SPARC: Visualization of genetically‐labeled vagal and spinal afferent subsets innervating the mouse lung

Abstract

Pulmonary functions are controlled by afferent nerves which convey peripheral information to the central nervous system. Cell bodies of these afferent nerves are found predominantly in the vagal ganglia (VG) with some in the dorsal root ganglia (DRG). These neurons are highly heterogeneous based on their developmental origins, anatomical sources, and physiochemical properties. Vagal ganglia are composed of nodose (placode origin), and jugular ganglia (neural crest origin). Most vagal afferent nerves innervating the lung are unmyelinated C‐fibers which are activated by capsaicin, the selective agonist of transient receptor potential vanilloid 1 (TRPV1). TRPV1 detects noxious stimuli and its activation results in defensive reflexes. Both nodose and jugular ganglia have TRPV1+ nociceptive C‐fiber but it is not currently known where they terminate within the lung. In addition, there is lack of information regarding pulmonary afferent nerves projecting from the DRG, which also are derived from the neural crest. Here, we used cell‐type specific Cre knock‐in strains in combination with injections with adeno‐associated viral vectors (AAV) carrying a Cre‐sensitive reporter allele to label specific subsets of vagal and DRG afferent nerves in lung. Pirt‐cre (marker for all sensory neurons), TRPV1‐cre (nociceptors) and Tac1‐cre (jugular, DRG) strains received unilateral injections of AAV9‐flex‐EGFP into nodose ganglia, and/or AAV9‐flex‐tdTomato into thoracic DRG (between T1‐T3). VG, DRG and lung were collected 4 weeks post‐injection and cryosectioned. Native fluorescent signals were amplified using anti‐DsRed and anti‐GFP immunoreactivity, and images were taken using an Andor Dragonfly spinning disk confocal microscope. Viral transfection was confirmed by expression of GFP in VG or tdTomato in DRG. Lung images were analyzed for VG/DRG nerve innervations based on diameters of airways (small: ≤175 µm, medium: 175‐376 µm, large: ≥376 µm). >95% of the conducting airways was innervated by vagal Pirt+ afferent nerves. About half of the conducting airways also had vagal Pirt+ fibers which projected out into the alveolar regions (mean distance 200±50 µm). Only 20% of the airways were innervated by Pirt+ DRG fibers, none of which projected into the alveolar regions. Vagal‐Pirt(+) nerves innervate most of the airways regardless of size, but DRG‐Pirt(+) nerves innervate mostly large diameter airways. ~75% of airways are innervated by vagal TRPV1+ afferents, some of which project into the alveolar regions. There is virtually no TRPV1+ innervation of the lung projected from the DRG. ~75% of large/medium sized airways were innervated by vagal originating nerves in Tac1‐cre, but none of these fibers projected into the alveolar spaces. Some large diameter airways were also innervated by Tac1+ fibers from DRG neurons. Together, our approach with the unilateral intraganglionic injections of AAV, into the VG and DRG, carrying a cre‐dependent reporter allele allows identification of specific subsets of afferent nerves in the VG and DRG innervating the lung.