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.

Activation of Intra-nodose Ganglion P2X7 Receptors Elicit Increases in Neuronal Activity.

Vagus nerve innervates several organs including the heart, stomach, and pancreas among others. Somas of sensory neurons that project through the vagal nerve are located in the nodose ganglion. The presence of purinergic receptors has been reported in neurons and satellite glial cells in several sensory ganglia. In the nodose ganglion, calcium depletion-induced increases in neuron activity can be partly reversed by P2X7 blockers applied directly into the ganglion. The later suggest a possible role of P2X7 receptors in the modulation of neuronal activity within this sensory ganglion. We aimed to characterize the response to P2X7 activation in nodose ganglion neurons under physiological conditions. Using an ex vivo preparation for electrophysiological recordings of the neural discharges of nodose ganglion neurons, we found that treatments with ATP induce transient neuronal activity increases. Also, we found a concentration-dependent increase in neural activity in response to Bz-ATP (ED50 = 0.62 mM, a selective P2X7 receptor agonist), with a clear desensitization pattern when applied every ~ 30 s. Electrophysiological recordings from isolated nodose ganglion neurons reveal no differences in the responses to Bz-ATP and ATP. Finally, we showed that the P2X7 receptor was expressed in the rat nodose ganglion, both in neurons and satellite glial cells. Additionally, a P2X7 receptor negative allosteric modulator decreased the duration of Bz-ATP-induced maximal responses without affecting their amplitude. Our results show the presence of functional P2X7 receptors under physiological conditions within the nodose ganglion of the rat, and suggest that ATP modulation of nodose ganglion activity may be in part mediated by the activation of P2X7 receptors.

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.

Identification of lung innervating sensory neurons and their target specificity.

Known as the gas exchange organ, the lung is also critical for responding to the aerosol environment in part through interaction with the nervous system. The diversity and specificity of lung innervating neurons remain poorly understood. Here, we interrogated the cell body location and molecular signature and projection pattern of lung innervating sensory neurons. Retrograde tracing from the lung coupled with whole tissue clearing highlighted neurons primarily in the vagal ganglia. Centrally, they project specifically to the nucleus of the solitary tract in the brainstem. Peripherally, they enter the lung alongside branching airways. Labeling of nociceptor Trpv1+ versus peptidergic Tac1+ vagal neurons showed shared and distinct terminal morphology and targeting to airway smooth muscles, vasculature including lymphatics, and alveoli. Notably, a small population of vagal neurons that are Calb1+ preferentially innervate pulmonary neuroendocrine cells, a demonstrated airway sensor population. This atlas of lung innervating neurons serves as a foundation for understanding their function in lung.

Pancreatic ß-Cells Communicate With Vagal Sensory Neurons.

BACKGROUND AND AIMS: 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. METHODS: We characterized the anatomical pattern of pancreatic sensory innervation by combining viral tracing, immunohistochemistry, and reporter mouse models. To assess the functional interactions of ß-cells with vagal sensory neurons, we recorded Ca2+ responses in individual nodose neurons in vivo while selectively stimulating ß-cells with chemogenetic and pharmacologic approaches. RESULTS: 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 in vivo to chemogenetic stimulation of ß-cells and to pancreas infusion with serotonin, but were not sensitive to insulin. Responses to chemogenetic and pharmacologic stimulation of ß-cells were blocked by a 5-HT3R antagonist and were enhanced by increasing serotonin levels in ß-cells. We further confirmed directly in living pancreas slices that sensory terminals in the islet were sensitive to serotonin. CONCLUSIONS: Our study establishes that pancreatic ß-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 ß-cells via vagal afferent nerves.

Unique Molecular Characteristics of Visceral Afferents Arising from Different Levels of the Neuraxis: Location of Afferent Somata Predicts Function and Stimulus Detection Modalities.

