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Abstract Background Previous studies suggest that transient receptor potential (TRP) channels and neurogenic inflammation may be involved in idiopathic pulmonary fibrosis (IPF)-related high cough sensitivity, although the details of mechanism are largely unknown. Here, we aimed to further explore the potential mechanism involved in IPF-related high cough sensitivity to capsaicin challenge in a guinea pig model of pulmonary fibrosis induced by bleomycin. Methods Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) were employed to measure the expression of TRP channel subfamily A, member 1 (TRPA1) and TRP vanilloid 1 (TRPV1), which may be involved in the cough reflex pathway. Immunohistochemical analysis and RT-qPCR were used to detect the expression of neuropeptides substance P (SP), Neurokinin-1 receptor (NK1R), and calcitonin gene-related peptide (CGRP) in lung tissues. Concentrations of nerve growth factor (NGF), SP, neurokinin A (NKA), neurokinin B (NKB), and brain-derived neurotrophic factor (BDNF) in lung tissue homogenates were measured by ELISA. Results Cough sensitivity to capsaicin was significantly higher in the model group than that of the sham group. RT-qPCR and immunohistochemical analysis showed that the expression of TRPA1 and TRPV1 in the jugular ganglion and nodal ganglion, and SP, NK1R, and CGRP in lung tissue was significantly higher in the model group than the control group. In addition, expression of TRP and neurogenic factors was positively correlated with cough sensitivity of the experimental animals. Conclusion Up-regulated expression of TRPA1 and TRPV1 in the cough reflex pathway and neurogenic inflammation might contribute to the IPF-related high cough sensitivity in guinea pig model.

Background: Previous studies suggest that transient receptor potential (TRP) channels and neurogenic inflammation may be involved in idiopathic pulmonary fibrosis (IPF)-related high cough sensitivity, although the details of mechanism are largely unknown. Here, we aimed to further explore the potential mechanism involved in IPF-related high cough sensitivity to capsaicin challenge in a guinea pig model of pulmonary fibrosis induced by bleomycin.Methods: Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) were employed to measure the expression of TRP channel subfamily A, member 1 (TRPA1) and TRP vanilloid 1 (TRPV1), which may be involved in the cough reflex pathway. Immunohistochemical analysis and RT-qPCR were used to detect the expression of neuropeptides substance P (SP), Neurokinin-1 receptor (NK1R), and calcitonin gene-related peptide (CGRP) in lung tissues. Concentrations of nerve growth factor (NGF), SP, neurokinin A (NKA), neurokinin B (NKB), and brain-derived neurotrophic factor (BDNF) in lung tissue homogenates were measured by ELISA.Results: Cough sensitivity to capsaicin was significantly higher in the model group than that of the sham group. RT-qPCR and immunohistochemical analysis showed that the expression of TRPA1 and TRPV1 in the jugular ganglion and nodal ganglion, and SP, NK1R, and CGRP in lung tissue was significantly higher in the model group than the control group. In addition, expression of TRP and neurogenic factors was positively correlated with cough sensitivity of the experimental animals.Conclusion: Up-regulated expression of TRPA1 and TRPV1 in the cough reflex pathway and neurogenic inflammation might contribute to the IPF-related high cough sensitivity in guinea pig model. [ABSTRACT FROM AUTHOR]

We have determined whether brain-derived neurotrophic factor immunoreactive (BDNF-ir) neurons in the vagal ganglia innervate the gastrointestinal tract. Many BDNF-ir neurons were medium in size and located throughout the jugular and nodose ganglia. When Fluorogold was injected into the wall of the cervical esophagus, many retrogradely Fluorogold-labeled neurons were found in both the jugular ganglion and the nodose ganglion. When Fluorogold was injected into the body of the stomach or applied to the cut end of the subdiaphragmatic vagus nerve, numerous Fluorogold-labeled neurons were found mostly in the nodose ganglion. Double-labeling combining immunohistochemistry for BDNF and retrograde tracing with Fluorogold showed that more than 90% of the neurons in the jugular ganglion and the nodose ganglion projecting to the cervical esophagus contained BDNF-like immunoreactivity. In the cases of both Fluorogold injection into the stomach and Fluorogold application to the subdiaphragmatic vagus nerve, almost all Fluorogold-labeled neurons in the nodose ganglion contained BDNF-like immunoreactivity. These results indicated that almost all vagal sensory neurons located in either the jugular ganglion or the nodose ganglion that innervate the gastrointestinal tract are BDNF-ir neurons. [ABSTRACT FROM AUTHOR]

