Whole-brain functional ultrasound imaging in awake head-fixed mice

Most brain functions engage a network of distributed regions. Full investigation of these functions thus requires assessment of whole brains; however, whole-brain functional imaging of behaving animals remains challenging. This protocol describes how to follow brain-wide activity in awake head-fixed mice using functional ultrasound imaging, a method that tracks cerebral blood volume dynamics. We describe how to set up a functional ultrasound imaging system with a provided acquisition software (miniScan), establish a chronic cranial window (timing surgery: similar to 3-4 h) and image brain-wide activity associated with a stimulus at high resolution (100 x 110 x 300 mu m and 10 Hz per brain slice, which takes similar to 45 min per imaging session). We include codes that enable data to be registered to a reference atlas, production of 3D activity maps, extraction of the activity traces of similar to 250 brain regions and, finally, combination of data from multiple sessions (timing analysis averages similar to 2 h). This protocol enables neuroscientists to observe global brain processes in mice