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 AAV8 as a highly effective vector, demonstrating strong neuronal tropism and anterograde/retrograde transgene expression, while AAVretro was ineffective. The CaMKII 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, including Cre/loxP and Tet-On/Tet-Off. 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.