF-1 ) and displacement of steady-state inactivation to more negative values of transmembrane potential only in a group of neurons from diabetic animals (DB2). In the other group (DB1), diabetes did not change this parameter (-58 pA pF-1 ). This change in sodium current did not cause an increase in membrane excitability, probably explainable by the alterations in sodium current kinetics, which are also induced by diabetes. Our data demonstrate that diabetes differently affects membrane properties of different nodose neuron subpopulations, which likely have pathophysiological implications for diabetes mellitus.

Ginger Constituent 6-Shogaol Attenuates Vincristine-Induced Activation of Mouse Gastroesophageal Vagal Afferent C-Fibers.

Chemotherapeutic agent-induced nausea and vomiting are the severe adverse effects that are induced by their stimulations on the peripheral and/or central emetic nerve pathways. Even though ginger has been widely used as an herbal medicine to treat emesis, mechanisms underlying its neuronal actions are still less clear. The present study aimed to determine the chemotherapeutic agent vincristine-induced effect on gastroesophageal vagal afferent nerve endings and the potential inhibitory role of ginger constituent 6-shogaol on such response. Two-photon neuron imaging studies were performed in ex vivo gastroesophageal-vagal preparations from Pirt-GCaMP6 transgenic mice. Vincristine was applied to the gastroesophageal vagal afferent nerve endings, and the evoked calcium influxes in their intact nodose ganglion neuron somas were recorded. The responsive nodose neuron population was first characterized, and the inhibitory effects of 5-HT3 antagonist palonosetron, TRPA1 antagonist HC-030031, and ginger constituent 6-shogaol were then determined. Vincristine application at gastroesophageal vagal afferent nerve endings elicited intensive calcium influxes in a sub-population of vagal ganglion neurons. These neurons were characterized by their positive responses to P2X2/3 receptor agonist α,β-methylene ATP and TRPA1 agonist cinnamaldehyde, suggesting their nociceptive placodal nodose C-fiber neuron lineages. Pretreatment with TRPA1 selective blocker HC-030031 inhibited vincristine-induced calcium influxes in gastroesophageal nodose C-fiber neurons, indicating that TRPA1 played a functional role in mediating vincristine-induced activation response. Such inhibitory effect was comparable to that from 5-HT3 receptor antagonist palonosetron. Alternatively, pretreatment with ginger constituent 6-shogaol significantly attenuated vincristine-induced activation response. The present study provides new evidence that chemotherapeutic agent vincristine directly activates vagal nodose nociceptive C-fiber neurons at their peripheral nerve endings in the upper gastrointestinal tract. This activation response requires both TRPA1 and 5-HT3 receptors and can be attenuated by ginger constituent 6-shogaol.

The Braking KV1/D‐type Potassium Channels Regulate the Excitability of Bronchopulmonary Vagal Afferent Nerves

