Protocol for cortical-wide field-of-view two-photon imaging with quick neonatal adeno-associated virus injection

We recently established a simple and versatile adeno-associated virus (AAV) induction approach that enables dense (>90% labeled neurons) and cortical-wide Ca²⁺ sensor expression. Here, we describe the stepwise protocol for neonatal AAV injection of a Ca²⁺ sensor. We also detail the steps for subsequent craniotomy to generate a chronic cranial window, followed by wide-field two-photon Ca²⁺ imaging in an awake mouse. This protocol serves as an alternative to the use of transgenic animals and offers translatable options for cortical-wide experiments. For complete details on the use and execution of this protocol, please refer to Ota et al. (2021).