SPARC: Intersectional labeling of vagal afferent nerve subsets using Cre and FLP dependent dual reporter strain

Abstract

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 nerves 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‐flanked STOP cassette and loxP‐flanked tdTomato). 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 also observed in the paratrigeminal nucleus (Pa5). In the lung, most TRPV1+ fibers expressing Tac1 were found in close proximity to the epithelial layer of bronchi and bronchioles. Whereas TRPV1+ fibers expressing P2X2 often projected away from the conducting airways into the alveolar tissue. Taken together, we have developed cell type specific‐dual reporter mouse models using a combinatorial/intersectional Cre and FLP strategy to successfully identify distinct nociceptive subtypes of afferent nerves in vagal ganglia, brainstem, and lung.