Viscera receive innervation from sensory ganglia located adjacent to multiple levels of the brainstem and spinal cord. Here we examined whether molecular profiling could be used to identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and nodose ganglia (NG) in male and female mice. Profiling of TL and LS bladder afferents was also performed. Visceral afferents were back-labeled using retrograde tracers injected into proximal and distal regions of colon or bladder, followed by single-cell qRT-PCR and analysis via an automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for colon) based on their established role in stimulus detection, regulation of sensitivity/function, or neuroimmune interaction. A total of 132 colon afferents (from NG, TL, and LS ganglia) and 128 bladder afferents (from TL and LS ganglia) were analyzed. Retrograde labeling from the colon showed that NG and TL afferents innervate proximal and distal regions of the colon, whereas 98% of LS afferents only project to distal regions. There were clusters of colon and bladder afferents, defined by mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was found. In addition, transcriptionally, NG colon afferents were almost completely segregated from colon TL and LS neurons. Furthermore, colon and bladder afferents expressed genes at similar levels, although different gene combinations defined the clusters. These results indicate that genes implicated in both homeostatic regulation and conscious sensations are found at all anatomic levels, suggesting that afferents from different portions of the neuraxis have overlapping functions.SIGNIFICANCE STATEMENT Visceral organs are innervated by sensory neurons whose cell bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, where sensation and pain are mediated by spinal afferents and fear and anxiety (the affective aspects of visceral pain) are the domain of nodose afferents. The transcriptomic analysis performed here reveals that genes implicated in both homeostatic regulation and pain are found in afferents across all ganglia types, suggesting that conscious sensation and homeostatic regulation are the result of convergence, and not segregation, of sensory input.

Different sensitivity of action potential generation to the rate of depolarization in vagal afferent A-fiber versus C-fiber neurons.

This study demonstrates that the action potential discharge in vagal afferent A-fiber neurons is about 20 times more sensitive to the rate of membrane depolarization compared to C-fiber neurons. The sensitivity of action potential generation to the depolarization rate in vagal sensory neurons is independent of the intensity of current stimuli but nearly abrogated by inhibiting the D-type potassium channel. These findings help better understand the mechanisms that control the activation of vagal afferent nerves.

Piezo2 channel in nodose ganglia neurons is essential in controlling hypertension in a pathway regulated directly by Nedd4-2.

Baroreflex plays a crucial role in regulation of arterial blood pressure (BP). Recently, Piezo1 and Piezo2, the mechanically-activated (MA) ion channels, have been identified as baroreceptors. However, the underlying molecular mechanism for regulating these baroreceptors in hypertension remains unknown. In this study, we used spontaneously hypertensive rats (SHR) and NG-Nitro-l-Arginine (L-NNA)- and Angiotensin II (Ang II)-induced hypertensive model rats to determine the role and mechanism of Piezo1 and Piezo2 in hypertension. We found that Piezo2 was dominantly expressed in baroreceptor nodose ganglia (NG) neurons and aortic nerve endings in Wistar-Kyoto (WKY) rats. The expression of Piezo2 not Piezo1 was significantly downregulated in these regions in SHR and hypertensive model rats. Electrophysiological results showed that the rapidly adapting mechanically-activated (RA-MA) currents and the responsive neuron numbers were significantly reduced in baroreceptor NG neurons in SHR. In WKY rats, the arterial BP was elevated by knocking down the expression of Piezo2 or inhibiting MA channel activity by GsMTx4 in NG. Knockdown of Piezo2 in NG also attenuated the baroreflex and increased serum norepinephrine (NE) concentration in WKY rats. Co-immunoprecipitation experiment suggested that Piezo2 interacted with Neural precursor cell-expressed developmentally downregulated gene 4 type 2 (Nedd4-2, also known as Nedd4L); Electrophysiological results showed that Nedd4-2 inhibited Piezo2 MA currents in co-expressed HEK293T cells. Additionally, Nedd4-2 was upregulated in NG baroreceptor neurons in SHR. Collectively, our results demonstrate that Piezo2 not Piezo1 may act as baroreceptor to regulate arterial BP in rats. Nedd4-2 induced downregulation of Piezo2 in baroreceptor NG neurons leads to hypertension in rats. Our findings provide a novel insight into the molecular mechanism for the regulation of baroreceptor Piezo2 and its critical role in the pathogenesis of hypertension.