The proper function of airway vagal afferent nerves is essential for the dynamic regulation of breathing and initiation of adequate airway defensive reflexes. A better understanding of the mechanisms controlling their activation bears physiological and pathological significance. The voltage‐gated potassium (KV) channels exert powerful controls on the neuronal excitability. Yet their expression and functions in the bronchopulmonary vagal afferent neurons remain largely unexplored. In this study we characterized the expression profile and inhibitory function of α‐dendrotoxin (α‐DTX)‐sensitive D‐type K+ channels composed of members of KV1subfamily of α‐subunits in mouse bronchopulmonary nodose neurons using a combination of single‐neurons RT‐PCR, patch clamp recording, ex‐vivo extracellular recording and two‐photon microscopic Ca2+ imaging techniques. The results showed that the vast majority of retrogradely labeled lung‐specific nodose neurons expressed Kcna1, 2 and 6 coding for the α‐DTX‐sensitive KV1.1, 1.2 and 1.6 α‐subunits, respectively. The D‐type K+ current (IK.D), defined as the α‐DTX‐sensitive K+ current, was recorded in 14/15 bronchopulmonary nodose neurons. It started to be activated at ‐65.7±4.3 mV with 50% channel activation at ‐24 ± 14 mV. IK.D opened rapidly in response to depolarization (activation time constants <10 ms at voltage ≥‐15 mV), and displayed no inactivation during 600 ms at subthreshold/ threshold voltages and little or slow inactivation at more positive potentials (≥‐5 mV). Inhibition of IK.D with 50 nM α‐DTX depolarized mouse pulmonary nodose neurons, increased input resistance, lowered the minimal depolarizing current needed to evoke an action potential, and increased the number and frequency of action potential firing in response to depolarizing current injections. Application of a bolus of 100 nM α‐DTX to the afferent terminals in the mouse lungs via trachea led to overt activation in 49% of capsaicin‐sensitive nodose neurons and in 32% of capsaicin‐insensitive nodose neurons as detected by two‐photon microscope. Further study with extracellular recording revealed that the application of an α‐DTX bolus evoked action potential discharges in 8/19 (42%) nodose C‐fibers terminating in the mouse lungs with a peak firing frequency of 4.4 ± 3.9 Hz. These results indicate that both the soma and terminals of bronchopulmonary nodose afferent nerves express functional IK.D characterized by low‐threshold, fast activation, and lack of or slow inactivation. With these unique biophysical properties, IK.D channels act as an activation brake on the airway vagal afferent nerves by stabilizing the resting membrane potential and by counterbalancing the subthreshold depolarizing forces. Down‐regulation of IK.D, as occurs in many inflammatory diseases, may result in a heightened excitability of airway vagal afferent nerves causing exaggerated dyspnea, excessive mucous secretion, bronchoconstriction and chronic unproductive coughs associated with airway inflammation.

High resolution retrograde tracing, morphological analysis, RNA sequencing and differential expression analysis of identified vagal afferent neurons innervating the muscle or mucosa in 3 different stomach regions in the rat

The functions of the stomach are powerfully influenced by the vagus nerve and vago‐vagal reflexes. The vagal afferent neurons that innervate the stomach and monitor its functional state have clearly demonstrated roles in appetite regulation, gastric accommodation and gastric emptying. In mammals with single compartment stomachs, including humans, the stomach can be divided into functional regions and the muscle and mucosal layers show region‐specific specializations. Electrophysiological and morphological studies suggest that the vagal sensory axons innervating the different regions and layers of the stomach wall have distinct properties and participate in different types of reflex controls. We therefore hypothesized that these functional and structural differences would be reflected in distinct gene expression profiles of afferent neurons, depending on the stomach region and tissue innervated. We developed methods to selectively retrogradely label afferent neurons innervating the muscle or mucosa in the corpus and antrum and the muscle in the fundus in the rat, using local microinjections of fluorescent nanospheres that diffuse minimally. Up to 30 microinjections of 30‐100nL of tracer beads were made into either the muscle or the mucosa in one stomach region in each rat under anesthesia. The animal was allowed to recover and kept for 1‐2 weeks before being humanely killed and tissues harvested. Labelled neurons were studied in situ in the nodose ganglion using confocal microscopy, immunohistochemistry and fluorescence in situ hybridization, and individual neurons collected after dissociation for RNAseq analysis. RNA sequencing was performed using pooled samples of 5‐12 neurons labelled from each region and deep sequencing (30‐50 million reads per sample), and then analyzed using minimal filtering to allow detection of genes expressed at low copy number.Nodose neurons labelled from the mucosa were significantly larger than those labelled from the muscle in both the corpus and antrum, whether quantified in situ or after dissociation, confirming that microinjections into the different stomach wall layers labelled different populations of neurons. Pairwise differential expression analyses of RNAseq data showed distinct mRNA expression profiles depending on the region innervated, and between muscle and mucosal innervating neurons in each region, based on Benjamini/Hochberg adjusted P values (Limma analysis tool). Some genes displayed region specific expression patterns while others showed tissue specific patterns (eg muscle vs mucosa) and some showed both. Among differentially expressed genes were some previously identified to specifically label gastric mucosal and muscle afferent neurons, such as somatostatin, CCKB receptor and GRP65. In addition, many other genes were differentially expressed and mRNA for genes known to be functional in vagal afferent neurons, such as ghrelin, leptin and galanin receptors, were detected. Differential expression patterns for various genes were confirmed in labelled neurons in situ using immunohistochemistry and/or in situhybridization. The data provide a rich resource for further studies of the visceral sensation in the different stomach regions and how region‐specific signaling might regulate appetite and gastric function in both normal and disease states.

