VIRAL STRATEGIES FOR 2-PHOTON IMAGING OF AVIAN RETINAL CIRCUITS

Avian vision relies on one of the most structurally and functionally elaborate retinas among vertebrates, enabling specialized behaviors such as flight navigation, exceptional spatial acuity, and complex color discrimination. Despite this sophistication, direct functional access to avian retinal circuits has remained limited, largely due to the longstanding challenge of achieving reliable expression of genetic tools in avian neurons.
Here, we present progress towards an experimental framework for functional imaging of avian retinal circuits using adeno-associated viral (AAV) gene delivery combined with 2-photon imaging. Building on recent validation of AAV mediated transduction in songbird retinal neurons (Seth et al., 2025; DOI:10.3389/fphys.2025.1549585), we extend this strategy to chick retina using a custom ocular injection strategy. Reporter expression reveals robust targeting of the outer and occasional access to inner retinal populations.
Using this framework, we demonstrate initial calcium and glutamate biosensor expression in defined retinal layers, establishing a functional entry point into retinal signaling. In parallel, we also demonstrate the initial validation of 2-photon recordings from cone synaptic terminals, offering direct optical access to light evoked activity at the first synapse of the avian visual system.
While biosensor expression is becoming increasingly consistent, reproducible recordings remain an active focus of optimization. Together, this work establishes a realistic and scalable path towards a system’s level investigation of avian retinal processing and lays the foundation for comparative studies of visual circuit function across avian species.