Longitudinal Resting-State fMRI of Awake Mice During Habituation: Stress, Head Motion, and Functional Connectivity

Awake mouse fMRI is a powerful tool for both neuroscience and translational research. To minimize head motion during scanning, habituation under physical restraint is commonly used. However, it remains unclear how stress levels and head motion evolve during habituation, particularly within the MRI environment. To address this, we repeatedly measured plasma corticosterone (CORT) levels in three groups of mice - controls, mice habituated outside the MRI magnet, and mice habituated within the fMRI environment - and acquired longitudinal resting-state fMRI data daily during an eight-day habituation period and again 15 days post-habituation at 15.2 T. We found that CORT levels initially increased by approximately twofold and gradually decreased during habituation outside the magnet, whereas in mice habituated within the fMRI environment, CORT levels increased two- to fourfold and remained elevated throughout the habituation period. One week after habituation, CORT levels returned to baseline in both groups. Throughout all resting-state fMRI scanning sessions, head motion and functional connectivity remained stable, likely due to the well-designed restraint cradle that permitted paw movement. These results suggest that additional habituation days do not further reduce stress, provided that head motion remains within acceptable limits.