Rapid neonatal AAV delivery for adult cortical two-photon imaging of genetically encoded sensors

Elucidating the mechanisms of the central nervous system necessitates the ability to precisely visualize, record, and manipulate specific neuronal populations within the living brain. Here, we report a fast and efficient technique for delivering adeno-associated virus (AAV) in neonatal pups for the robust co-expression of reporters and sensors in cortical neuronal subpopulations under different promoters and transgenic mouse lines. This approach has lower training requirements than adult AAV injections, and it enables a 40- to 50-fold increase in injection throughput per animal with high reproducibility. Pup-injected adult mice show reduced signals of neuroinflammation compared to adult-injected mice. Importantly, adult pup-injected mice have stable and high expression of multiple AAVs suitable for structure-function two-photon imaging in local cortical circuits of awake, behaving adult animals. This approach offers a fast, efficient, and robust method of introducing multiple AAVs for investigating neuronal structure and function in defined cortical neuronal populations in the living brain.