Contributing mechanisms underlying desensitization of cholecystokinin-induced activation of primary nodose ganglia neurons.

Cholecystokinin (CCK) is a gut-derived peptide that potently promotes satiety and facilitates gastric function in part by activating G protein-coupled CCK1 receptors on primary vagal afferent neurons. CCK signaling is dynamic and rapidly desensitizes, due to decreases in either receptor function and the resulting signal cascade, ion channel effectors, or both. Here we report a decay-time analytical approach using fluorescent calcium imaging that relates peak and steady-state calcium responses in dissociated vagal afferent neurons, enabling discrimination between receptor and ion channel effector functions. We found desensitization of CCK-induced activation was predictable, consistent across cells, and strongly concentration dependent. The decay-time constant (tau) was inversely proportional to CCK concentration, apparently reflecting the extent of receptor activation. To test this possibility, we directly manipulated the ion channel effector(s) with either decreased bath calcium or the broad-spectrum pore blocker ruthenium red. Conductance inhibition diminished the magnitude of the CCK responses without altering decay kinetics, confirming changes in tau reflect changes in receptor function selectively. Next, we investigated the contributions of the PKC and PKA signaling cascades on the magnitude and decay-time constants of CCK calcium responses. While inhibition of either PKC or PKA increased CCK calcium response magnitude, only general PKC inhibition significantly decreased the decay-time constant. These findings suggest that PKC alters CCK receptor signaling dynamics, while PKA alters the ion channel effector of the CCK response. This analytical approach should prove useful in understanding receptor/effector changes underlying acute desensitization of G-protein coupled signaling and provide insight into CCK receptor dynamics.

A novel role for the extracellular matrix glycoprotein-Tenascin-X in gastric function.

KEY POINTS: Tenascin X (TNX) functions in the extracellular matrix of skin and joints where it maintains correct intercellular connections and tissue architecture TNX is associated exclusively with vagal-afferent endings and some myenteric neurones in mouse and human stomach, respectively. TNX-deficient mice have accelerated gastric emptying and hypersensitivity of gastric vagal mechanoreceptors that can be normalized by an inhibitor of vagal-afferent sensitivity. Cultured nodose ganglion neurones showed no changes in response to capsaicin, cholecystokinin and potassium chloride in TNX-deficient mice. TNX-deficient patients have upper gastric dysfunction consistent with those in a mouse model. Our translational studies suggest that abnormal gastric sensory function may explain the upper gut symptoms present in TNX deficient patients, thus making it important to study gastric physiology. TNX deficiency should be evaluated routinely in patients with connective tissue abnormalities, which will enable a better understanding of its role and allow targeted treatment. For example, inhibitors of vagal afferents-baclofen could be beneficial in patients. These hypotheses need confirmation via targeted clinical trials. ABSTRACT: Tenascin-X (TNX) is a glycoprotein that regulates tissue structure via anti-adhesive interactions with collagen in the extracellular matrix. TNX deficiency causes a phenotype similar to hypermobility Ehlers-Danlos syndrome involving joint hypermobility, skin hyperelasticity, pain and gastrointestinal dysfunction. Previously, we have shown that TNX is required for neural control of the bowel by a specific subtype of mainly cholinergic enteric neurones and regulates sprouting and sensitivity of nociceptive sensory endings in mouse colon. These findings correlate with symptoms shown by TNX-deficient patients and mice. We aimed to identify whether TNX is similarly present in neural structures found in mouse and human gastric tissue. We then determined whether TNX has a functional role, specifically in gastric motor and sensory function and nodose ganglia neurones. We report that TNX was present in calretinin-immunoreactive extrinsic nerve endings in mouse and human stomach. TNX deficient mice had accelerated gastric emptying and markedly increased vagal afferent responses to gastric distension that could be rescued with GABAB receptor agonist. There were no changes in nodose ganglia excitability in TNX deficient mice, suggesting that vagal afferent responses are probably the result of altered peripheral mechanosensitivity. In TNXB-deficient patients, significantly greater symptoms of reflux, indigestion and abdominal pain were reported. In the present study, we report the first role for TNX in gastric function. Further studies are required in TNX deficient patients to determine whether symptoms can be relieved using GABAB agonists.