Abstract: We have determined the localization of calcitonin gene-related peptide-immunoreactive (CGRP-ir) and calretinin-ir neurons in the vagal ganglia that innervate the cervical or subdiaphragmatic esophagus. Many CGRP-ir neurons were found exclusively in the jugular ganglion located in the cranial cavity. Calretinin-ir neurons were distributed throughout the vagal ganglia. Injection of Fluorogold into the cervical esophagus resulted in many Fluorogold-labeled neurons in the jugular and nodose ganglia. Injection of Fluorogold into the subdiaphragmatic esophagus resulted in many Fluorogold-labeled neurons, with most in the nodose ganglion. In the case of Fluorogold injection into the cervical esophagus, double-labeling combining immunohistochemistry and retrograde tracing showed that about 40% of the Fluorogold-labeled neurons in the jugular ganglion express CGRP-like immunoreactivity, and about 20% of the Fluorogold-labeled neurons in both the jugular and nodose ganglia express calretinin-like immunoreactivity. In the case of injection into the subdiaphragmatic esophagus, only a few Fluorogold-labeled neurons express CGRP-like immunoreactivity or calretinin-like immunoreactivity in the vagal ganglia. These results indicate that the cervical esophagus receives projections from many CGRP-ir neurons in the jugular ganglion and from calretinin-ir neurons in the jugular and nodose ganglia, while the subdiaphragmatic esophagus receives projections from only a few CGRP-ir and calretinin-ir neurons in the vagal ganglia. [Copyright &y& Elsevier]

We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed AΔ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aß-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as ~50% of the nodose neurons. GFRα2 was expressed in ~50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract. [ABSTRACT FROM AUTHOR]

Abstract: We have studied the connections of calcitonin gene-related peptide immunoreactive (CGRP-ir) sensory neurons in the ganglia of the vagus nerve. Many CGRP-ir neurons were identified in the jugular ganglion located in the cranial cavity, while fewer CGRP-ir neurons were found in the nodose ganglion located at the level of the jugular foramen. Application of Fluorogold to the cut end of the cervical vagus nerve resulted in many Fluorogold-labeled neurons in both the jugular and the nodose ganglia. Application of Fluorogold to the cut end of the subdiaphragmatic vagus nerve resulted in Fluorogold-labeled neurons mostly in the nodose ganglion with only a few labeled neurons in the jugular ganglion. Injection of Fluorogold into the heart resulted in Fluorogold-labeled neurons in both the jugular and the nodose ganglia. Double labeling combining CGRP immunohistochemistry and Fluorogold retrograde tracing showed that in cases of both the application of Fluorogold to the cut end of the cervical vagus nerve and the injection of Fluorogold into the heart, about 40% of the Fluorogold-labeled neurons in the jugular ganglion expressed CGRP-like immunoreactivity. These results indicate that many CGRP-ir neurons in the jugular ganglion innervate the cervical and thoracic visceral organs, including the heart, but only a few CGRP-ir neurons project to the abdominal visceral organs. [Copyright &y& Elsevier]

Abstract: TRPM8 and TRPA1 are cold-activated transient receptor potential (TRP) cation channels. TRPM8 is activated by moderate cooling, while TRPA1 is activated by extreme, noxious cold temperatures. These cold receptors are expressed in different subpopulations of primary afferent neurons. TRPA1 is co-expressed in a subpopulation of somatosensory neurons expressing TRPV1, which is activated by heat. However, the distribution and co-expression of these channels in the nodose–petrosal ganglion complex, which contains the jugular (JG), petrosal (PG), and nodose ganglia (NG) (mainly involved in putative somatic, chemo- and somato-sensation, and somato and visceral sensation, respectively), remain unknown. Here, we conducted in situ hybridization analysis of the rat nodose–petrosal ganglion complex using specific riboprobes for TRPM8, TRPA1, and TRPV1 to compare the features of the cranial sensory ganglia. Hybridization signals for TRPA1 were diffusely observed throughout these ganglia, whereas TRPM8 transcripts were seen in the JG and PG but not in the NG. We retrogradely labeled cranial nerve X with Fast Blue (fluorescent dye) and found TRPM8 transcripts in the jugular-vagal ganglion but not the NG neurons. TRPA1 transcripts were not detected in TRPM8-expressing neurons but were present in the subpopulation of TRPV1-expressing visceral sensory neurons. Taken together, these findings support that in the vagal system the expression of cold-activated TRP channels differs between nodose- and jugular-ganglion neurons suggesting different mechanisms of cold-transduction and that the TRPA1 distribution is consistent with its proposed function as a cold-sensing receptor in the visceral system. [Copyright &y& Elsevier]

