Identification of lung innervating sensory neurons and their target specificity

Abstract

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

Keywords: innervation; interoception; lung; nerves; vagal ganglia.

Figure 1.

Lung innervating sensory cell bodies reside in the vagal ganglia and project to the nucleus of the solitary tract. A: intratracheal instillation scheme. B–D: Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis protocol (CUBIC) cleared vagal ganglia (VG) and nerve tracts from Rosa-lxl-tdTom (Ai14) mice with intratracheal instillation of rAAV-retro-cre. PN, pharyngeal nerve. Quantification in C shows the number of tdTomato+ neurons in the VG and the T3-T4 dorsal root ganglia (DRG) after instillation of rAAV-retro-cre. Thick nerve tract in D projects to lung. E: CUBIC cleared whole brainstem of Ai14 mice with intratracheal instillation of rAAV2-retro-cre, showing nerve terminal signals in bilateral nTS. F–I: sections (40 μm) of brainstem medulla at either rostral (F and G) or caudal (H and I) levels showing nerve signals. Dashed circles outline the brainstem. Boxed regions in F and H are magnified in G and I, respectively. nTS, nucleus of the solitary tract; AP, area postrema; CC, central canal. Scale bars = 250 μm in B, 100 μm in D, 500 μm in E, and 50 μm in F and H.

Figure 2.

Sensory innervation pattern of the respiratory tract and lung. A and B: confocal max projection of Vglut2-cre; Ai14 vagal ganglia. tdTomato labels both large and small neurons (arrowheads in B). C–G: representative Vglut2-cre; Ai14 respiratory tract with vagal ganglia and nerve bundles. H: diagram depicting the consensus pattern of respiratory tract innervation by vagal sensory nerves. I–K: light sheet images of Vglut2-cre; Ai14 whole cranial lung lobes from 2 mice at similar age. L–N: light sheet images of Nkx2-1^GFP; Vglut2-cre; Ai14 whole cranial lung lobes from 2 mice at similar age. GN, glossopharyngeal nerve; PN, pharyngeal nerve; SLN, superior laryngeal nerve; RLN, recurrent laryngeal nerve. Left and right side of the mouse is labeled in E and H. Scale bars = 100 μm in A and B and 1 mm in E and I–N.

Figure 3.

Transcriptional signature of lung innervating vagal neurons. A–L: lung innervating vagal neurons are retrogradely labeled with rAAV-retro-cre into Ai14 lungs (tdTom). RNAscope was performed on vagal ganglia sections for overlap between tdTom (magenta) and Vglut2, Trpv1, Tac1, Calb1, Piezo1, Piezo2, Trpa1, Runx3, Vip, Tmc3, Glp1r, and Gabra1 (green), individually as labeled. Arrowheads point to neurons with double labeling. Scale bar = 50 μm.

Figure 4.

Vagal neuron labeling and brainstem projection of Vglut2-cre, Trpv1-cre, Tac1-cre and Calb1-cre mice after unilateral vagal ganglia injection of AAV-flex-tdTomato. A: schematic for AAV-flex-tdTomato vagal microinjection. B–I: representative Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis protocol (CUBIC)-cleared vagal ganglia (B–E) and whole brainstem (F–I) after AAV-flex-tdTomato vagal microinjection to the respective cre lines as labeled. F–I: autofluorescence green channel was used as background to outline the brainstem. nTS, the nucleus of solitary tract; TS, the solitary tract; AP, the area postrema; Pa5, the paratrigeminal nucleus; Sp5, the spinal trigeminal nucleus, CC, central canal. Scale bars = 200 μm in B–E and 500 μm in F–I; n = 5 animals for Vglut2-cre, n = 6 animals for TrpV1-cre, and n = 5 animals for Tac1-cre and Calb1-cre brainstem light sheet imaging and analysis.

Figure 5.

