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

Nat Protoc. 2021 Jul;16(7):3547-3571. doi: 10.1038/s41596-021-00548-8. Epub 2021 Jun 4.


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: ~3-4 h) and image brain-wide activity associated with a stimulus at high resolution (100 × 110 × 300 µm and 10 Hz per brain slice, which takes ~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 ~250 brain regions and, finally, combination of data from multiple sessions (timing analysis averages ~2 h). This protocol enables neuroscientists to observe global brain processes in mice.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Artifacts
  • Brain / diagnostic imaging*
  • Brain / physiology*
  • Head
  • Mice, Inbred C57BL
  • Time Factors
  • Ultrasonography*
  • Wakefulness / physiology*