Inducible nitric oxide synthase-derived nitric oxide reduces vagal satiety signalling in obese mice.

KEY POINTS: Obesity is associated with disrupted satiety regulation. Mice with diet-induced obesity have reduced vagal afferent neuronal excitability and a decreased afferent response to satiety signals. A low grade inflammation occurs in obesity with increased expression of inducible nitric oxide synthase (iNOS). Inhibition of iNOS in diet-induced obese mice restored vagal afferent neuronal excitability, increased the afferent response to satiety mediators and distention of the gut, and reduced short-term energy intake. A prolonged inhibition of iNOS reduced energy intake and body weight gain during the first week, and reduced amounts of epididymal fat after 3 weeks. We identified a novel pathway underlying disrupted satiety regulation in obesity. Blocking of this pathway might be clinically useful for the management of obesity. ABSTRACT: Vagal afferents regulate feeding by transmitting satiety signals to the brain. Mice with diet-induced obesity have reduced vagal afferent sensitivity to satiety signals. We investigated whether inducible nitric oxide synthase (iNOS)-derived NO contributed to this reduction. C57BL/6J mice were fed a high- or low-fat diet for 6-8 weeks. Nodose ganglia and jejunum were analysed by immunoblotting for iNOS expression; NO production was measured using a fluorometric assay. Nodose neuron excitability and intestinal afferent sensitivity were evaluated by whole-cell patch clamp and in vitro afferent recording, respectively. Expression of iNOS and production of NO were increased in nodose ganglia and the small intestine in obese mice. Inhibition of iNOS in obese mice by pre-treatment with an iNOS inhibitor increased nodose neuron excitability via 2-pore-domain K+ channel leak currents, restored afferent sensitivity to satiety signals and reduced short-term energy intake. Obese mice given the iNOS inhibitor daily for 3 weeks had reduced energy intake and decreased body weight gain during the first week, compared to mice given saline, and lower amounts of epididymal fat at the end of 3 weeks. Inhibition of iNOS or blocking the action of iNOS-derived NO on vagal afferent pathways might comprise therapeutic strategies for hyperphagia and obesity.

Central vagal afferent endings mediate reduction of food intake by melanocortin-3/4 receptor agonist.

Injection of the melanocortin-3/4 receptor agonist melanotan-II (MTII) into the nucleus of the solitary tract (NTS) produces rapid and sustained reduction of food intake. Melanocortin-4 receptors (MC4Rs) are expressed by vagal afferent endings in the NTS, but it is not known whether these endings participate in MTII-induced reduction of food intake. In experiments described here, we evaluated the contribution of central vagal afferent endings in MTII-induced reduction of food intake. Examination of rat hindbrain sections revealed that neuronal processes expressing immunoreactivity for the endogenous MC4R agonist α-melanoctyte-stimulating hormone course parallel and wrap around anterogradely labeled vagal afferent endings in the NTS and thus are aptly positioned to activate vagal afferent MC4Rs. Furthermore, MTII and endogenous MC4R agonists increased protein kinase A (PKA)-catalyzed phosphorylation of synapsin I in vagal afferent endings, an effect known to increase synaptic strength by enhancing neurotransmitter release in other neural systems. Hindbrain injection of a PKA inhibitor, KT5720, significantly attenuated MTII-induced reduction of food intake and the increase in synapsin I phosphorylation. Finally, unilateral nodose ganglion removal, resulting in degeneration of vagal afferent endings in the ipsilateral NTS, abolished MTII-induced synapsin I phosphorylation ipsilateral to nodose ganglion removal. Moreover, reduction of food intake following MTII injection into the NTS ipsilateral to nodose ganglion removal was significantly attenuated, whereas the response to MTII was not diminished when injected into the contralateral NTS. Altogether, our results suggest that reduction of food intake following hindbrain MC4R activation is mediated by central vagal afferent endings.

