Nodose Neurons Research Articles

Sphingosine-1-phosphate activates mouse vagal airway afferent C-fibres via S1PR3 receptors.

Sphingosine-1-phosphate (S1P) strongly activates mouse vagal C-fibres in the airways. Airway-specific nodose and jugular C-fibre neurons express mRNA coding for the S1P receptor S1PR3. S1P activation of nodose C-fibres is inhibited by a S1PR3 antagonist. S1P activation of nodose C-fibres does not occur in S1PR3 knockout mice. We evaluated the effect of sphingosine-1-phosphate (S1P), a lipid that is elevated during airway inflammatory conditions like asthma, for its ability to stimulate vagal afferent C-fibres in mouse lungs. Single cell RT-PCR on lung-specific vagal afferent neurons revealed that both TRPV1-expressing and TRPV1-non-expressing nodose neurons express mRNA coding for the S1P receptor S1PR3. TRPV1-expressing airway-specific jugular ganglion neurons also express S1PR3 mRNA. S1PR1 and S1PR2 mRNAs were also found to be expressed but only in a limited subset (32% and 22%, respectively) of airway-specific vagal sensory neurons; whereas S1PR4 and S1PR5 were rarely expressed. We used large scale two-photon imaging of the nodose ganglia from our ex vivo preparation isolated from Pirt-Cre;R26-GCaMP6s transgenic mice, which allows for simultaneous monitoring of calcium transients in ∼1000 neuronal cell bodies in the ganglia during tracheal perfusion with S1P (10μM). We found that S1P in the lungs strongly activated 81.5% of nodose fibres, 70% of which were also activated by capsaicin. Single fibre electrophysiological recordings confirmed that S1P evoked action potential (AP) generation in a concentration-dependent manner (0.1-10μM). Action potential generation by S1P in nodose C-fibres was effectively inhibited by the S1PR3 antagonist TY 52156 (10μM). Finally, in S1PR3 knockout mice, S1P was not able to activate any of the airway nodose C-fibres analysed. These results support the hypothesis that S1P may play a role in evoking C-fibre-mediated airway sensations and reflexes that are associated with airway inflammatory diseases.

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

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. 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.

Activity in nodose ganglia neurons after treatment with CP 55,940 and cholecystokinin.

Previous work has shown that cannabinoids increase feeding, while cholecystokinin (CCK) has an anorexigenic effect on food intake. Receptors for these hormones are located on cell bodies of vagal afferent nerves in the nodose ganglia. An interaction between CCK and endocannabinoid receptors has been suggested. The purpose of these studies is to explore the effect of pretreatment with a cannabinoid agonist, CP 55,940, on nodose neuron activation by CCK. To determine the effect of CP 55,940 and CCK on neuron activation, rats were anesthetized and nodose ganglia were excised. The neurons were dissociated and placed in culture on coverslips. The cells were treated with media; CP 55,940; CCK; CP 55,940 followed by CCK; or AM 251, a CB1 receptor antagonist, and CP 55,940 followed by CCK. Immunohistochemistry was performed to stain the cells for cFos as a measure of cell activation. Neurons were identified using neurofilament immunoreactivity. The neurons on each slip were counted using fluorescence imaging, and the number of neurons that were cFos positive was counted in order to calculate the percentage of activated neurons per coverslip. Pretreatment with CP 55,940 decreased the percentage of neurons expressing cFos‐immunoreactivity in response to CCK. This observation suggests that cannabinoids inhibit CCK activation of nodose ganglion neurons.

Abstract P323: TMEM16A and TMEM16B Contribute to Mice Cholecystokinin Response in Sex-Specific Manner

High fat diet (HFD) leads to obesity and the metabolic syndrome. However, the effect are less pronounced in females than in males. We suggest that a gender difference is in their response to the satiety peptide Cholecystokinin (CCK). We previously reported that the Ca 2+ -activated Cl - channel (CaCC) subunit Ano2 /TMEM16B is essential for the CCK-induced current in intestinal nodose neurons in male mice, and is down regulated in obese mice fed on HFD. We hypothesize that the CCK-induced current in female mice is mediated predominantly by Ano1 /TMEM16A, the alternative subunit of TMEM16B. We studied the CCK-induced current in nodose neurons from female C57BL/6 mice using whole cell patch-clamp. We found that 10nM of CCK induces an inward current in these neurons with a current density averaging 13.4±2.5 pA/pF (n=7), and this current is inhibited to 3.1±1.2 pA/pF (n=7, p<0.01) by TMEM16A specific inhibitor T16A inhi-A01 . We further tested whether HFD which effectively suppressed the expression of TMEM16B in male mice, would also decrease TMEM16A in female mice. Our results show that the CCK-induced TMEM16A current in nodose neurons is not decreased in female, but tend to increase from 12.5±1.6 (n=10) in control to 17.4±4.1 pA/pF (n=11, p=0.298) in female mice fed on HFD. Concurrently the mRNA level of Ano1 in mice fed on HFD also show a trend to increase by 1.33±0.41 fold (n=2 pairs of mice, p=0.37) compared to mice on regular chow. Our findings indicate that activation of CCK-sensitive Ano1 /TMEM16A may partially protect female mice from HFD induced obesity and metabolic syndrome.

