Fluorescent Based Tracing of Sensory Nerve Subtypes in Vagal Ganglia and Lung

Abstract

Cell bodies of airway afferent nerves originate from vagal ganglia and vagal afferent nerves project to the respiratory tract. The inputs from these nerves converge at the brainstem to regulate respiratory reflexes. These vagal afferent nerves can be classified into various subtypes based on the origins of cell bodies in vagal ganglia, conduction velocity, size and stimuli required for activation. The polymodal nature of vagal afferent nerves complicates the study of respiratory reflexes. Transient receptor potential vanilloid 1 (TRPV1) is one of the ion channels expressed in bronchopulmonary C‐fibers. Tachykinin 1 (Tac1) encodes for a neuropeptide, Substance P, which is released from sensory nerve terminals in jugular originated afferent nerves. 5HT3 is expressed in nodose originated nociceptive C‐fibers. In the present study, we focused on defining afferent nerves subtypes in vagal ganglia based on ion channel/receptor expression as well as innervation into respiratory tract using a transgenic mouse model.The Cre‐LoxP reporter system was used to target specific populations of afferent nerve subtypes and to control red fluorescent protein (tdTomato) expression. Trpv1‐Cre, Tac1‐Cre and 5ht3‐Cre strains were bred with Floxed Ai9 ROSA tdTomato; thus, expressing red fluorescent in Trpv1, Tac1 and 5ht3 positive neurons. Mice (6 to 8‐week‐old) were euthanized and tissues were collected. Lungs were inflated with 3 % low‐melting agarose solution and sectioned at 100 μm using a vibratome. Immunohistochemistry was done using anti‐E‐Cadherin and anti‐dsRed antibodies. Vagal ganglia were dissected and cryo‐sectioned at 20 μm. Immunohistochemistry was done using anti‐Trpv1 antibody to compare with native tdTomato expression. Images were taken using Olympus FV1200 confocal microscope.Native tdTomato expression for Trpv1 positive afferent nerves were seen in both jugular and nodose ganglia. tdTomato tagged for Tac1 positive afferent nerves were mostly expressed in jugular and some in nodose ganglia, while 5ht3 positive afferent nerves were exclusively in nodose ganglia. The native tdTomato expression of 5ht3 positive afferent nerves did not completely overlap with anti‐TRPV1 staining. We further visualized the nerve endings that projected from vagal ganglia into lung and the tdTomato expression patterns of the nerve endings were different depending on strains. With Trpv1 strain, nerve endings spread out in all the lobes and those nerve endings resided in the epithelial layer, mainly around the bronchi.Overall, we were able to identify the distinct locations of cell bodies in vagal ganglia and nerve endings in lung of various subsets of airway afferent nerves using Cre‐LoxP reporter system. This information may be valuable to investigate the highly complicated mechanism of respiratory reflexes.Support or Funding InformationNIH Common Fund SPARC OT2This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.