Viral targeting of sensory nerves of the external ear in a murine model

Vagus Nerve Stimulation is used to treat a variety of medical conditions including stroke, tinnitus, and opioid withdrawal. However, because implantation of the device is invasive and involves surgical risks, stimulation of the auricular branch of the vagus nerve (ABVN) has been proposed as a non-invasive alternative. The ABVN is a purely sensory nerve that innervates a small portion of the external ear. Unfortunately, little work has been done to understand the function of the complex, overlapping nerves of the external ear, partly because methods to target the sensory nerves of the external ear have been limited. The goal of this study was to test whether we could selectively label sensory nerves of the external ear in mice using a novel adeno-associated virus (AAV). A Cre-dependent fluorescent reporter was packed in AAV-PHP.S, an AAV with enhanced tropism for peripheral sensory neurons, and was injected retro-orbitally into 9 adult mice. The mice transgenically expressed Cre recombinase under the control of a promoter (Pirt) which is expressed primarily in peripheral sensory neurons. One week after the injection, we transcardially perfused the mice and harvested the sensory ganglia for cranial nerves V, VII, IX, X, cervical dorsal root ganglia, and external ears. Immunohistochemistry was used to visualize reporter expression within the ganglia and the peripheral nerve fibers. We used anti-RFP to visualize the tdTomato reporter and β-III tubulin to label the nerve fibers within the external ear. After clearing the external ear, we imaged via lightsheet microscopy and confocal microscopy. We quantified the extent of nerve fibers with tdTomato expression in the external ear by dividing the virus labeled area by the β-III tubulin labeled area. The jugular ganglion (which houses the cell-bodies of the ABVN), nodose-petrosal ganglia, and nodose-petrosal-jugular complex all showed relatively similar incidence of virus labeling, specifically an average of 16%, 25%, and 22% of neurons in these ganglia were labeled, respectively. On the other hand, approximately 44% of the sensory afferents of the ear displayed labeling with tdTomato. Overall, these findings are a key step to a better understanding of the innervation of the rodent external ear and understanding the function of the ABVN. Future directions include targeted transfection of each ganglion that innervates the external ear. Funding Sources: This study was supported by NIH/NINDS (R01NS131493) and NIH/NIDCD (R01DC018733), and The Buoniconti Fund This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.