Parapheromones Suppress Chemotherapy Side Effects

The cytotoxic drugs used in chemotherapy are often accompanied by nausea and vomiting. Despite the use of antiemetic drugs, chemotherapy-induced nausea and vomiting (CINV) remain significant side effects for cancer patients and are associated with serotonin type 3 receptor (5-HT3R) activation in the brainstem. Farnesol and nerolidol are sesquiterpene alcohols found in essential oils of plants such as roses, citronella, and lemon grass and are used as antiemetic parapheromones. Medicinal plants often are effective in treating gastrointestinal disorders, including CINV, although the mechanism of action remains unclear. In the current work, the antiemetic efficacy of the naturally occurring racemic mixture of farnesol (m-farnesol) and nerolidol (m-nerolidol) against cisplatin CINV was tested using the pica behavior (consumption of nonnutritive substances) of rats. Animals treated with m-farnesol or m-nerolidol consumed a smaller amount of kaolin than of saline-treated control animals. This result is consistent with the antiemetic efficacy of farnesol and nerolidol. Compared with controls, m-farnesol- but not m-nerolidol-treated animals consumed more food and lost less body weight. Thus, farnesol effectively reduced appetite suppression and weight loss induced by cisplatin. In separate experiments, isomers of farnesol and nerolidol were tested on 5-HT-induced responses of acutely isolated nodose neurons using patch-clamp methods. All the tested constituents inhibited 5-HT3R-mediated current in a noncompetitive manner. Thus, both farnesol and nerolidol may exert antiemetic efficacy by inhibiting 5-HT signaling in cranial visceral afferents, resulting in interruption of emetogenic signaling; however, nerolidol failed to suppress cisplatin-induced anorexia and weight loss, suggesting that additional mechanisms may contribute.

Spontaneous activities in baroreflex afferent pathway contribute dominant role in parasympathetic neurocontrol of blood pressure regulation.

To study the dominant role of parasympathetic inputs at cellular level of baroreflex afferent pathway and underlying mechanism in neurocontrol of blood pressure regulation. Whole-cell patch-clamp and animal study were conducted. For the first time, we demonstrated the spontaneous activities from resting membrane potential in myelinated A- and Ah-type baroreceptor neurons (BRNs, the 1st-order), but not in unmyelinated C-types, using vagus-nodose slice of adult female rats. These data were further supported by the notion that the spontaneous synaptic currents could only be seen in the pharmacologically and electrophysiologically defined myelinated A- and Ah-type baroreceptive neurons (the 2nd-order) of NTS using brainstem slice of adult female rats. The greater frequency and the larger amplitude of the spontaneous excitatory postsynaptic currents (EPSCs) compared with the inhibitory postsynaptic currents (IPSCs) were only observed in Ah-types. The ratio of EPSCs:IPSCs was estimated at 3:1 and higher. These results confirmed that the afferent-specific spontaneous activities were generated from baroreflex afferent pathway in female-specific subpopulation of myelinated Ah-type BRNs in nodose and baroreceptive neurons in NTS, which provided a novel insight into the dominant role of sex-specific baroreflex-evoked parasympathetic drives in retaining a stable and lower blood pressure status in healthy subjects, particularly in females. The data from current investigations establish a new concept for the role of Ah-type baroreceptor/baroreceptive neurons in controlling blood pressure stability and provide a new pathway for pharmacological intervention for hypertension and cardiovascular diseases.

Increased TASK channel-mediated currents underlie high-fat diet induced vagal afferent dysfunction.

We have previously demonstrated that satiety sensing vagal afferent neurons are less responsive to meal-related stimuli in obesity because of reduced electrical excitability. As leak K+ currents are key determinants of membrane excitability, we hypothesized that leak K+ currents are increased in vagal afferents during obesity. Diet-induced obesity was induced by feeding C57Bl/6J mice a high-fat diet (HFF) (60% energy from fat) for 8-10 wk. In vitro extracellular recordings were performed on jejunal afferent nerves. Whole cell patch-clamp recordings were performed on mouse nodose ganglion neurons. Leak K+ currents were isolated using ion substitution and pharmacological blockers. mRNA for TWIK-related acid-sensitive K+ (TASK) subunits was measured using quantitative real-time PCR. Intestinal afferent responses to nutrient (oleate) and non-nutrient (ATP) stimuli were significantly decreased in HFF mice. Voltage clamp experiments revealed the presence of a voltage-insensitive resting potassium conductance that was increased by external alkaline pH and halothane, known properties of TASK currents. In HFF neurons, leak K+ current was approximately doubled and was reduced by TASK1 and TASK3 inhibitors. The halothane sensitive current was similarly increased. Quantitative PCR revealed the presence of mRNA encoding TASK1 (KCNK3) and TASK3 (KCNK9) channels in nodose neurons. TASK3 transcript was significantly increased in HFF mice. The reduction in vagal afferent excitability in obesity is due in part to an increase of resting (leak) K+ conductance. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a therapeutic target for obesity treatment. NEW & NOTEWORTHY This study characterized the electrophysiological properties and gene expression of the TWIK-related acid-sensitive K+ (TASK) channel in vagal afferent neurons. TASK conductance was increased and contributed to decreased excitability in diet-induced obesity. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a promising therapeutic target.

