Systematic evaluation of AAV administration strategies for targeting Phox2b + primary visceral sensory neurons

Visceral sensory neurons (VSNs) play an essential role in interoception, but it remains challenging to selectively access them using genetic tools due to their anatomical location and shared molecular markers with central autonomic circuits. Although transgenic models visualizing Phox2b-expressing neurons have been developed to genetically label VSNs, concurrent Phox2b expression in the hindbrain limits their selectivity. To overcome these limitations, we engineered several Phox2b promoter-driven adeno-associated virus (AAV) vectors and evaluated the effects of capsid serotype, delivery route, and injection timing. Using systemic delivery of AAV-PHP.S-Phox2b-Cre, we found that postnatal day 8 is an optimal time window for efficient and selective labeling of VSNs with a relatively low dose. The result contrasts with neonatal administration, which led to extensive ectopic labeling at the same dose. Unexpectedly, lingual submucosal delivery of AAV-retro-Phox2b-Cre, initially intended to label tongue-innervating sensory neurons, also labeled most vagal neurons. These labeled neurons were AAV-retro capsid-specific, because local delivery of AAV-PHP.S-Phox2b-Cre labeled cells only near the injection site. Altogether, this study provides practical parameters for selective genetic access to VSNs using AAV.