The lungs and esophagus are innervated by sensory neurons with somata in the nodose, jugular, and dorsal root ganglion. These sensory ganglia are derived from embryonic placode (nodose) and neural crest tissues (jugular and dorsal root ganglia; DRG). We addressed the hypothesis that the neuron's embryonic origin (e.g., placode vs. neural crest) plays a greater role in determining particular aspects of its phenotype than the environment in which it innervates (e.g., lungs vs. esophagus). This hypothesis was tested using a combination of extracellular and patch-clamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to α,β-methylene ATP with action potential discharge that was sensitive to the P2X3 (P2X2/3) selective receptor antagonist A-317491. The somata of lung- and esophagus-specific sensory fibers were identified using retrograde tracing with a fluorescent dye. Esophageal- and lung-traced neurons from placodal tissue (nodose neurons) responded similarly to α,β-methylene ATP (30 µM) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of α,β-methylene ATP resulted in only a transient rapidly inactivating current or no detectable current. It has been shown previously that only activation of P2X213 heteromeric receptors produce sustained currents, whereas homomeric P2X3 receptor activation produces a rapidly inactivating current. Consistent with this, single-cell RT-PCR analysis revealed that the nodose ganglion neurons innervating the lungs and esophagus expressed mRNA for P2X2 and P2X3 subunits, whereas the vast majority of jugular and dorsal root ganglia innervating these tissues expressed only P2X3 mRNA with little to no P2X2 mRNA expression. We conclude that the responsiveness of C-fibers innervating the lungs and esophagus to ATP and other purinergic agonists is determined more by their embryonic origin than by the environment of the tissue they ultimately innervate. [ABSTRACT FROM AUTHOR]

Abstract: Immunohistochemistry for brain-derived neurotrophic factor (BDNF) was performed on the rat vagal and glossopharyngeal sensory ganglia. In the jugular, petrosal and nodose ganglia, 56.1±5.5%, 52.4±9.4% and 80.0±3.0% of sensory neurons, respectively, were immunoreactive for BDNF. These neurons were small- to medium-sized and observed throughout the ganglia. In the solitary tract nucleus, the neuropil showed BDNF immunoreactivity. A double immunofluorescence method demonstrated that BDNF-immunoreactive neurons were also immunoreactive for calcitonin gene-related peptide (CGRP), P2X3 receptor, the capsaicin receptor (VR1) or vanilloid receptor 1-like receptor (VRL-1) in the jugular (CGRP, 43.5%; P2X3 receptor, 51.1%; VR1, 71.7%; VRL-1, 0.5%), petrosal (CGRP, 33.2%; P2X3 receptor, 58.4%; VR1, 54.2%; VRL-1, 23.3%) and nodose ganglia (CGRP, 1.8%; P2X3 receptor, 49.1%; VR1, 70.7%; VRL-1, 11.5%). The co-expression with tyrosine hydroxylase was also detected in the petrosal (2.9%) and nodose ganglia (2.2%). However, BDNF-immunoreactive neurons were devoid of parvalbumin in these ganglia. The present findings suggest that BDNF-containing vagal and glossopharyngeal sensory neurons have nociceptive and chemoreceptive functions. [Copyright &y& Elsevier]

Abstract: ASIC3-immunoreactivity (ir) was examined in the rat vagal and glossopharyngeal sensory ganglia. In the jugular, petrosal and nodose ganglia, 24.8%, 30.8% and 20.6% of sensory neurons, respectively, were immunoreactive for ASIC3. These neurons were observed throughout the ganglia. A double immunofluorescence method demonstrated that many ASIC3-immunoreactive (ir) neurons co-expressed calcitonin gene-related peptide (CGRP)- or vanilloid receptor subtype 1 (VRL-1)-ir in the jugular (CGRP, 77.8%; VRL-1, 28.0%) and petrosal ganglia (CGRP, 61.7%; VRL-1, 21.5%). In the nodose ganglion, however, such neurons were relatively rare (CGRP, 6.3%; VRL-1, 0.4%). ASIC3-ir neurons were mostly devoid of tyrosine hydroxylase in these ganglia. However, some ASIC3-ir neurons co-expressed calbindin D-28k in the petrosal (5.5%) and nodose ganglia (3.8%). These findings may suggest that ASIC3-containing neurons have a wide variety of sensory modalities in the vagal and glossopharyngeal sensory ganglia. [Copyright &y& Elsevier]