Roles of Nav1.9 in inflammatory Mediator‐induced Activation of Vagal C‐fibers Innervating Mouse Lungs

We used NaV1.9 knockout (KO) mice to evaluate the role of NaV1.9 in chemical activation of vagal C‐fiber terminals in the mouse lungs. Single‐cell rt‐PCR of lung‐labeled neurons revealed that the vast majority of TRPV1 expressing vagal sensory neurons innervating the lungs express NaV1.9 mRNA. The mouse lungs were isolated with the vagus nerves and vagal ganglia intact. Action potentials (APs) were recorded using extracellular electrodes positioned near the relevant cell bodies in the vagal ganglion. The stimuli were applied to the receptive field via perfusion through the mouse trachea. The PAR1 agonist, TFLLR(10mM) evoked 270 ± 62 APs in wild type mice (n=23), but only 85 ± 38 action potentials in NaV1.9 ‐/‐ mice (P< 0.01, n=29). PAR1 is a GPCR stimulus, so we next evaluated a,b‐methylene ATP (1ml, 1µM) that stimulates nodose C‐fibers in the mouse lung via activation of the ionotropic P2X2/3 receptor. a,b‐Methylene ATP evoked 93 ± 18 APs in wildtype mice (n=33), compared to only 13 ± 4 APs the NaV1.9 ‐/‐ mice (P=0.0001, n= 35). The C‐fibers of both WT and NaV1.9 ‐/‐ were equally activated by rapid punctate mechanical stimulation of the receptive field with Von Frey fibers. At the patched clamped cell soma, there was no difference in active or passive electrophysiological properties of nodose neurons isolated from WT versus KO animals; rheobases were also the same with AP threshold averaging 0.34 ± 0.1 and 0.29 ± 0.06 mV/ms, respectively. However, bath‐applied ATP was more effective in evoking action potentials in neurons isolated from WT (40 % responding) vs. KO (0 % responding). The data reveal that NaV1.9 is important in the activation of nodose C‐fiber terminals evoked by inflammatory mediators that act via GPCRs or ionotropic receptors but less critical in the activation by rapid punctate mechanical or electrical stimulation. NaV1.9 provides an intriguing target for treating symptoms caused by inflammatory mediator‐induced C‐fiber activation in the airways, e.g. chronic coughing and excessive reflex bronchospasm and secretions associated with inflammatory airway disease.

KV 1/D-type potassium channels inhibit the excitability of bronchopulmonary vagal afferent nerves.

The KV1/D‐type potassium current (I D) is an important determinant of neuronal excitability. This study explored whether and how I D channels regulate the activation of bronchopulmonary vagal afferent nerves. The single‐neuron RT‐PCR assay revealed that nearly all mouse bronchopulmonary nodose neurons expressed the transcripts of α‐dendrotoxin (α‐DTX)‐sensitive, I D channel‐forming KV1.1, KV1.2 and/or KV1.6 α‐subunits, with the expression of KV1.6 being most prevalent. Patch‐clamp recordings showed that I D, defined as the α‐DTX‐sensitive K+ current, activated at voltages slightly more negative than the resting membrane potential in lung‐specific nodose neurons and displayed little inactivation at subthreshold voltages. Inhibition of I D channels by α‐DTX depolarized the lung‐specific nodose neurons and caused an increase in input resistance, decrease in rheobase, as well as increase in action potential number and firing frequency in response to suprathreshold current steps. Application of α‐DTX to the lungs via trachea in the mouse ex vivo vagally innervated trachea–lungs preparation led to action potential discharges in nearly half of bronchopulmonary nodose afferent nerve fibres, including nodose C‐fibres, as detected by the two‐photon microscopic Ca2+ imaging technique and extracellular electrophysiological recordings. In conclusion, I D channels act as a critical brake on the activation of bronchopulmonary vagal afferent nerves by stabilizing the membrane potential, counterbalancing the subthreshold depolarization and promoting the adaptation of action potential firings. Down‐regulation of I D channels, as occurs in various inflammatory diseases, may contribute to the enhanced C‐fibre activity in airway diseases that are associated with excessive coughing, dyspnoea, and reflex bronchospasm and secretions.Key points The α‐dendrotoxin (α‐DTX)‐sensitive D‐type K+ current (I D) is an important determinant of neuronal excitability.Nearly all bronchopulmonary nodose afferent neurons in the mouse express I D and the transcripts of α‐DTX‐sensitive, I D channel‐forming KV1.1, KV1.2 and/or KV1.6 α‐subunits.Inhibition of I D channels by α‐DTX depolarizes the bronchopulmonary nodose neurons, reduces the minimal depolarizing current needed to evoke an action potential (AP) and increases AP number and AP firing frequency in response to suprathreshold stimulations.Application of α‐DTX to the lungs ex vivo elicits AP discharges in about half of bronchopulmonary nodose C‐fibre terminals. Our novel finding that I D channels act as a critical brake on the activation of bronchopulmonary vagal afferent nerves suggests that their down‐regulation, as occurs in various inflammatory diseases, may contribute to the enhanced C‐fibre activity in airway inflammation associated with excessive respiratory symptoms.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization.

