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 remains poorly understood. Here, we interrogated the cell body location, 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.

TLR7 is expressed by support cells, but not sensory neurons, in ganglia

Background Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. Methods TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. Results In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. Conclusion TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7’s neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7’s primary role in neuronal tissues is not related to antiviral immunity.

Differential mechanisms of cold sensitivity in mouse trigeminal and vagal ganglion neurons

Thermal signals are critical elements in the operation of interoceptive and exteroceptive neural circuits, essential for triggering thermally-driven reflexes and conscious behaviors. A fraction of cutaneous and visceral sensory endings are activated by cold temperatures. Compared to somatic (DRG and TG) neurons, little is known about the mechanisms underlying cold sensitivity of visceral vagal neurons. We used pharmacological and genetic tools for a side-by-side characterization of cold-sensitive (CS) neurons in adult mouse trigeminal (TG) and vagal ganglia (VG). We found that CS neurons are more abundant in VG than in TG. In both ganglia, sensitivity to cold varied widely and was enhanced by the potassium channel blocker 4-AP. The majority of CS neurons in VG co-express TRPA1 markers and cold-evoked responses are severely blunted in Trpa1 KO mice, with little impact of TRPM8 deletion or pharmacological TRPM8 blockade. Consistent with these findings, the expression of TRPM8-positive neurons was low in VG and restricted to the rostral jugular ganglion. In vivo retrograde labelling of airway-innervating vagal neurons demonstrated their enhanced cold sensitivity and a higher expression of TRPA1 compared to neurons innervating the stomach wall. In contrast, the majority of CS TG neurons co-express TRPM8 markers and their cold sensitivity is reduced after TRPM8 deletion or blockade. However, pharmacological or genetic reduction of TRPA1 showed that these channels contribute significantly to their cold sensitivity in TG. In both ganglia, a fraction of CS neuron respond to cooling by a mechanism independent of TRPA1 or TRPM8 yet to be characterized.

TLR7 Is Expressed by Support Cells, but Not Sensory Neurons, in Ganglia

Background: Toll like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded development of a mechanistic understanding of TLR7 function in nervous tissues.Methods: TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout mice, and in guinea pigs. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. Pulmonary afferent neurons in vagal ganglia were also specifically tested since respiratory viruses are a common TLR7 ligand. Guinea pig vagal afferents were labeled by intranasal instillation of wheat germ agglutinin, isolated using flow cytometry and analyzed for TLR7 by RT-PCR. The effects of influenza A infection on TLR7 expression in sensory ganglia and in spleen were also assessed.Results: In situ probes detected TLR7 in the spleens and in small support cells adjacent to sensory neurons in dorsal root and vagal ganglia in wild type mice and guinea pig, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage and satellite glial cell marker Iba1, but was absent in sensory nerves in vagal and dorsal root ganglia in both mice and guinea pigs. In contrast, a TLR7 antibody labeled small and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Wheat germ agglutinin-positive cells sorted by flow cytometry expressed both TLR7 and Iba1, indicating that TLR7-expressing support cells sort alongside neurons despite ganglia dissociation. Influenza A infection caused significant weight loss and upregulation of TLR7 in spleens, but not in vagal ganglia, in mice.Conclusion: TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7’s neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7’s role in neuronal tissues is not primarily related to antiviral immunity.

Inferior vagal ganglion galaninergic response to gastric ulcers

Galanin is a neuropeptide widely expressed in central and peripheral nerves and is known to be engaged in neuronal responses to pathological changes. Stomach ulcerations are one of the most common gastrointestinal disorders. Impaired stomach function in peptic ulcer disease suggests changes in autonomic nerve reflexes controlled by the inferior vagal ganglion, resulting in stomach dysfunction. In this paper, changes in the galaninergic response of inferior vagal neurons to gastric ulceration in a pig model of the disease were analyzed based on the authors’ previous studies. The study was performed on 24 animals (12 control and 12 experimental). Gastric ulcers were induced by submucosal injections of 40% acetic acid solution into stomach submucosa and bilateral inferior vagal ganglia were collected one week afterwards. The number of galanin-immunoreactive perikarya in each ganglion was counted to determine fold-changes between both groups of animals and Q-PCR was applied to verify the changes in relative expression level of mRNA encoding both galanin and its receptor subtypes: GalR1, GalR2, GalR3. The results revealed a 2.72-fold increase in the number of galanin-immunoreactive perikarya compared with the controls. Q-PCR revealed that all studied genes were expressed in examined ganglia in both groups of animals. Statistical analysis revealed a 4.63-fold increase in galanin and a 1.45-fold increase in GalR3 mRNA as compared with the controls. No differences were observed between the groups for GalR1 or GalR2. The current study confirmed changes in the galaninergic inferior vagal ganglion response to stomach ulcerations and demonstrated, for the first time, the expression of mRNA encoding all galanin receptor subtypes in the porcine inferior vagal ganglia.

The Role of the Placodes in the Development of the Glossopharyngeal, Vagal, and Trigeminal Ganglia

The epibranchial placodes combine with the neural crest to form the inferior and superior ganglia of the glossopharyngeal and vagal cranial nerves, respectively. By comparison, the single trigeminal ganglion is composed of both neural crest and placodal cells. The steps that lead up to these events include gastrulation and the embryology of the notochord, neural crest, and the placodes. Each of these steps is reviewed in some detail. In previous reviews in this series, the embryology related to the olfactory, otic, and lens placodes, and to the geniculate ganglia has been discussed.1-3 However, the somewhat unusual embryology of the 2 ganglia of cranial nerves IX and X was only briefly mentioned as was the development of the trigeminal ganglion.4 This present review revisits these events and specifically focuses on how these ganglia develop.Learning Objective: The reader will learn the unusual development of the superior and inferior glossopharyngeal and the vagal ganglia as well as review the varied steps in the embryology that proceeds these events. By comparison, the development of the single trigeminal ganglion is presented and the differences in its development from that of the ganglia of cranial nerves IX and X are emphasized.