Abstract: The co-expression of calretinin with parvalbumin and calbindin D-28k was examined in the rat cranial and spinal sensory ganglia by triple immunofluorescence method. In the trigeminal and nodose ganglia, 9% and 5% of calretinin-immunoreactive neurons, respectively, also contained both parvalbumin- and calbindin D-28k immunoreactivity. These neurons had large cell bodies. In the trigeminal ganglion, they were restricted to the caudal portion. Such neurons were evenly distributed throughout the nodose ganglion. The co-expression could not be detected in the dorsal root, jugular or petrosal ganglia. Nerve fibers which co-expressed all the three calcium-binding proteins were observed in the inferior alveolar nerve but not the infraorbital nerve or palate. In the periodontal ligament, these nerve fibers formed Ruffini-like endings. These findings suggest that (1) the co-expression in trigeminal neurons is intimately related to their peripheral receptive fields; (2) the three calcium-binding proteins (calretinin, parvalbumin, calbindin D-28k) co-expressed in the trigeminal neurons may have mechanoreceptive function in the periodontal ligament. [Copyright &y& Elsevier]

Abstract: Immunohistochemistry for delta-opioid receptor (DOR) was performed on the rat cranial sensory ganglia. The immunoreactivity was detected in 16%, 19% and 11% of neurons in the trigeminal, jugular and petrosal ganglia, respectively. The nodose ganglion was devoid of such neurons. DOR-immunoreactive (IR) neurons were mostly small to medium-sized (trigeminal, range = 62–851 μm2, mean ± SD = 359 ± 175 μm2; jugular, range = 120–854 μm2, mean ± SD = 409 ± 196 μm2; petrosal, range = 167–1146 μm2, mean ± SD = 423 ± 233 μm2). Double immunofluorescence method revealed that all DOR-IR neurons were also immunoreactive for calcitonin gene-related peptide. The cutaneous and mucosal epithelia in the oro-facial region, tooth pulp, taste bud and carotid body were innervated by DOR-IR nerve fibers. In the brainstem, IR nerve terminals were located in the superficial medullary dorsal horn and dorsomedial part of the subnucleus oralis as well as the solitary tract nucleus. The present study suggests that DOR-IR neurons may be associated with nociceptive and/or chemoreceptive function in the cranial sensory ganglia. [Copyright &y& Elsevier]

Abstract: Peptide 19 (PEP 19) is a 7.6-kDa polypeptide which binds to calmodulin and inhibits calcium–calmodulin signaling. In this study, PEP 19-immunoreactivity (PEP 19-IR) was examined in the rat vagal and glossopharyngeal sensory ganglia. Twenty-nine percent, 59%, and 41% of sensory neurons contained PEP 19-IR in the jugular, petrosal, and nodose ganglia, respectively. These neurons were of various sizes (jugular, mean ± SD = 635.8 ± 392.6 μm2, range = 105.9–1695.9 μm2; petrosal, mean ± SD = 370.9 ± 228.5 μm2, range = 57.7–1662.7 μm2; nodose, mean ± SD = 380.5 ± 157 μm2, range = 87.5–950.4 μm2) and scattered throughout these ganglia. Double immunofluorescence method revealed that PEP 19-IR neurons which had parvalbumin-IR were rare in the ganglia (jugular, 4%; petrosal, 10%; nodose, 8%). PEP 19-IR neurons which contained calbindin D-28k were abundant in the petrosal (20%) and nodose (22%) ganglia but not in the jugular ganglion (8%). Retrograde tracing method indicated that many PEP 19-IR neurons projected to the circumvallate papilla and soft palate. In the soft palate, taste buds were innervated by PEP 19-IR nerve fibers. The present study suggests that PEP 19-IR neurons include chemoreceptors in the vagal and glossopharyngeal sensory ganglia. [Copyright &y& Elsevier]

Immunohistochemistry for two nociceptive transducers, the vanilloid receptor 1 (VR1) and vanilloid receptor 1-like receptor (VRL-1), was performed on the vagal sensory ganglia. In the jugular ganglion, VR1-immunoreactive (IR) neurons were small to medium-sized (range 49.7–1125.6 μm2, mean±S.D. 407.7±219.7 μm2), whereas VRL-1-IR neurons were medium-sized to large (range 223.6–1341.1 μm2, mean±S.D. 584.3±253.5 μm2). In the nodose ganglion, VR1- and VRL-1-IR neurons were mostly small to medium-sized (VR1: range 148.5–1464.4 μm2, mean±S.D. 554.3±207.4 μm2; VRL-1: range 161.7–1166.2 μm2, mean±S.D. 541.9±186.2 μm2). The double immunofluorescence method revealed that co-expression of VR1-immunoreactivity among VRL-1-IR neurons was more abundant in the nodose ganglion (63%) than in the jugular ganglion (4%). The present study suggests that co-expression of VR1 and VRL-1 may be more common in visceral sensory neurons than in somatic sensory neurons. [Copyright &y& Elsevier]