Vagal sensory neurons project to proximal and distal airways and vasculature within the lung. A–H’: sections (99 μm) of the lung showing vagal nerve projections near proximal airways (A–D’) or distal airways (bronchioles, E–H’) within the lung following vagal injection of AAV-flex-tdTomato into the respective cre lines as labeled. Airway smooth muscles were marked by airway smooth muscle (SMA) staining. The alveolar region was marked by staining of Podoplanin (PDPN) expressed by alveolar type I cells. Scale bars = 500 μm (A-–D) and 200 μm (E–H). I–P’: sections (99 μm) of the lung showing vagal nerve projections near arteries/veins marked by Von Willebrand Factor (VWF; I–L’) or lymphatic vessels marked by lymphatic vessel endothelial hyaluronan receptor-1 (LYVE; M–P’) following vagal injection of AAV-flex-tdTomato into respective cre lines as labeled. All scale bars = 100 μm in I–P; n = 3 animals for Vglut2-cre and n = 4 animals for TrpV1-cre, Tac1-cre, and Calb1-cre staining and analysis.

Figure 6.

Vagal sensory neurons project to airway smooth muscles, the alveolar region and pulmonary neuroendocrine cells. A–C’: sections (99 μm) of the lung showing thin nerve fibers adjacent to airway smooth muscles marked by SM22 following vagal injection of AAV-flex-tdTomato into respective cre lines as labeled. D–F’: thick, branched nerve terminals near airway smooth muscle marked by airway smooth muscle (SMA). This subepithelial nerve terminal morphology was observed following injection of AAV-flex-tdTomato into Vglut2-cre mice (D and D’), in Vglut2-cre; Ai14 mice from direct genetic cross (E and E’), and in Adcyap1-cre; Ai14 mice from direct genetic cross (F and F’). G–J’: sections (99 μm) of the alveolar region showing thin nerve fibers on selected alveolar type 1 (AT₁) cells, marked by receptor for advanced glycation end products (RAGE), and alveolar type 2 (AT₂) cells, marked by pro-surfactant protein C (Pro-SPC), following vagal injection of AAV-flex-tdTomato into Vglut2-cre (G–H’) or Trpv1-cre (I–J’) mice. K–N’: sections (99 μm) of the lung showing vagal nerve projections contacting PNECs, labeled by calcitonin gene-related peptide (CGRP), following vagal injection of AAV-flex-tdTomato into respective lines as labeled. Vglut2, Tac1 and Calb1 projections intercalate between PNECs from the basal to apical side (K′-K′′, M′-M′′ and N′-N′′; Supplemental Videos S4, S6, and S7). Trpv1-cre vagal sensory nerves only contact the basal side of PNEC clusters (arrowheads in L’–L”) and do not intercalate between PNECs (Supplemental Video S5). Arrowhead in N’ indicates CGRP+ nerve fibers passing above PNEC clusters. Scale bars = 50 μm in A–J; 10 μm in K–N’; n = 3 animals for Vglut2-cre and n = 4 animals for TrpV1-cre, Tac1-cre, and Calb1-cre staining and analysis.

Figure 7.

Summary of Vglut2-, Trpv1-, Tac1-, and Calb1-expressing vagal sensory neuron projection patterns in lung. Within the lung, Vglut2-cre-, Tac1-cre-, and Trpv1-cre-labeled thin nerve fibers on airway smooth muscles. Vglut2-cre and Adcyap1-cre labeled nerves with branched and knob-like terminals on airway smooth muscles. Vglut2-cre- and Calb1-cre-labeled nerve fibers that innervate the epithelium at pulmonary neuroendocrine cell (PNEC) clusters, intercalating between PNECs. Trpv1-cre-labeled nerve fibers contact the basal side of PNEC clusters. Vglut2-cre- and Trpv1-cre-labeled nerves were found in the alveolar region near junction with distal airways. A sparse density of Vglut2-cre- and Trpv1-cre-labeled nerve fibers project to vasculature including large vessels and lymphatics. Also see Supplemental Table S3 for summary.