# Longitudinal resting-state fMRI of awake mice during habituation: stress, head motion, and functional connectivity

## TL;DR

This study tracks stress and brain activity in awake mice during MRI habituation, finding that stress levels stabilize after initial increases.

## Contribution

The study provides novel longitudinal insights into stress and motion during awake mouse fMRI habituation.

## Key findings

- CORT levels increased twofold during habituation outside the MRI and fourfold within the MRI environment.
- Head motion and functional connectivity remained stable due to a restraint cradle allowing paw movement.
- Stress levels returned to baseline one week after habituation regardless of MRI exposure.

## Abstract

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, for our experimental setup, extending the number of habituation days does not further reduce stress in the MRI environment, provided that head motion remains within acceptable limits.