Adeno Associated Virus Mediated Neural Tracing of TRPV1‐Expressing Airway Afferent Nerves

Abstract

The various sensory nerve subtypes deliver different types of information to brainstem in which the integration of the information takes place to regulate respiratory reflexes. The bronchopulmonary C‐fibers, the main sensory afferent regulating respiratory reflexes, arise from vagal ganglia and those are known to innervate the nucleus of the solitary tract in the medulla. However, the innervation patterns may vary depending on the subsets of vagal afferents. Vagal afferent nerve subtypes express particular ion channels/receptors which are stimulated by various stimuli resulting in different information being conveyed to the brainstem. The variation makes it difficult to understand how distinct peripheral vagal afferent inputs are processed in the central nervous system.In the current study, we determined the central projections of Transient receptor potential vanilloid 1 (TRPV1)‐expressing vagal sensory afferent innervating the airways using AAV mediated retrograde neural tracing. TRPV1 is expressed on nociceptive afferents involved in defensive/protective reflexes (e.g. cough, apnea). TRPV1‐Cre mice were used, along with a rAAV2 vector with a flex‐tdTomato reporter. In the presence of cre the AAV will be recombined to express tdTomato. Without cre the AAV does not express the reporter. Mice (6 to 8‐week‐old) received intratracheal instillation of AAV virus and housed for three weeks. Vagal ganglia and brainstem were collected and cryosectioned sequentially. Immunohistochemistry was performed and images were taken using Olympus FV1200 confocal microscope.As a result, we observed tdTomato labeled cells and nerves in vagal ganglia. With immunohistochemistry using anti‐DsRed and anti‐TRPV1 antibodies, we confirmed that the red fluorescent cells and nerves are TRPV1 positive. In medulla, we were able to see tdTomato labeled projections starting from caudal (approximately −780 μm to obex) to rostral (approximately +210 μm to obex) ends of medulla. The tdTomato labeled projections are mainly located in the nucleus of solitary tract (NTS) as well as along the solitary tract. The tdTomato labeled projections were seen mainly in gelatinous NTS and dorsolateral NTS, as well as in area postrema. These data suggest that airway nociceptor central terminals are located within a specific region of the NTS.Support or Funding InformationThis study is funded by National Institutes of Health Common Fund SPARC OT2 (2016–2019): “Functional mapping of peripheral and central circuits for airway protection and breathing”This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.