Publications

Action potential (AP) conduction depends on voltage-gated sodium channels, of which there are nine subtypes. The vagus nerve, comprising sensory afferent fibers and efferent parasympathetic fibers, provides autonomic regulation of visceral organs, but the voltage-gated sodium channels (Na(V)1) subtypes involved in its AP conduction are poorly defined. We studied the A- and C-waves of electrically stimulated compound action potentials (CAPs) of the mouse and rat vagus nerves with and without Na(V)1 inhibitor administration: tetrodotoxin (TTX), PF-05089771 (mouse Na(V)1.7), ProTX-II (Na(V)1.7), ICA-121341 (Na(V)1.1, Na(V)1.3, and Na(V)1.6), LSN-3049227 (Na(V)1.2, Na(V)1.6, and Na(V)1.7), and A-803467 (Na(V)1.8). We show that TTX-sensitive Na(V)1 channels are essential for all vagal AP conduction. PF-05089771 but not ICA-121341 inhibited the mouse A-wave, which was abolished by LSN-3049227, suggesting roles for Na(V)1.7 and Na(V)1.2. The mouse C-wave was abolished by LSN-3049227 and a combination of PF-05089771 and ICA-121341, suggesting roles for Na(V)1.7 and Na(V)1.6. The rat A-wave was inhibited by ProTX-II, ICA-121341, and a combination of these inhibitors but only abolished by LSN-3049227, suggesting roles for Na(V)1.7, Na(V)1.6, and Na(V)1.2. The rat C-wave was abolished by LSN-3049227 and a combination of ProTX-II and ICA-121341, suggesting roles for Na(V)1.7 and Na(V)1.6. A-803467 also inhibited the mouse and rat CAP suggesting a cooperative role for the TTX-resistant Na(V)1.8. Overall, our data demonstrate that multiple Na(V)1 subtypes contribute to vagal CAPs, with Na(V)1.7 and Na(V)1.8 playing predominant roles and Na(V)1.6 and Na(V)1.2 contributing to a different extent based on nerve fiber type and species. Inhibition of these Na(V)1 may impact autonomic regulation of visceral organs.NEW & NOTEWORTHY Distinct Na(V)1 channels are involved in action potential (AP) initiation and conduction from afferent terminals within specific organs. Here, we have identified the Na(V)1 necessary for AP conduction in the entire murine and rat vagus nerve. We show TTX-sensitive channels are essential for all AP conduction, predominantly Na(V)1.7 with Na(V)1.2 and Na(V)1.6 playing lesser roles depending on the species and fiber type. In addition, we show that Na(V)1.8 is also essential for most axonal AP conduction.