Menthol Enhances the Desensitization of Human α3ß4 Nicotinic Acetylcholine Receptors.

The α3ß4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3ß4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3ß4 nAChRs by an allosteric action.

KATP channels in the nodose ganglia mediate the orexigenic actions of ghrelin.

Ghrelin, a hunger signalling peptide derived from the peripheral tissues, overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. Using in vivo and in vitro electrophysiological techniques, we show that ghrelin hyperpolarizes neurons and inhibits currents evoked by leptin and CCK-8. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition. The inhibitory actions of ghrelin were also abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a-Gαi -PI3K-Erk1/2-KATP pathway. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways. Ghrelin is the only known hunger signal derived from the peripheral tissues. Ghrelin overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. The mechanisms by which ghrelin reduces the sensory signals evoked by anorexigenic hormones, which act via the vagus nerve to stimulate feeding, are unknown. Patch clamp recordings of isolated rat vagal neurons show that ghrelin hyperpolarizes neurons by activating K(+) conductance. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition in vitro and in vivo. Patch clamp studies show that ghrelin inhibits currents evoked by leptin and CCK-8, which operate through independent ionic channels. The inhibitory actions of ghrelin were abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. In vivo gene silencing of PI3K and Erk1/2 in the nodose ganglia prevented ghrelin inhibition of leptin- or CCK-8-evoked vagal firing. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a-Gαi -PI3K-Erk1/2-KATP pathway. The resulting hyperpolarization renders the neurons less responsive to signals evoked by anorexigenic hormones. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways.

Glucagon directly interacts with vagal afferent nodose ganglion neurons to induce Ca(2+) signaling via glucagon receptors.

Glucagon is released from the pancreatic islets postprandially and under hypoglycemic and cold conditions, and regulates glucose metabolism, feeding, energy expenditure and heat production, the functions partly controlled by the brain. Peripheral glucagon could signal to the brain via passing through the blood-brain barrier and/or acting on the vagal afferent. However, the latter remains to be determined. The present study aimed to clarify whether glucagon directly interacts with the nodose ganglion (NG) neurons of vagal afferent nerves in mice. In vivo study showed that intraperitoneal injection of glucagon induced phosphorylation of extracellular signal regulated kinase 1 and 2 (ERK1/2), cellular activation makers, in NG neurons. In fura-2 microfluorometric studies, glucagon increased cytosolic Ca(2+) concentration ([Ca(2+)]i) in single NG neurons. The glucagon-induced [Ca(2+)]i increases were suppressed by a glucagon receptor antagonist, des-His(1)-[Glu(9)]-Glucagon (1-29) amide, and the glucagon receptor mRNA was expressed in NG neurons. The majority of glucagon-responsive NG neurons exhibited [Ca(2+)]i responses to insulin and cholecystokinin-8, the hormones that are secreted postprandially and implicated in satiety. These results demonstrate that glucagon, by interacting with the glucagon receptor, directly activates vagal afferent nerves, possibly being relayed to the signaling to the brain and formation of satiety.

Circadian variation in gastric vagal afferent mechanosensitivity.