Sphingosine-1-phosphate selectively activates vagal afferent C-fiber subtype in guinea pig esophagus.

Activation and sensitization of visceral afferent nerves by inflammatory mediators play important roles in visceral nociception. Sphingosine-1-phosphate (S1P) is a lipid with intracellular and extracellular functions. Extracellularly, it can act as an autacoid via interactions with S1P receptors. The present study aims to determine the effect of S1P on esophageal vagal afferent nerve functions. Extracellular single-unit recordings were performed in ex vivo guinea pig esophageal-vagal preparations. The action potentials (APs) evoked by mechanical distension and chemical perfusions applied to the vagal afferent nerve endings in the esophagus were recorded at their intact neuronal cell bodies in either nodose or jugular ganglia. The effects of S1P and its receptor subtype agonists on vagal afferents were recorded and compared. The expression of S1P receptors (S1PR1-3) in esophageal-labeled vagal nodose and jugular neurons was studied by single-cell RT-PCR. Sphingosine-1-phosphate evoked AP discharges in almost all esophageal jugular but not nodose C-fibers without changing their responses to esophageal distension. Esophageal-labeled vagal nodose and jugular neurons highly expressed transcripts of S1PR1 and S1PR3. Agonists of S1PR1 and S1PR3 each partially mimicked S1P-induced effect in jugular C-fibers, suggesting that these receptors may contribute partially to S1P-induced activation effect on esophageal jugular C-fiber subtype. These data, for the first time, demonstrated a selective activation effect of S1P on vagal afferent nerve subtype in the gastrointestinal tract. This may help to better understand its role in visceral inflammatory nociception.

Different role of TTX-sensitive voltage-gated sodium channel (NaV 1) subtypes in action potential initiation and conduction in vagal airway nociceptors.

The action potential initiation in the nerve terminals and its subsequent conduction along the axons of afferent nerves are not necessarily dependent on the same voltage-gated sodium channel (NaV 1) subunits. The action potential initiation in jugular C-fibres within airway tissues is not blocked by TTX; nonetheless, conduction of action potentials along the vagal axons of these nerves is often dependent on TTX-sensitive channels. This is not the case for nodose airway Aδ-fibres and C-fibres, where both action potential initiation and conduction is abolished by TTX or selective NaV 1.7 blockers. The difference between the initiation of action potentials within the airways vs. conduction along the axons should be considered when developing NaV 1 blocking drugs for topical application to the respiratory tract. The action potential (AP) initiation in the nerve terminals and its subsequent AP conduction along the axons do not necessarily depend on the same subtypes of voltage-gated sodium channels (NaV 1s). We evaluated the role of TTX-sensitive and TTX-resistant NaV 1s in vagal afferent nociceptor nerves derived from jugular and nodose ganglia innervating the respiratory system. Single cell RT-PCR was performed on vagal afferent neurons retrogradely labelled from the guinea pig trachea. Almost all of the jugular neurons expressed the TTX-sensitive channel NaV 1.7 along with TTX-resistant NaV 1.8 and NaV 1.9. Tracheal nodose neurons also expressed NaV 1.7 but, less frequently, NaV 1.8 and NaV 1.9. NaV 1.6 were expressed in ∼40% of the jugular and 25% of nodose tracheal neurons. Other NaV 1 α subunits were only rarely expressed. Single fibre recordings were made from the vagal nodose and jugular nerve fibres innervating the trachea or lung in the isolated perfused vagally-innervated preparations that allowed for selective drug delivery to the nerve terminal compartment (AP initiation) or to the desheathed vagus nerve (AP conduction). AP initiation in jugular C-fibres was unaffected by TTX, although it was inhibited by NaV 1.8 blocker (PF-01247324) and abolished by combination of TTX and PF-01247324. However, AP conduction in the majority of jugular C-fibres was abolished by TTX. By contrast, both AP initiation and conduction in nodose nociceptors was abolished by TTX or selective NaV 1.7 blockers. Distinction between the effect of a drug with respect to inhibiting AP in the nerve terminals within the airways vs. at conduction sites along the vagus nerve is relevant to therapeutic strategies involving inhaled NaV 1 blocking drugs.

