GoKawiilBrainwide fUSI measurements
Experiments were conducted in 5 C57/BL6 mice (3 male, 2 female), 13–34 weeks of age. All experimental procedures were conducted according to the UK Animals Scientific Procedures Act (1986). Experiments were performed at University College London, under a Project License released by the Home Office following appropriate ethics review.
Surgery
Mice were first implanted with a headplate and cranial window under surgical anaesthesia in sterile conditions. The cranial window replaced a dorsal section of the skull (∼8 mm in medial–lateral and ~5 mm in anterior–posterior) with 90-μm-thick ultrasound-permeable polymethylpentene (PMP) film (TPX DX845 PMP, Goodfellow Cambridge) attached to the skull with cyanoacrylate glue. The PMP film was then covered with Kwik-Cast (World Precision Instruments), except during imaging sessions. After surgery, mice were allowed to recover for seven days before starting habituation. Mice were then habituated to handling and head fixation in the rig across several days, as session duration progressively increased.
Histology
Mice were perfused with PBS, paraformaldehyde and a gel containing TRITC–Dextran (Sigma-Aldrich) to perform blood vessel labelling. We imaged full 3D stacks of the brains in a custom-made serial section62 two-photon63 tomography microscope. Images were acquired using ScanImage (Vidrio Technologies) and the hardware coordinated using BakingTray. We extracted the signal in green and red channels, for autofluorescence and labelled blood vessels.
fUSI recordings
Recordings were performed in 5 mice across different days for up to 10 weeks. On a given day, we sequentially recorded 2–3 sessions in different coronal planes, lasting ~45 min each. The final dataset consisted of 138 such sessions for a total of 101 h of recordings, ranging from 17 h to 24 h per mouse. Because each plane did not contain all regions, the exact number of recording sessions for each brain region differed (median by animal by region: 11 ± 7.7 s.d.; see Supplementary Fig. 1 for all numbers).
In each recording session, we head-fixed the mice by securing the headplate to a post placed 10 cm from three video displays (Adafruit, LP097QX1, 60 Hz refresh rate) arranged at right angles to span 270° in azimuth and ∼70° in elevation. The screens were uniform grey or black. In some sessions (13 out of 138), the screens switched from grey to black, or from black to grey halfway through the recording session. The mice were placed on a running wheel. Four infrared cameras and the wheel rotary encoder were used to monitor their spontaneous behaviour.
We covered the PMP film with ultrasound gel and positioned the ultrasound transducer above it (128-element linear array, 100 μm pitch, 8 mm focal length, 15 MHz central frequency, model L22-Xtech, Vermon). Doppler signals from the transducer were acquired using an ultrasound system (Vantage 128, Verasonics) controlled by a custom MATLAB-based user interface (Alan Urban Consulting) recording continuously at 500 Hz.
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