QUETZAL: AN OPTIMIZED SINGLE-AAV, DUAL-FLUOROPHORE CONSTRUCT FOR SUBCELLULAR FUNCTIONAL IMAGING

Calcium imaging is a powerful approach for monitoring neuronal activity in living tissues. Among genetically encoded calcium indicators, GCaMP is widely used due to its high sensitivity and robust green fluorescence upon calcium binding. However, interpreting GCaMP signals during awake behavior remains challenging. Motion artifacts can introduce fluorescence fluctuations unrelated to true calcium events, due to shifts in the focal plane. These motion-induced fluctuations are exacerbated when imaging fine subcellular structures such as dendrites and dendritic spines, where even small displacements can substantially distort measured signals. To address these limitations, we developed Quetzal, a fusion construct combining jGCaMP8m with the bright red fluorescent protein mScarlet. By integrating both fluorophores into a single fusion protein, Quetzal ensures precise co-localization of the green calcium-dependent signal and the red calcium-independent structural signal within the same cellular compartment. This design enables improved motion correction of the GCaMP signal using the red structural fluorophore as a stable reference. To further optimize the construct for subcellular imaging, we created a Quetzal construct with an added actin-binding domain, facilitating expression in actin-rich structures such as dendritic spines. Using Quetzal, we performed two-photon imaging of individual spines in the retrosplenial cortex during an associative learning task in which an auditory cue was paired with an aversive air puff. Together, this study shows that we can longitudinally track individual spines and their calcium dynamics during learning in the retrosplenial cortex.