• Human jugular ganglion had sensory neurons with small to medium-sized cell bodies. • These neurons contained TRPV1, TRPV2, SP, CGRP and VGLUT2. • Free nerve endings contained the TRPVs and neuropeptides in external auditory canal. • TRPV- and neuropeptide-containing vagal nociceptors probably innervate the periphery. The jugular ganglion (JG) contains sensory neurons of the vagus nerve which innervate somatic and visceral structures in cranial and cervical regions. In this study, the number of sensory neurons in the human JG was investigated. And, the morphology of sensory neurons in the human JG and nodose ganglion (NG) was compared. The estimated number of JG neurons was 2721.8–9301.1 (average number of sensory neurons ± S.D. = 7975.1 ± 3312.8). There was no significant difference in sizes of the neuronal cell body and nucleus within the JG (cell body, 1128.8 ± 99.7 μ m2; nucleus, 127.7 ± 20.8 μ m2) and NG (cell body, 963.8 ± 225.7 μ m2; nucleus, 123.2 ± 32.3 μ m2). These findings indicate that most of sensory neurons show the similar morphology in the JG and NG. Our immunohistochemical method also demonstrated the distribution of ion channels, neurotransmitter agents and calcium-binding proteins in the human JG. Numerous JG neurons were immunoreactive for transient receptor potential cation channel subfamily V member 1 (TRPV1, mean ± SD = 19.9 ± 11.5 %) and calcitonin gene-related peptide (CGRP, 28.4 ± 6.7 %). A moderate number of JG neurons contained TRPV2 (12.0 ± 4.7 %), substance P (SP, 15.7 ± 6.9 %) and secreted protein, acidic and rich in cysteine-like 1 (SPARCL1, 14.6 ± 7.4 %). A few JG neurons had vesicular glutamate transporter 2 (VGLUT2, 5.6 ± 2.9 %) and parvalbumin (PV, 2.3 ± 1.4 %). SP- and TRPV2-containing JG neurons had mainly small and medium-sized cell bodies, respectively. TRPV1- and VGLUT2- containing JG neurons were small to medium-sized. CGRP- and SPARCL1-containing JG neurons were of various cell body sizes. Sensory neurons in the human JG were mostly free of vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY). In the external auditory canal skin, subepithelial nerve fibers contained TRPV1, TRPV2, SP, CGRP and VGLUT2. Perivascular nerve fibers also had TRPV1, TRPV2, SP, CGRP, VIP, NPY and TH. However, PV- and SPARCL1-containing nerve endings could not be seen in the external auditory canal. It is likely that sensory neurons in the human JG can transduce nociceptive and mechanoreceptive information from the external auditory canal. Theses neurons may be also associated with neurogenic inflammation in the external auditory canal and ear-cough reflex through the vagus nerve. [ABSTRACT FROM AUTHOR]

Sensory functions of the vagus nerve are critical for conscious perceptions and for monitoring visceral functions in the cardio-pulmonary and gastrointestinal systems. Here, we present a comprehensive identification, classification, and validation of the neuron types in the neural crest (jugular) and placode (nodose) derived vagal ganglia by single-cell RNA sequencing (scRNA-seq) transcriptomic analysis. Our results reveal major differences between neurons derived from different embryonic origins. Jugular neurons exhibit fundamental similarities to the somatosensory spinal neurons, including major types, such as C-low threshold mechanoreceptors (C-LTMRs), A-LTMRs, Aδ-nociceptors, and cold-, and mechano-heat C-nociceptors. In contrast, the nodose ganglion contains 18 distinct types dedicated to surveying the physiological state of the internal body. Our results reveal a vast diversity of vagal neuron types, including many previously unanticipated types, as well as proposed types that are consistent with chemoreceptors, nutrient detectors, baroreceptors, and stretch and volume mechanoreceptors of the respiratory, gastrointestinal, and cardiovascular systems. • A comprehensive molecular identification of neuronal types in vagal ganglion complex • Prdm12 + jugular ganglion neurons share features with spinal somatosensory neurons • Phox2b + viscerosensory nodose neurons are molecularly versatile and highly specialized • Nodose neuron types are consistent with chemo-, baro-, stretch-, tension-, and volume-sensors Visceral sensory neurons are necessary for the control of organ functions, but knowledge on the complexity of neuron types involved is missing. Kupari et al. molecularly identify jugular and nodose ganglion neurons and find a large diversity of neuron types that are consistent with the numerous sensory functions of the vagus nerve. [ABSTRACT FROM AUTHOR]