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

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

Aerosolized adenosine 5'-triphosphate (ATP) induces cough and bronchoconstriction by activating vagal sensory fibers' P2X3 and P2X2/3 receptors (P2X3R and P2X2/3R). The goal of this study is to determine the effect of these receptors on the superior laryngeal nerve (SLN)-mediated cardiorespiratory responses to ATP challenge. We compared the cardiorespiratory responses to intralaryngeal perfusion of either ATP or α,β-methylene ATP in rat pups before and after 1) intralaryngeal perfusion of A-317491 (a P2X3R and P2X2/3R antagonist); 2) bilateral section of the SLN; and 3) peri-SLN treatment with capsaicin (to block conduction in superior laryngeal C-fibers, SLCFs) or A-317491. The immunoreactivity (IR) of P2X3R and P2X2R was determined in laryngeal sensory neurons of the nodose/jugular ganglia. Lastly, a whole cell patch clamp recording was used to determine ATP- or α,β-methylene ATP (α,β-mATP)-induced currents without and with A-317491 treatment. It was found that intralaryngeal perfusion of both ATP and α,β-mATP induced immediate apnea, hypertension, and bradycardia. The apnea was eliminated and the hypertension and bradycardia were blunted by intralaryngeal perfusion of A-317491 and peri-SLN treatment with either A-317491 or capsaicin, although all of the cardiorespiratory responses were abolished by bilateral section of the SLN. P2X3R- and P2X2R-IR were observed in nodose and jugular ganglionic neurons labeled by fluoro-gold (FG). ATP- and α,β-mATP-induced currents recorded in laryngeal C-neurons were reduced by 75% and 95%, respectively, by the application of A-317491. It is concluded that in anesthetized rat pups, the cardiorespiratory responses to intralaryngeal perfusion of either ATP or α,β-mATP are largely mediated by the activation of SLCFs' P2X3R-P2X2/3R.NEW & NOTEWORTHY Aerosolized ATP induces cough and bronchoconstriction via activating P2X3 and P2X2/3 receptors (P2X3R and P2X2/3R) localized on vagal pulmonary sensory fibers. The superior laryngeal nerve (SLN), particularly SLN C-fibers (SLCFs), is involved in generating apnea, hypertension, and bradycardia. This study demonstrates for the first time that either ATP or α,β-mATP applied onto the laryngeal mucosa elicit these cardiorespiratory responses predominately through the activation of P2X3R-P2X2/3R localized on SLCFs.

Capsaicin-Sensitive Vagal Afferent Nerve-Mediated Interoceptive Signals in the Esophagus.