Sexually Dimorphic Ano2 Expression in Nodose Neurons Determines CCK‐mediated Satiation and Obesity in Heterozygote Male Mice

The gut peptide cholecystokinin (CCK) contributes to neurally mediated post prandial satiety. Our previous results indicate that high fat diet downregulates the CCK‐sensitive anoctamins (Ano1 and Ano2) that function as calcium‐activated chloride channels (CaCCs) in intestinal vagal afferents. Using Cre‐loxP system, we generated heterozygote (HET) Nav1.8CreAno2fl/WT mice. The endogenous Scn10a promoter drives Cre expression (Nav1.8Cre) selectively in sensory neurons of the nodose, dorsal root and trigeminal ganglia where loxP sites flanked Ano2 exon 12. The male HET mice exhibited an obese phenotype and decreased sensitivity to CCK. To assess the Ano2 expression in HET vs. control (Nav1.8Cre+/−) mice, we first did qRT‐PCR on whole nodose ganglia obtained under anesthesia at 50 weeks of age and found no significant difference in Ano2 mRNA expression (n=4, p>0.05). Ano2 gene may be expressed in non‐neuronal cells of nodose ganglia while Cre expression is selective to neurons. We measured Ano2 mRNAs in nodose neurons cultured for 16–18 hours using single cell qRT‐PCR. Cre positive neurons were 57% (12 of 21) in HET and 74% (29 of 39) in control, indicating a lower efficiency of Cre expression in HET. Correspondingly, Ano2 positive neurons were 48% (10 of 21) and 62% (24 of 39), respectively, indicating the Ano2 allelic deletion in HET. The combined Cre and Ano2 positivity in HET was 29% (6 of 21) vs. 54% (21 of 39) in control. Most importantly the Ano2 mRNA levels in HET neurons (n=10) were 20% of the levels measured in control neurons (n=24, p<0.05) (0.20±0.16 vs. 1.00±0.57). The Ano2 expression in individual nodose neurons of female mice did not show a reduction or even higher in HET (n=27) vs. control (n=28) (2.58±1.48 vs. 1.42±1.27, p>0.05). The female HET mice remained CCK sensitive indicating a sexual dimorphism. We conclude that differences in expression of Ano2 mRNA support our findings of CCK insensitivity and selective obesity in male but not female HET mice (HL14388).Support or Funding InformationNIH HL14388This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxytocin Reduction of Nodose Ganglion Neurons Potassium Currents is Enhanced in Chronic Intermittent Hypoxia (CIH)

Oxytocin (OT) from the hypothalamus is increased in several cardiorespiratory nuclei and the circulation in response to a variety of stimuli and stressors, including hypoxia. Oxytocin within the nucleus tractus solitarii (nTS) enhances synaptic transmission, action potential (AP) discharge, and cardiac baroreflex gain to lower heart rate. The baroreceptor reflex is mediated through afferent neurons whose somas originate in the nodose ganglia and project centrally to the nTS. Nodose neurons express the OT receptor, and its activation elevates intracellular calcium; the influence of OT on other ion channels, especially during hypoxia, is not known. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. Recent studies suggest enhancing OT in the dorsal vagal complex during CIH may reduce blood pressure. This study was undertaken to determine the mechanism(s) by which OT modulates sensory afferent‐nTS mediated reflexes normally and after CIH. Nodose neurons of 5–6 wk old male Sprague‐Dawley rats were isolated after 10 d of Normoxia (21% O2, Norm) or CIH (alternating 21% & 6% O2, 8 hr/day). Two‐5 hrs after isolation, we recorded outward potassium currents (IK) and APs by the patch clamp method. To examine IK, neurons were held at −60 mV and currents were evoked by depolarizing voltages (−70 to +60 mV, 140 ms). The steady state IK‐voltage relationship was analyzed. AP discharge was evoked by injecting 50 pA depolarizing steps (−20 to 280 pA, 200 ms); the current‐discharge relationship was evaluated. IK and APs were examined during baseline and after 3 min of vehicle or OT (300, 600, or 1000 nM). Vehicle did not alter IK in Norm (n=6) and CIH (n=4) cells, confirming lack of time‐dependent IK changes. In Norm cells, OT decreased IK at voltages greater than +40 mV (300 nM, n=8) and +60 mV (600 nM, n=7). After CIH, OT (300 nM, n=5) also decreased IK at +40 mV and greater. OT at 600 nM (n=5) attenuated IK at significantly lower voltages (+20 mV) in CIH vs. Norm. OT‐sensitive currents at +60 mV were greater in CIH than Norm at 300 and 600 nM. In Norm and CIH groups (n=4, 5), OT at 1000 nM had variable IK responses which overall were not significantly different from baseline. Fitting IKs to Boltzmann functions demonstrated a reduction in half‐activation during OT in Norm cells. In Norm and CIH cells, application of the K+ channel blocker tetraethylammonium (TEA, 10 mM) decreased IK and blocked the OT‐induced reduction in IK (600 nM, n=3). Depolarizing nodose neurons with up to 280 pA injection induced APs in Norm (n=7) and CIH (n=5) neurons, and 600 nM OT did not alter the number of APs during a given current step. Yet, 600 nM OT decreased AP threshold and tended to increase AP half‐width in CIH but not Norm cells, consistent with the reduction in IK by OT in CIH. In Norm cells, vehicle and OT did not alter holding currents measured at −60 mV, but after CIH 600 nM OT induced modest depolarizing inward currents. Taken together, these data demonstrate OT induces changes in ionic currents in nodose ganglia cells that are consistent with increasing excitability in CIH, and may also shed light on the bradycardic and anti‐hypertensive action of OT.Support or Funding InformationRO1 HL 128454 & HL 098602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiple tetrodotoxin(TTX)‐sensitive NaV1 channels regulate action potential initiation in the nerve terminals of vagal nodose C‐fibers innervating the esophagus.

