Retro-Orbital Delivery of AAVs for CNS Wide Astrocyte Targeting

Viral vector-mediated astrocyte targeting in live mice is a popular and valuable method to investigate astrocyte function in the context of intact neural circuits and complex brain physiology. Targeted genetic manipulation and functional investigation of this cell population can be accomplished by utilizing cell type–specific promoters to drive adeno-associated virus (AAV)-mediated transgene expression specifically in astrocytes. Here, we provide a comprehensive protocol for non-invasive retro-orbital (RO) administration of blood–brain barrier (BBB)-crossing AAVs in neonatal and adult mice, such as AAV-PHP.B, AAV-PHP.eB, and AAV.CAP-B22, which results in central nervous system (CNS)-wide transduction. Key procedures outlined include the preparation of AAV solutions for injection, a modified two-handed injection technique for precise and consistent RO injections, and a training strategy to practice mock RO injections using non-toxic dyes. This protocol serves as a valuable resource for researchers interested in exploring the roles of astrocytes in brain functions and neurological disorders.