SPARC: Intersectional Labeling of Vagal Afferent Nerve Subsets Using Cre and Flp Dependent Dual Reporter Strain
Authors: Kim Seol-Hee , Hadley Stephen , Taylor-Clark Thomas 
Published: April 2020

Respiratory reflexes are tightly controlled by distinct types of vagal sensory afferent nerves. Afferent nerves can be categorized by their conduction velocity, axon diameter, myelination, responses to stimuli, ion channels/receptor expression, and embryological origin. Some airway vagal afferent nerve are sensitive to noxious stimuli (i.e. nociceptive). TRPV1, the capsaicin receptor, is selectively expressed on most vagal nociceptors. Vagal ganglia are comprised of jugular ganglia (neural crest origin) and nodose ganglia (placodal origin). Tac1 encodes Tachykinin1, the substance P precursor protein, and is mainly expressed in the jugular ganglia. P2X2, a purinergic receptor, is exclusively expressed in nodose ganglia. Previous data suggests that activation of jugular nociceptors and nodose nociceptors causes distinct reflexes. Here, we used intersectional genetics to simultaneously visualize nodose and jugular nociceptors.

We generated a transgenic mouse model with dual recombinase responsive allele using Cre‐Lox and Flp‐FRT (Flippase‐Flp recognition target) systems. With the ROSA26‐RC::FLTG allele, cells with only Flp show tdTomato expression (by excising of FRT flanked STOP cassette) while cells with both Flp and Cre have GFP expression (by excising of both FRT‐ and loxP‐flanked STOP cassette). We generated TRPV1‐Flp:Tac1‐Cre:ROSA and TRPV1‐Flp:P2X2‐Cre:ROSA strains. Offspring (6 to 8 weeks old) were used for characterization. Vagal ganglia, lung and brainstem were collected and fixed with 4% paraformaldehyde. Tissue were cryosectioned for immunohistochemistry.

In TRPV1‐Flp:Tac1‐Cre:ROSA, the majority of tdTomato expressing cells were in nodose ganglia and GFP expressing neurons were in jugular ganglia. In TRPV1‐Flp:P2X2‐Cre:ROSA, as expected, GFP expressing neurons were only observed in nodose ganglia while tdTomato expressing neurons were observed in both nodose and jugular ganglia. Reporter expressing neurons in both strains overlapped with anti‐TRPV1 antibody staining indicating they were all nociceptive. In the brainstem, reporter expression was mainly in the nucleus of solitary tract (nTS) in both strains. In TRPV1‐Flp:Tac1‐Cre:ROSA strain, GFP expression (jugular nociceptors) was observed in the nTS as well as paratrigeminal nucleus (Pa5). GFP labeled nerves were seen underneath the epithelial layer of airways in the lung. The majority of tdTomato labeled afferent nerve terminals traveled through the lung tissue with with GFP (+) nerves.

Taken together, we developed cell type specific‐dual reporter mouse models using Cre and Flp strategy in combination with the RC::FLTG strain that showed successfully labeled neurons and nerves that are specific to distinct nociceptive subtypes of afferent nerves in vagal ganglia, brainstem and lung. This allows the detailed visualization of complex architecture of various afferent nerves simultaneously in both peripheral organs and central nervous system.

Support or Funding Information

This 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” (grant no. OT2OD023854).