Vagal afferent C‐fibers innervating the esophagus detect inflammatory and noxious stimuli and contribute to dysregulation of esophageal reflexes and sensations in diseases. The knowledge of voltage‐gated sodium channels (NaV1 subunits) regulating action potential initiation in the nerve terminal of esophageal C‐fibers would aid development of therapies aimed at inhibiting unwanted activation of these nociceptors. Extracellular single fiber recordings were made from the neurons in vagal nodose ganglia projecting mechanosensitive C‐fibers into the esophagus in the isolated vagally innervated guinea pig esophagus preparation. The drugs were perfused through the esophageal lumen (mucosa was removed to facilitate diffusion into the muscle wall containing nerve terminals) in order to allow the drug access to the nerve terminals in the esophageal wall but not to their conducting axons in the laryngeal recurrent and vagus nerves. Esophageal distention with 60–100mmHg (20s) was used as a mechanical stimulus. Single cell RT‐PCR was performed on nodose neurons retrogradely labeled from the esophagus. Mechanical response to esophageal distention in nodose C‐fibers was highly reproducible (two consecutive control distentions evoked 62±5 and 61±4 action potentials, respectively, n=39). TTX 0.1–1μM completely abolished mechanical response in 85%(24/28) C‐fibers. In the remaining 4 C‐fibers only approximately 30% of the mechanical response persisted and was abolished by 1mM lidocaine. scRT‐PCR revealed various combinations of TTX‐sensitive NaV1 subunits NaV1.7, 1.2, 1.6, 1.1 and 1.3 in the nodose TRPV1‐positive (C‐fiber) neurons traced from the esophagus. The NaV1.7 blocker PF‐05089771 0.3–30μM dose‐dependently inhibited mechanical response (n=4). At 3μM PF‐05089771 the response was inhibited by 40±8% (P<0.05). Another NaV1.7 blocker, Compound 801 (J Pharmacol Exp Ther. 361:172–80) also dose‐dependently inhibited mechanical response (n=8). At 1μM Compound 801 the response was inhibited by 39±8% (P<0.05). In the concentrations 10‐fold of those needed to completely block guinea pig NaV1.7 PF‐05089771 30μM and Compound 801 10μM inhibited mechanical response by 60+9% (N=4, P<0.05) and 52±12% (N=8, P<0.01), respectively, showing a robust (>40%) NaV1.7‐independet component of nodose C‐fiber mechanical response. NaV1.1, 1.2 and 1.3 group blocker ICA‐121431 (0.1–10μM) dose‐dependently inhibited mechanical response (N=4). At 1μM ICA‐121431 the response was inhibited by 31±6% (P<0.05). Combination of Compound 801 10μM and ICA‐121431 10μM nearly abolished mechanical response (inhibition by 91±5%, N=7, P<0.01). We conclude that multiple TTX‐sensitive NaV1 channels regulate action potential initiation in the nerve terminals of vagal nodose C‐fibers innervating the esophagus.Support or Funding InformationSupported by Eli Lilly and SPARC DK116311.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of Airway Afferent Vagal C‐fibers by Sphingosine‐1‐Phosphate

Airway inflammation leads to a release of myriad inflammatory mediators and autacoids. At the site of inflammation these molecules in theory can activate airway C‐fibers leading to the observed symptoms of reflex bronchoconstriction, mucus secretion, as well as sensations of dyspnea and urge‐to‐cough. Relatively little progress has been made to understand which among the plethora of mediators that arise during airway inflammation are most responsible for C‐fiber activation. Here we show that shingosine‐1‐phosphate, a lipid that is elevated during airway inflammatory conditions like asthma, strongly activates airway vagal C‐fibers. We employed an ex‐vivo mouse trachea/lung‐vagus nerve preparation in which action potential discharge in single afferent nerves terminating in the lung can be evaluated with extracellular recording from an electrode placed in the vagal sensory (nodose/jugular) ganglion. Drugs were delivered into the lungs directly to the receptive field. S1P (10μM) strongly stimulated, in a concentration dependent manner, 62% (n=21 out of 34 tested) of vagal C‐fibers terminating in the trachea/lungs (CV=0.63±0.04m/s), (426±71 APs, 7.2±4 Hz peak Freq). An antagonist to S1Pr3, TY 52156 (10μM) significantly blocked the stimulatory effect of S1P on S1P positive C‐fibers (214±53 APs in control vs 56±18 APs in presence of TY 52156, n=7 p=0.01). We developed an imaging technique using 2‐photon microscopy and Pirt‐GCamp6 mice to simultaneously monitor the activity of hundreds of vagal ganglion neurons in the trachea/lung‐ganglia preparation upon drug delivery into the lungs. About 40% of capsaicin (1μM) positive neurons responded to S1P (10μM). Single cell RT‐PCR revealed that ~90% of nodose ganglia neurons retrogradely labeled from the airways that express TRPV1 (denoting C‐fiber neurons) also express the S1P receptor, S1Pr3. In an RNA seq transcription analysis specifically of capsaicin‐sensitive nodose neurons we found S1Pr3 was selectively and strongly expressed. In counts per million the mRNA expression was 0, 8, 376, 3, 0 for S1Pr1, 2, 3, 4 and 5, receptors respectively. Jugular neurons expressed relatively little S1Pr receptors. We hypothesize that S1P may contribute to vagal (nodose) C‐fiber activation in airway inflammatory diseases.Support or Funding InformationRO1 HL137807This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A310 THE INHIBITION OF PTP1B REVERSES IMPAIRED VAGAL AFFERENT SENSITIVITY CAUSED BY DIET-INDUCED OBESITY, REDUCING NITRIC OXIDE AND 2 PORE DOMAIN K+ CONDUCTANCE

