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). [Display omitted] •Fast, cell-resolution and wide field-of-view (FOV) Ca2+ imaging in an awake mouse•Dense and cortical-wide Ca2+ sensor expression with quick neonatal AAV injection•Transcranial evaluation of the Ca2+ sensor expression with one-photon macro imaging•Making a chronic large cranial window for the wide FOV two-photon imaging 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 Ca 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.