Inhalation of noxious irritants activates nociceptive sensory afferent nerves innervating the airways, inducing reflex regulation of autonomic networks and the modulation of respiratory drive and cardiovascular (CV) parameters such as heart rate and blood pressure. In healthy mammals, irritant-evoked pulmonary-cardiac reflexes cause parasympathetic-mediated bradycardia. However, in spontaneously hypertensive (SH) rats, irritant inhalation also increases sympathetic drive to the heart. This remodeled pulmonary-cardiac reflex may contribute to cardiovascular risk caused by inhalation of air pollutants/irritants in susceptible individuals with cardiovascular disease (CVD). Previous studies have shown that the cooling mimic l-menthol, an agonist for the cold-sensitive transient receptor potential melastatin 8 (TRPM8), can alleviate nasal inflammatory symptoms and respiratory reflexes evoked by irritants. Here, we investigated the impact of inhalation of TRPM8 agonists l-menthol and WS-12 on pulmonary-cardiac reflexes evoked by inhalation of the irritant allyl isothiocyanate (AITC) using radiotelemetry. l-Menthol, but not its inactive analog d-menthol, significantly reduced the AITC-evoked reflex tachycardia and premature ventricular contractions (PVCs) in SH rats but had no effect on the AITC-evoked bradycardia in either SH or normotensive Wistar-Kyoto (WKY) rats. WS-12 reduced AITC-evoked tachycardia and PVCs in SH rats, but this more potent TRPM8 agonist also reduced AITC-evoked bradycardia. l-Menthol had no effect on heart rate when given alone, whereas WS-12 evoked a minor bradycardia in WKY rats. We conclude that stimulation of TRPM8-expressing afferents within the airways reduces irritant-evoked pulmonary-cardiac reflexes, especially the aberrant reflex tachyarrhythmia in SH rats. Airway menthol treatment may be an effective therapy for reducing pollution-associated CV exacerbations.NEW & NOTEWORTHY Irritant-evoked pulmonary-cardiac reflexes are remodeled in spontaneously hypertensive (SH) rats-causing de novo sympathetic reflexes that drive tachyarrhythmia. This remodeling may contribute to air pollution-associated risk in susceptible individuals with cardiovascular disease. We found that inhalation of TRPM8 agonists, l-menthol and WS-12, but not the inactive analog d-menthol, selectively reduces the reflex tachyarrhythmia evoked by allyl isothiocyanate (AITC) inhalation in SH rats. Use of menthol may protect susceptible individuals from pollution-associated CV exacerbations.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that is activated by electrophilic irritants, oxidative stress, cold temperature, and GPCR signaling. TRPA1 expression has been primarily identified in subsets of nociceptive sensory afferents and is considered a target for future analgesics. Nevertheless, TRPA1 has been implicated in other cell types including keratinocytes, epithelium, enterochromaffin cells, endothelium, astrocytes, and CNS neurons. Here, we developed a knock-in mouse that expresses the recombinase FlpO in TRPA1-expressing cells. We crossed the TRPA1Flp mouse with the R26ai65f mouse that expresses tdTomato in a Flp-sensitive manner. We found tdTomato expression correlated well with TRPA1 mRNA expression and sensitivity to TRPA1 agonists in subsets of TRPV1 (transient receptor potential vanilloid receptor type 1)-expressing neurons in the vagal ganglia and dorsal root ganglia (DRGs), although tdTomato expression efficiency was limited in DRG. We observed tdTomato-expressing afferent fibers centrally (in the medulla and spinal cord) and peripherally in the esophagus, gut, airways, bladder, and skin. Furthermore, chemogenetic activation of TRPA1-expressing nerves in the paw evoked flinching behavior. tdTomato expression was very limited in other cell types. We found tdTomato in subepithelial cells in the gut mucosa but not in enterochromaffin cells. tdTomato was also observed in supporting cells within the cochlea, but not in hair cells. Lastly, tdTomato was occasionally observed in neurons in the somatomotor cortex and the piriform area, but not in astrocytes or vascular endothelium. Thus, this novel mouse strain may be useful for mapping and manipulating TRPA1-expressing cells and deciphering the role of TRPA1 in physiological and pathophysiological processes.

Airway function is under constant neurophysiological control, in order to maximize airflow and gas exchange and to protect the airways from aspiration, damage, and infection. There are multiple sensory nerve subtypes, whose disparate functions provide a wide array of sensory information into the CNS. Activation of these subtypes triggers specific reflexes, including cough and alterations in autonomic efferent control of airway smooth muscle, secretory cells, and vasculature. Importantly, every aspect of these reflex arcs can be impacted and altered by local inflammation caused by chronic lung disease such as asthma, bronchitis, and infections. Excessive and inappropriate activity in sensory and autonomic nerves within the airways is thought to contribute to the morbidity and symptoms associated with lung disease.

This virtual workshop was convened by the National Heart, Lung, and Blood Institute, in partnership with the Office of Strategic Coordination of the Office of the National Institutes of Health Director, and held September 2 to 3, 2020. The intent was to assemble a multidisciplinary group of experts in basic, translational, and clinical research in neuroscience and cardiopulmonary disorders to identify knowledge gaps, guide future research efforts, and foster multidisciplinary collaborations pertaining to autonomic neural mechanisms of cardiopulmonary regulation. The group critically evaluated the current state of knowledge of the roles that the autonomic nervous system plays in regulation of cardiopulmonary function in health and in pathophysiology of arrhythmias, heart failure, sleep and circadian dysfunction, and breathing disorders. Opportunities to leverage the Common Fund s SPARC (Stimulating Peripheral Activity to Relieve Conditions) program were characterized as related to nonpharmacologic neuromodulation and device-based therapies. Common themes discussed include knowledge gaps, research priorities, and approaches to develop novel predictive markers of autonomic dysfunction. Approaches to precisely target neural pathophysiological mechanisms to herald new therapies for arrhythmias, heart failure, sleep and circadian rhythm physiology, and breathing disorders were also detailed.