AbstractBackgroundWe have demonstrated that diet-induced obesity impairs vagal afferents and that the effects of high concentrations of leptin on vagal afferents in vitro, mimic the effects of obesity. One of downstream molecules in leptin signaling is protein tyrosine phosphatase 1B (PTP1B), which plays a role in negative regulation.AimsWe hypothesized that inhibition of PTP1B would prevent an impairment of vagal afferents caused by diet-induced obesity/hyperleptinemiaMethodsDiet-induced obese (DIO) mice were fed diets composed of 60% or 10% kCal fat for 12–16 weeks. Nodose neurons from DIO mice or standard diet fed mice were incubated overnight with leptin (100nM) and PTP1B inhibitor (10mM). Current and voltage clamp were performed to assess membrane excitability and two-pore domain K+ (K2P) conductance, respectively. NO was measured in culture media using Nitrate/Nitrite fluorometric Assay kit. Media was collected after cell incubation for 24 hrs.ResultsLeptin (100nM) incubation reduced the excitability of vagal afferents. PTP1B inhibitor reversed this inhibitory effect of leptin. PTP1B inhibitor significantly decreased rheobase (100.8 ± 18.6 pA, n=12, leptin) vs. 64.7 ± 5.2 pA (n=15, Leptin + PTP1B inhibitor), *p=0.0497) and significantly increased input resistance (353.8 ± 36.2 MΩ (n=12, leptin) vs. 508.2 ± 52.7 MΩ (n=15, Leptin + PTP1B inhibitor), *p=0.0305). Leptin significantly increased K2P conductance (0.280 ± 0.022 nS (n=17, control) vs. 0.377 ± 0.035 nS (n=16, leptin), *p=0.0108). Inhibition of PTP1B significantly decreased the conductance (0.249 ± 0.013 pA (n=14, Leptin + PTP1B inhibitor), **p=0.0028) in leptin-incubated neuron. In mice fed a HFF diet, there is reduction in vagal excitability, which was reversed by the inhibition of PTP1B. PTP1B inhibitor significantly decreased rheobase (112.1 ± 14.6 pA (n=14, leptin) vs. 70.8 ± 8.1 pA (n=12, HFF + PTP1B inhibitor), *p=0.0268). Rheobase was not significantly different in LFF. Inhibition of PTP1B significantly decreased K2P conductance both in LFF and HFF neurons (LFF; **p=0.0050; HFF; **p=0.0041). PTP1B inhibitor significantly reduced NO fluorescence in leptin-incubated media (33.1 ± 2.2 (Leptin) vs. 12.2 ± 1.0 (Leptin+PTP1B inhibitor), ***<0.0001, n=6) and in neurons from HFF mice (43.4 ± 0.7 (HFF) vs. 29.9 ± 0.6 (HFF+PTP1B inhibitor), ***<0.0001 n=6), but not in LFF (27.8 ± 1.3 (LFF) vs. 25.8 ± 0.8 (LFF+PTP1B inhibitor), NS n=6).ConclusionsIinhibition of PTP1B reverses the effects of HFF and elevated leptin on NO production and K2P conductance in nodose ganglion neurons. Consistent with this, the inhibitory effects of leptin and diet-induced obesity on the excitability of vagal afferent neuron were also reversed by PTP1B blockade. We suggest PTP1B may be a pivotal regulator and potential drug target in obesity.Funding AgenciesCIHR

Distinct Expression of Phenotypic Markers in Placodes- and Neural Crest-Derived Afferent Neurons Innervating the Rat Stomach.

