SPARC: AAV‐Mediated Reporter Expression in Vagal Afferent Subsets Using Cell‐type Specific Cre Mice
Authors: Kim Seol-Hee , Hadley Stephen , Patil Mayur , Kollarik Marian , Taylor-Clark Thomas 
Published: April 2020

Vagal afferent nerves carry information from peripheral organs to the brainstem, where they synapse with central networks that govern physiological function through reflexes. Vagal afferent nerves are known to control respiratory functions. There are various subtypes of afferent nerves depending on its their developmental origin, expression of certain ion channels/receptors, stimuli and conduction velocities. Here, we used cell type specific Cre knock‐in strains in combination with AAV carrying Cre‐sensitive reporter allele to label specific subsets of vagal afferent nerves and determine their brainstem projection.

In this study, Trpv1 and Tac1 Cre strains were crossed with Floxed Rosa‐Ai9 tdTomato to generate mouse models expressing tdTomato in TRPV1 or Tac1 expressing cells/nerves in the presence of Cre. First, unilateral vagal injection of AAV9‐Flex‐EGFP (Cre sensitive) was carried out in TRPV1‐ and Tac1‐CreRosa strains. Next, unilateral vagal injection of AAV9‐hSyn‐EGFP (constitutively active) and AAV9‐Flex‐tdTomao (Cre‐dependent tdTomato expression) was carried out into the TRPV1‐Cre strain. Mice (6 to 8‐week‐old) received vagal injection of AAV virus and housed for four weeks. Vagal ganglia and brainstem were collected and cryosectioned sequentially. Immunohistochemistry was performed using anti‐Trpv1, anti‐DsRed and anti‐GFP, and images were taken using confocal microscope.

We observed tdTomato and EGFP labeled‐neurons in vagal ganglia of TRPV1‐ and Tac1‐CreRosa strains that received AAV‐Flex‐EGFP injection. 62% of tdTomato expressing cells also expressed EGFP, and less than 1% of EGFP(+) cells were negative for tdTomato in the TRPV1‐CreRosa strain. In the Tac1‐CreRosa strain, the majority of tdTomato and EGFP expression was seen in jugular ganglion. 60% of tdTomato expressing neurons co‐expressed EGFP. In medulla of both TRPV1‐Cre and Tac1‐CreRosa strains, the majority of EGFP expressing nerves were seen in the nucleus of solitary tract (nTS) and in the area postrema (AP) and paratrigeminal nucleus (Pa5). In TRPV1‐CreRosa strain, EGFP+ nerves mainly innervated SolG, SolDL and SolC in the nTS. In Tac1‐CreRosa strain, some EGFP+ nerves were observed in SolG, SolDL and SolC in caudal nTS, while others were seen in SolM, SolV and SolVL subnuclei in the rostral nTS. Next, unilateral intraganglionic co‐injection of AAV9‐hSyn‐EGFP and AAV9‐Flex‐tdTomato, caused tdTomato in 70% of EGFP(+) vagal neurons, while only 2% expressed tdTomato without EGFP. In the brainstem, most of the nociceptive vagal afferents (tdTomato+ population) innervated SolG, SolDL and SolC. Non‐nociceptive population (only expressing EGFP expression) was observed in SolM, SolV and SolVL subnuclei.

Taken together, our approach with the unilateral intraganglionic injections of AAV, into the vagal ganglia, carrying Cre‐dependent reporter allele allows the visualization of specific subsets of neurons/nerves in the vagal ganglia and the brainstem.

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).