Food intake is coordinated to cellular metabolism by clock gene expression with a master clock in the suprachiasmatic nucleus synchronized by light exposure. Gastric vagal afferents play a role in regulating food intake, but it is unknown whether they exhibit circadian variation in their mechanosensitivity. We aimed to determine whether gastric vagal afferents express clock genes and whether their response to mechanical stimuli oscillates throughout the light/dark cycle. Nodose ganglia were collected from 8-week-old female C57BL/6 mice every 3 h starting at lights off (1800 h) to quantify Bmal1, Per1, Per2, and Nr1d1 mRNA by qRT-PCR. Additionally in vitro single-fiber recordings of gastric vagal mechanoreceptors were taken at all time points. Per1, Per2, Bmal1, and Nr1d1 mRNA is expressed in the nodose ganglia and levels oscillated over a 24 h period. In mice fed ad libitum, gastric content was 3 times higher at 0000 h and 0300 h than 1200 h. The response of tension receptors to 3 g stretch was reduced by up to 70% at 2100 h, 0000 h, and 0300 h compared with 1200 h. Gastric mucosal receptor response to stroking with a 50 mg von Frey hair was 3 times greater at 1200 h and 1500 h than the response at 0000 h. Similar findings were obtained in mice fasted for 6 h or maintained in darkness for 3 d before study. Therefore, these changes do not result from food intake or the light/dark cycle. Thus, gastric vagal mechanoreceptors display circadian rhythm, which may act to control food intake differentially at different times of the day.

Diet-dependent modulation of gastro-oesphageal vagal afferent mechanosensitivity by endogenous nitric oxide.

Neuronal nitric oxide (NO) plays an important role in gastric motor activity and modulates the mechanosensitivity of gastro-oesophageal vagal afferents. Effects of NO on food intake are dependent on feeding status. We sought to determine the effect of NO on gastro-oesophageal vagal afferent activity in the normally fed and food-restricted states and the second messenger pathways mediating these effects. Eight week old female C56BL/6 mice were fed ad libitum or food restricted for 14 h. An in vitro preparation was used to determine the functional effects of NO and the second messenger pathways involved. Expression of NO signal transduction molecules in vagal afferents was determined by reverse-transcription polymerase chain reaction (RT-PCR). Endogenous NO and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited vagal mucosal afferent responses to tactile stimuli in mice fed ad libitum. After a 14 h fast endogenous NO and SNAP potentiated tension and mucosal afferent responses to mechanical stimulation. The excitatory effect of NO was blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. After a 14 h fast expression of NADPH oxidase 2 (NOX2) mRNA in whole nodose ganglia was significantly reduced and the excitatory effect of NO on gastro-oesophageal vagal afferents was lost. Under fasting conditions the inhibitory effect of NO was blocked with the hyperpolarisation-activated cyclic nucleotide-gated (HCN) channel blocker ivabradine and mRNA expression of HCN3 in the nodose ganglia was elevated. In conclusion, the role of NO in the peripheral modulation of gastro-oesophageal vagal afferents is dynamic and dependent on feeding status.

Gastric vagal afferent modulation by leptin is influenced by food intake status.

Energy intake is strongly influenced by vagal afferent signals from the stomach, and is also modulated by leptin. Leptin may be secreted from gastric epithelial cells, so we aimed to determine the direct effect of leptin on gastric vagal afferents under different feeding conditions. Female C57BL/6 mice were fed standard laboratory diet, high-fat diet or were food restricted. The expression of leptin receptor (Lep-R) and its signal transduction molecules in vagal afferents was determined by retrograde tracing and reverse-transcription polymerase chain reaction, and the relationship between leptin-immunopositive cells and gastric vagal afferent endings determined by anterograde tracing and leptin immunohistochemistry. An in vitro preparation was used to determine the functional effects of leptin on gastric vagal afferents and the second messenger pathways involved. Leptin potentiated vagal mucosal afferent responses to tactile stimuli, and epithelial cells expressing leptin were found close to vagal mucosal endings. After fasting or diet-induced obesity, potentiation of mucosal afferents by leptin was lost and Lep-R expression reduced in the cell bodies of gastric mucosal afferents. These effects in diet-induced obese mice were accompanied by a reduction in anatomical vagal innervation of the gastric mucosa. In striking contrast, after fasting or diet-induced obesity, leptin actually inhibited responses to distension in tension receptors. The inhibitory effect on gastric tension receptors was mediated through phosphatidylinositol 3-kinase-dependent activation of large-conductance calcium-activated potassium channels. The excitatory effect of leptin on gastric mucosal vagal afferents was mediated by phospholipase C-dependent activation of canonical transient receptor potential channels. These data suggest the effect of leptin on gastric vagal afferent excitability is dynamic and related to the feeding state. Paradoxically, in obesity, leptin may reduce responses to gastric distension following food intake.

