Achieving cell-type specific transduction with adeno-associated viral vectors in pigeons

Birds are valuable models for studying learning, cognition, song, and vision, yet tools for controlling and recording brain activity with millisecond precision remain underutilized in avian research. Advances in methods such as chemogenetics, optogenetics, and in vivo imaging have transformed rodent studies but require gene delivery techniques, like adeno-associated viruses (AAVs), in non-transgenic species. This study validates AAV tools for precise gene expression in pigeons. We identified both AAV1 and AAV8 as usable vectors, demonstrating strong neuronal tropism and anterograde/retrograde transgene expression, while AAVretro was ineffective. The CaMKIIα promoter and mDLX enhancer enabled cell-type-specific expression, targeting predominantly excitatory and inhibitory neurons, respectively. Additionally, we established proof of concept for the expression of NpHR (a chloride pump) and demonstrated the functionality of conditional gene expression systems using the Cre-loxP system. These advancements expand the genetic toolkit for pigeons, facilitating precise manipulation of neural circuits and enabling future studies on complex avian behaviors and brain functions. By bridging molecular tools and avian neuroscience, this work paves the way for comparative and translational research, offering insights into the neural basis of cognition and sensory processing in birds. • AAV8 enables efficient and non-toxic neuronal gene delivery in pigeons. • CaMKIIα and mDLX systems drive cell-type-preferential gene expression. • Cre/loxP system validated for conditional expression. • AAVretro shows limited utility for retrograde labeling in pigeons. • Expands the genetic toolkit for avian neuroscience applications.