The airways are densely innervated by sensory afferent nerves, whose activation regulates respiration and triggers defensive reflexes (e.g., cough, bronchospasm). Airway innervation is heterogeneous, and distinct afferent subsets have distinct functional responses. However, little is known of the innervation patterns of subsets within the lung. A neuroanatomical map is critical for understanding afferent activation under physiological and pathophysiological conditions. Here, we quantified the innervation of the mouse lung by vagal and dorsal root ganglion (DRG) sensory subsets defined by the expression of Pirt (all afferents), 5HT(3) (vagal nodose afferents), Tac1 (tachykinergic afferents), and transient receptor potential vanilloid 1 channel (TRPV1; defensive/nociceptive afferents) using Cre-mediated reporter expression. We found that vagal afferents innervate almost all conducting airways and project into the alveolar region, whereas DRG afferents only innervate large airways. Of the two vagal ganglia, only nodose afferents project into the alveolar region, but both nodose and jugular afferents innervate conducting airways throughout the lung. Many afferents that project into the alveolar region express TRPV1. Few DRG afferents expressed TRPV1. Approximately 25% of blood vessels were innervated by vagal afferents (many were Tac1+). Approximately 10% of blood vessels had DRG afferents (some were Tac1+), but this was restricted to large vessels. Lastly, innervation of neuroepithelial bodies (NEBs) correlated with the cell number within the bodies. In conclusion, functionally distinct sensory subsets have distinct innervation patterns within the conducting airways, alveoli and blood vessels. Physiologic (e.g., stretch) and pathophysiological (e.g., inflammation, edema) stimuli likely vary throughout these regions. Our data provide a neuroanatomical basis for understanding afferent responses in vivo.

The lower airways (larynx to alveoli) are protected by a complex array of neural networks that regulate respiration and airway function. Harmful stimuli trigger defensive responses such as apnea, cough and bronchospasm by activating a subpopulation of sensory afferent nerves (termed nociceptors) which are found throughout the airways. Airway nociceptive fibers are projected from the nodose vagal ganglia, the jugular vagal ganglia and the dorsal root ganglia, which are derived from distinct embryological sources: the former from the epibranchial placodes, the latter two from the neural crest. Embryological source determines nociceptive gene expression of receptors and neurotransmitters and recent evidence suggests that placode- and neural crest-derived nociceptors have distinct stimuli sensitivity, innervation patterns and functions. Improved understanding of the function of each subset in specific reflexes has substantial implications for therapeutic targeting of the neuronal components of airway disease such as asthma, viral infections and chronic obstructive pulmonary disease.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, nonselective cation channel expressed on nociceptive sensory nerves whose activation elicits nocifensive responses (e.g. pain). TRPA1 is activated by electrophiles found in foods and pollution, or produced during inflammation and oxidative stress, via covalent modification of reactive cysteines, but the mechanism underlying electrophilic activation of TRPA1 is poorly understood. Here we studied TRPA1 activation by the irreversible electrophiles iodoacetamide and N-ethylmaleimide (NEM) following transient expression in HEK293 cells. We found that in Ca(2+) imaging studies C621 is critical for electrophile-induced TRPA1 activation, but the role of C665 in TRPA1 activation is dependent on the size of the electrophile. We identified slower TRPA1 activation in whole-cell recordings compared to studies with intact cells, which is rescued by pipette solution supplementation with the antioxidant glutathione. Single-channel recordings identified two distinct electrophilic-induced TRPA1 activation phases: a partial activation that, in some channels, switched to full activation with continued electrophile exposure. Full activation but not the initial activation was regulated by C665. Fitting of open time distributions suggests that full activation correlated with an additional (and long) exponential component, thus suggesting the phases are manifestations of distinct activation states. Our results suggest that distinct NEM-induced TRPA1 activation states are evoked by sequential modification of C621 then C665.