Visceral pain is initiated by activation of primary afferent neurons among which the capsaicin-sensitive (TRPV1-positive) neurons play an important role. The stomach is a common source of visceral pain. Similar to other organs, the stomach receives dual spinal and vagal afferent innervation. Developmentally, spinal dorsal root ganglia (DRG) and vagal jugular neurons originate from embryonic neural crest and vagal nodose neurons originate from placodes. In thoracic organs the neural crest- and placodes-derived TRPV1-positive neurons have distinct phenotypes differing in activation profile, neurotrophic regulation and reflex responses. It is unknown to whether such distinction exists in the stomach. We hypothesized that gastric neural crest- and placodes-derived TRPV1-positive neurons express phenotypic markers indicative of placodes and neural crest phenotypes. Gastric DRG and vagal neurons were retrogradely traced by DiI injected into the rat stomach wall. Single-cell RT-PCR was performed on traced gastric neurons. Retrograde tracing demonstrated that vagal gastric neurons locate exclusively into the nodose portion of the rat jugular/petrosal/nodose complex. Gastric DRG TRPV1-positive neurons preferentially expressed markers PPT-A, TrkA and GFRα3 typical for neural crest-derived TRPV1-positive visceral neurons. In contrast, gastric nodose TRPV1-positive neurons preferentially expressed markers P2X2 and TrkB typical for placodes-derived TRPV1-positive visceral neurons. Differential expression of neural crest and placodes markers was less pronounced in TRPV1-negative DRG and nodose populations. There are phenotypic distinctions between the neural crest-derived DRG and placodes-derived vagal nodose TRPV1-positive neurons innervating the rat stomach that are similar to those described in thoracic organs.

Distinct and common expression of receptors for inflammatory mediators in vagal nodose versus jugular capsaicin-sensitive/TRPV1-positive neurons detected by low input RNA sequencing.

Capsaicin-sensitive sensory C-fibers derived from vagal ganglia innervate the visceral organs, and respond to inflammatory mediators and noxious stimuli. These neurons play an important role in maintenance of visceral homeostasis, and contribute to the symptoms of visceral inflammatory diseases. Vagal sensory neurons are located in two ganglia, the jugular ganglia (derived from the neural crest), and the nodose ganglia (from the epibranchial placodes). The functional difference, especially in response to immune mediators, between jugular and nodose neurons is not fully understood. In this study, we microscopically isolated murine nodose and jugular capsaicin-sensitive / Trpv1-expressing C-fiber neurons and performed transcriptome profiling using ultra-low input RNA sequencing. RNAseq detected genes with significantly differential expression in jugular and nodose neurons, which were mostly involved in neural functions. Transcriptional regulators, including Cited1, Hoxb5 and Prdm12 showed distinct expression patterns in the two C-fiber neuronal populations. Common and specific expression of immune receptor proteins was characterized in each neuronal type. The expression of immune receptors that have received little or no attention from vagal sensory biologists is highlighted including receptors for certain chemokines (CXCLs), interleukins (IL-4) and interferons (IFNα, IFNγ). Stimulation of immune receptors with their cognate ligands led to activation of the C-fibers in isolated functional assays.

Abstract 020: Heterozygote Knockout of TMEM16B in Intestinal Vagal Afferents Causes Cholecystokinin Insensitivity and Obesity in Male but Not Female Mice

Cholecystokinin (CCK) is a well-known satiety peptide that inhibits food intake. The CCK induced satiety signal in intestinal vagal afferents is attenuated in mice on a High Fat Diet (HFD). We have shown that down regulation of a Ca 2+ -activated Cl - channel (CaCC) downstream of CCK receptors is responsible for the neuronal insensitivity to CCK. We also found that the CaCC subunit Ano2/TMEM16B is essential for the CCK-induced current in nodose neurons. In this study we tested the hypothesis that reduction of this subunit in vivo contributes to weight gain. One allele of the Ano2/TMEM16B was knocked out in sensory neurons by crossing the ano2 fl/ fl mice with Nav1.8Cre mice to generate the Nav1.8Cre;ano2 fl /wt mice. The Cre negative littermates were used as control. We found that CCK-induced suppression of food intake is eliminated in male Nav1.8Cre;ano2 fl /wt mice. Food intake measured over 4 hours was 1.21±0.11g (n=5) in male wild type (wt) mice injected with saline and was reduced to 0.77±0.18 g (n=7, p&lt;0.05) in mice injected with CCK-8 (3μg/kg). Those values were 0.63±0.15 g (n=6) in saline injected and 0.91±0.13 g (n=7, p&gt;0.05) in CCK injected male Nav1.8Cre;ano2 fl /wt mice. However, the CCK injection did not affect food intake in either female wt or Nav1.8Cre;ano2 fl /wt mice. The male Nav1.8/ano2 fl/wt mice were on the average 5.5g heavier than wt mice at 40 weeks of age (39.8±1.4 g, n=13 vs 34.3±1.4 g, n=14, p&lt;0.01). Body weight of females was significantly lower than in males but was not different between wt and Nav1.8Cre;ano2 fl /wt mice (30.4±1.0 g, n=14 vs 28.5±1.0 g, n=11, p=0.20 respectively). Single cell mRNA level of Ano2 and CCK-induced TMEM16 currents in nodose neurons were reduced significantly in male Nav1.8Cre;ano2 fl/wt mice compared to Cre negative controls, but such changes were not seen in female mice. We conclude that heterozygote knockout of Ano2/TMEM16B specifically in sensory neurons causes neuronal insensitivity to CCK and excessive weight gain in male but not female mice. Reduction of this subunit may contribute to the HFD induced obesity. The reason for the phenotype and allele expression variability between sexes is unclear.

Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion.

Pulmonary neuroendocrine cells (PNECs) are the only innervated airway epithelial cells. To what extent neural innervation regulates PNEC secretion and function is unknown. Here, we discover that neurotrophin 4 (NT4) plays an essential role in mucus overproduction after early life allergen exposure by orchestrating PNEC innervation and secretion of GABA. We found that PNECs were the only cellular source of GABA in airways. In addition, PNECs expressed NT4 as a target-derived mechanism underlying PNEC innervation during development. Early life allergen exposure elevated the level of NT4 and caused PNEC hyperinnervation and nodose neuron hyperactivity. Associated with aberrant PNEC innervation, the authors discovered that GABA hypersecretion was required for the induction of mucin Muc5ac expression. In contrast, NT4-/- mice were protected from allergen-induced mucus overproduction and changes along the nerve-PNEC axis without any defects in inflammation. Last, GABA installation restored mucus overproduction in NT4-/- mice after early life allergen exposure. Together, our findings provide the first evidence for NT4-dependent neural regulation of PNEC secretion of GABA in a neonatal disease model. Targeting the nerve-PNEC axis may be a valid treatment strategy for mucus overproduction in airway diseases, such as childhood asthma.-Barrios, J., Patel, K. R., Aven, L., Achey, R., Minns, M. S., Lee, Y., Trinkaus-Randall, V. E., Ai, X. Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion.

Thoracic spinal cord and cervical vagosympathetic neuromodulation obtund nodose sensory transduction of myocardial ischemia.

Autonomic regulation therapy involving either vagus nerve stimulation (VNS) or spinal cord stimulation (SCS) represents emerging bioelectronic therapies for heart disease. The objective of this study was to determine if VNS and/or SCS modulate primary cardiac afferent sensory transduction of the ischemic myocardium. Using extracellular recordings in 19 anesthetized canines, of 88 neurons evaluated, 36 ventricular-related nodose ganglia sensory neurons were identified by their functional activity responses to epicardial touch, chemical activation of their sensory neurites (epicardial veratridine) and great vessel (descending aorta or inferior vena cava) occlusion. Neural responses to 1min left anterior descending (LAD) coronary artery occlusion (CAO) were then evaluated. These interventions were then studied following either: i) SCS [T1-T3 spinal level; 50Hz, 90% motor threshold] or ii) cervical VNS [15-20Hz; 1.2× threshold]. LAD occlusion activated 66% of identified nodose ventricular sensory neurons (0.33±0.08-0.79±0.20Hz; baseline to CAO; p<0.002). Basal activity of cardiac-related nodose neurons was differentially reduced by VNS (0.31±0.11 to 0.05±0.02Hz; p<0.05) as compared to SCS (0.36±0.12 to 0.28±0.14, p=0.59), with their activity response to transient LAD CAO being suppressed by either SCS (0.85±0.39-0.11±0.04Hz; p<0.03) or VNS (0.75±0.27-0.12±0.05Hz; p<0.04). VNS did not alter evoked neural responses of cardiac-related nodose neurons to great vessel occlusion. Both VNS and SCS obtund ventricular ischemia induced enhancement of nodose afferent neuronal inputs to the medulla.

Lipopolysaccharide Induced Activation of VRAC (LRRC8) in Nodose Neurons Is Dependent on TLR4 Receptor Mediated ROS Generation

The Volume‐Regulated Anion Channel and its essential subunit Leucine Rich Repeats Containing protein 8a (VRAC/LRRC8) is a widely expressed Cl− channel critical for cell volume regulation. This channel is activated by osmotic gradient and ionic strength (Voss et al., 2014, Qiu et al., 2014, Syeda et al., 2016). Using patch‐clamp techniques, we reported that channel activation is also induced in the majority of vagal afferent neurons isolated from mouse (C57BL/6) nodose ganglia by hypoosmolarity and by extracellular low pH (Wang et al., 2017). We have also observed that this current is activated by lipopolysaccharide (LPS) at 10μg/ml. Here we tested the hypothesis that the response to LPS is dependent on toll‐like receptor 4 (TLR4) and is mediated by intracellular reactive oxygen species (ROS). The current was reduced from 26.7±5.4 pA/pF (n=5) in control peptide‐treated neurons to 6.4±2.1 pA/pF (n=6, p&lt;0.05) in neurons treated with the TLR4 inhibitor peptide VIPER. Application of LPS at a lower dose (100ng/ml) increased intracellular neuronal ROS measured by dihydroethidium within several minutes. The cell permeable hydrogen peroxide scavenger PEG‐catalase decreased the LPS‐induced current from 10.2±2.5 pA/pF (n=7) to 2.3±1.2 pA/pF (n=5, p&lt;0.05). We conclude that LPS induces activation of the VRAC/LRRC8 channel in nodose neurons via TLR4 receptor‐mediated ROS generation. The results indicate a diverse functional role of this channel beyond volume homeostasis with potential implications in high fat diet‐ or LPS‐induced decrease in satiety signaling in intestinal vagal afferents.Support or Funding InformationHL14388