Heartburn and non-cardiac chest pain are the predominant symptoms in many esophageal disorders, such as gastroesophageal reflux disease (GERD), non-erosive reflux disease (NERD), functional heartburn and chest pain, and eosinophilic esophagitis (EoE). At present, neuronal mechanisms underlying the process of interoceptive signals in the esophagus are still less clear. Noxious stimuli can activate a subpopulation of primary afferent neurons at their nerve terminals in the esophagus. The evoked action potentials are transmitted through both the spinal and vagal pathways to their central terminals, which synapse with the neurons in the central nervous system to induce esophageal nociception. Over the last few decades, progress has been made in our understanding on the peripheral and central neuronal mechanisms of esophageal nociception. In this review, we focus on the roles of capsaicin-sensitive vagal primary afferent nodose and jugular C-fiber neurons in processing nociceptive signals in the esophagus. We briefly compare their distinctive phenotypic features and functional responses to mechanical and chemical stimulations in the esophagus. Then, we summarize activation and/or sensitization effects of acid, inflammatory cells (eosinophils and mast cells), and mediators (ATP, 5-HT, bradykinin, adenosine, S1P) on these two nociceptive C-fiber subtypes. Lastly, we discuss the potential roles of capsaicin-sensitive esophageal afferent nerves in processing esophageal sensation and nociception. A better knowledge of the mechanism of nociceptive signal processes in primary afferent nerves in the esophagus will help to develop novel treatment approaches to relieve esophageal nociceptive symptoms, especially those that are refractory to proton pump inhibitors.

Effect of high-fat diet on mechanosensitive transient receptor potential channel activation in vagal afferent neurons.

Mechanical stimulation of the gastrointestinal tract is an important stimulus of satiety and can be transduced by transient receptor potential (TRP) channels. Several studies have revealed attenuated vagally-mediated satiety responses including mechanosensitivity in diet-induced obesity; however, ion channels underlying this hyposensitivity have not been fully understood. This study aimed to examine the effect of chronic high-fat diet on activation of selected mechanosensitive TRP channels in vagal afferents. C57/BL6 mice were fed on either a high-fat or low-fat diet for 6-8weeks. An increase in the intracellular calcium to hypotonic solution and activators of TRPV1, TRPV4, and TRPA1 was measured in nodose neurons using Ca2+-imaging techniques. Jejunal afferent nerve firing induced by mechanical stimulation and TRP channel agonists was measured using invitro extracellular multiunit afferent recording. In high-fat diet-fed mice, we observed reduced calcium influx and jejunal afferent response induced by mechanical stimuli and agonists of TRPV4 and TRPA1, but not TRPV1. Our data show diet-induced obesity disrupts the activation of TRPV4 and TRPA1, at both the cellular level and the level of nerve terminals in the small intestine, which may partly explain reduced mechanosensitivity of vagal afferents and may contribute to decreased gut-brain satiety signaling in obesity.

Pancreatic β-Cells Communicate With Vagal Sensory Neurons

Destroying visceral sensory nerves impacts pancreatic islet function, glucose metabolism, and diabetes onset, but how islet endocrine cells interact with sensory neurons has not been studied. We characterized the anatomical pattern of pancreatic sensory innervation by combining viral tracing, immunohistochemistry, and reporter mouse models. To assess the functional interactions of β-cells with vagal sensory neurons, we recorded Ca2+ responses in individual nodose neurons invivo while selectively stimulating β-cells with chemogenetic and pharmacologic approaches. We found that pancreatic islets are innervated by vagal sensory axons expressing Phox2b, substance P, calcitonin-gene related peptide, and the serotonin receptor 5-HT3R. Centrally, vagal neurons projecting to the pancreas terminate in the commissural nucleus of the solitary tract. Nodose neurons responded invivo to chemogenetic stimulation of β-cells and to pancreas infusion with serotonin, but were not sensitive to insulin. Responses to chemogenetic and pharmacologic stimulation of β-cells were blocked by a 5-HT3R antagonist and were enhanced by increasing serotonin levels in β-cells. We further confirmed directly in living pancreas slices that sensory terminals in the islet were sensitive to serotonin. Our study establishes that pancreatic β-cells communicate with vagal sensory neurons, likely using serotonin signaling as a transduction mechanism. Serotonin is coreleased with insulin and may therefore convey information about the secretory state of β-cells via vagal afferent nerves.