Physiological effects of a habituation procedure for functional MRI in awake mice using a cryogenic radiofrequency probe

•We developed head-fixation implements for fMRI in awake mice using CryoProbe.•Physiological examination suggested at least seven days of habituation was necessary for fMRI in awake mice.•Our method did not use anesthesia to hold mice in an animal bed for MRI.•The method achieved high-SNR fMRI in awake mice using CryoProbe. Functional magnetic resonance imaging (fMRI) in mice is typically performed under anesthesia due to difficulties in holding the head of awake mice stably with a conventional three-point fixation method that uses a tooth-bar and earplugs. Although some studies have succeeded in fMRI in awake mice by attaching a head-post on the skull, this cannot be applied to fMRI using a high signal-to-noise ratio (SNR) cryogenic MRI-detector, CryoProbe, because it covers the head of a mouse closely. We developed head-fixation implements for awake mice that are applicable to fMRI using CryoProbe. A head-bar was surgically attached to the skull of a mouse that was then habituated to a mock fMRI-environment, two hours/day for eight days with physiological examinations of body-weight, fecal weight, electromyogram (EMG), and electrocardiogram. EMG power decreased with just one day of habituation, whereas heart rate decreased after at least seven days of habituation. Estimated head motions of awake mice during fMRI were significantly smaller than a voxel size. Unexpectedly, temporal SNR of fMRI signals for awake mice was higher than that for anesthetized mice held by a conventional method. Functional connectivity in the brain of both anesthetized and awake mice showed bilateral and unilateral networks. fMRI using CryoProbe had been performed on anesthetized mice previously. Our method does not use anesthetics during habituation or fMRI. Our method would be beneficial for translational research using fMRI in mice and humans because human fMRI is typically performed without anesthetics.