Irritant inhalation is associated with increased incidence of atrial fibrillation (AF) and stroke. Irritant inhalation acutely regulates cardiac function via autonomic reflexes. Increases in parasympathetic and sympathetic reflexes may increase atrial susceptibility to ectopic activity and the initiation of arrhythmia such as AF. Both age and hypertension are risk factors for AF. We have shown that irritant-evoked pulmonary-cardiac reflexes are remodeled in spontaneously hypertensive (SH) rats to include a sympathetic component in addition to the parasympathetic reflex observed in normotensive Wistar-Kyoto (WKY) rats. Here, we analyzed P wave morphology in 15-week old WKY and SH rats during inhalation of the transient receptor potential ankyrin 1 agonist allyl isothiocyanate (AITC). P Wave morphology was normal during vehicle inhalation but was variably modulated by AITC. AITC increased RR intervals (RRi), PR intervals, and the P Wave duration. In SH rats only, AITC inhalation increased the occurrence of negative P waves. The incidence of AITC-evoked negative P waves in SH rats was dependent on RRi, increasing during bradycardic and tachycardic cardiac cycles. Inhibition of both parasympathetic (using atropine) and sympathetic (using atenolol) components of the pulmonary-cardiac reflex decreased the incidence of negative P waves. Lastly, the probability of evoking a negative P Wave was increased by the occurrence of preceding negative P waves. We conclude that the remodeled irritant-evoked pulmonary-cardiac reflex in SH rats provides a substrate for altered P Wave morphologies. These are likely ectopic atrial beats that could provide a trigger for AF initiation in structurally remodeled atria.

Action potentials depend on voltage-gated sodium channels (Na(V)1s), which have nine α subtypes. Na(V)1 inhibition is a target for pathologies involving excitable cells such as pain. However, because Na(V)1 subtypes are widely expressed, inhibitors may inhibit regulatory sensory systems. Here, we investigated specific Na(V)1s and their inhibition in mouse esophageal mechanoreceptors-non-nociceptive vagal sensory afferents that are stimulated by low threshold mechanical distension, which regulate esophageal motility. Using single fiber electrophysiology, we found mechanoreceptor responses to esophageal distension were abolished by tetrodotoxin. Single-cell RT-PCR revealed that esophageal-labeled TRPV1-negative vagal neurons expressed multiple tetrodotoxin-sensitive Na(V)1s: Na(V)1.7 (almost all neurons) and Na(V)1.1, Na(V)1.2, and Na(V)1.6 (in ∼50% of neurons). Inhibition of Na(V)1.7, using PF-05089771, had a small inhibitory effect on mechanoreceptor responses to distension. Inhibition of Na(V)1.1 and Na(V)1.6, using ICA-121341, had a similar small inhibitory effect. The combination of PF-05089771 and ICA-121341 inhibited but did not eliminate mechanoreceptor responses. Inhibition of Na(V)1.2, Na(V)1.6, and Na(V)1.7 using LSN-3049227 inhibited but did not eliminate mechanoreceptor responses. Thus, all four tetrodotoxin-sensitive Na(V)1s contribute to action potential initiation from esophageal mechanoreceptors terminals. This is different to those Na(V)1s necessary for vagal action potential conduction, as demonstrated using GCaMP6s imaging of esophageal vagal neurons during electrical stimulation. Tetrodotoxin-sensitive conduction was abolished in many esophageal neurons by PF-05089771 alone, indicating a critical role of Na(V)1.7. In summary, multiple Na(V)1 subtypes contribute to electrical signaling in esophageal mechanoreceptors. Thus, inhibition of individual Na(V)1s would likely have minimal effect on afferent regulation of esophageal motility.