Serotype-dependent differences in AAV cellular transduction rates in the hypothalamus of Arctic ground squirrels

Adeno-associated viral (AAV) vectors are essential tools for molecular and circuit neuroscience, yet serotype tropism varies across brain regions and species, requiring empirical validation in each new biological context. This need is especially critical in non-model organisms, where molecular tools remain underdeveloped and access to research subjects is limited. No studies have evaluated AAV performance in the Arctic ground squirrel (AGS, Urocitellus parryii), a hibernating species increasingly used to investigate neural mechanisms of torpor, thermogenesis, and seasonal energy balance.

We compared cellular transduction rates of AAV serotypes 1, 8, 9, and DJ in the AGS hypothalamus using the human synapsin (hSyn) promoter. To maximize data collection from a limited experimental population, we used a within-animal, contralateral stereotaxic injection design with recombinant AAV vectors expressing EGFP or mCherry, analyzed four weeks post-injection.

All serotypes produced clear and reproducible reporter expression. AAV1 produced significantly greater cellular transduction rates than AAV-DJ (17.2% ± 3.5% vs 8.4% ± 2.9%, paired t-test, p = 0.032). AAV8 and AAV9 showed transduction rates of 22.8% ± 0.6% and 20.1% ± 1.5%, respectively. Reporter expression was predominantly neuronal across all serotypes, consistent with the hSyn promoter.

Prior AAV characterization in ground squirrels has been limited to 13-lined ground squirrels; no systematic serotype comparison has been performed in AGS.

These findings provide the first characterization of AAV-mediated gene delivery in the AGS brain and establish a methodological foundation for future molecular interrogation of hypothalamic circuits in this extreme mammalian hibernator.