Selective inhibition of vagal afferent nerve pathways regulating cough using Nav 1.7 shRNA silencing in guinea pig nodose ganglia.

Adeno-associated virus delivery systems and short hairpin RNA (shRNA) were used to selectively silence the voltage-gated sodium channel NaV 1.7 in the nodose ganglia of guinea pigs. The cough reflex in these animals was subsequently assessed. NaV 1.7 shRNA was delivered to the majority of nodose ganglia neurons [50-60% transfection rate determined by green fluorescent protein (GFP) gene cotransfection] and action potential conduction in the nodose vagal nerve fibers, as evaluated using an extracellular recording technique, was markedly and significantly reduced. By contrast, <5% of neurons in the jugular vagal ganglia neurons were transfected, and action potential conduction in the jugular vagal nerve fibers was unchanged. The control virus (with GFP expression) was without effect on action potential discharge and conduction in either ganglia. In vivo, NaV 1.7 silencing in the nodose ganglia nearly abolished cough evoked by mechanically probing the tracheal mucosa in anesthetized guinea pigs. Stimuli such as capsaicin and bradykinin that are known to stimulate both nodose and jugular C-fibers evoked coughing in conscious animals was unaffected by NaV 1.7 silencing in the nodose ganglia. Nodose C-fiber selective stimuli including adenosine, 2-methyl-5-HT, and ATP all failed to evoke coughing upon aerosol challenge. These results indicate that cough is independently regulated by two vagal afferent nerve subtypes in guinea pigs, with nodose Aδ fibers regulating cough evoked mechanically from the trachea and bradykinin- and capsaicin-evoked cough regulated by C-fibers arising from the jugular ganglia.

A role for ATP in bronchoconstriction-induced activation of guinea pig vagal intrapulmonary C-fibres.

Activation of vagal afferent sensory C-fibres in the lungs leads to reflex responses that produce many of the symptoms associated with airway allergy. There are two subtypes of respiratory C-fibres whose cell bodies reside within two distinct ganglia, the nodose and jugular, and whose properties allow for differing responses to stimuli. We here used extracellular recording of action potentials in an ex vivo isolated, perfused lung-nerve preparation to study the electrical activity of nodose C-fibres in response to bronchoconstriction. We found that treatment with both histamine and methacholine caused strong increases in tracheal perfusion pressure that were accompanied by action potential discharge in nodose, but not in jugular C-fibres. Both the increase in tracheal perfusion pressure and action potential discharge in response to histamine were significantly reduced by functionally antagonizing the smooth muscle contraction with isoproterenol, or by blocking myosin light chain kinase with ML-7. We further found that pretreatment with AF-353 or 2',3'-O-(2,4,6-Trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), structurally distinct P2X3 and P2X2/3 purinoceptor antagonists, blocked the bronchoconstriction-induced nodose C-fibre discharge. Likewise, treatment with the ATPase apyrase, in the presence of the adenosine A1 and A2 receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and SCH 58261, blocked the C-fibre response to histamine, without inhibiting the bronchoconstriction. These results suggest that ATP released within the tissues in response to bronchoconstriction plays a pivotal role in the mechanical activation of nodose C-fibres.