Longitudinal Awake Mouse fMRI During Voluntary Locomotion Using Zero TE Imaging and a Novel Treadmill Training Protocol

PURPOSE: Functional MRI (fMRI) in awake rodents presents countless valuable opportunities for researchers to probe questions that may not be accessible through anesthetized models, such as voluntary locomotion. The commonly used echo planar imaging (EPI) sequence is highly sensitive to motion that occurs even outside of the imaging plane. Recently, zero echo time sequences have been adopted for fMRI to address this challenge. METHODS: This study proposes a robust and reproducible protocol for longitudinal imaging of awake mice during spontaneous locomotion, using an implanted headpiece, incremental training, zero TE fMRI, reinforcement learning, and a custom treadmill module. Locomotion is known to have wide-ranging effects on brain activity and can alter neurovascular coupling, making it critical to understand this aspect of natural behavior. RESULTS: We present results from 10 trained mice across three different fMRI scanning sessions, finding minimal head motion across scans (average framewise displacement matched anesthetized EPI (p > 0.05)), consistent resting-state functional connectivity across subjects and scans, and evidence of a minimal stress response at the group and individual level. We also demonstrate little difference on signal quality during locomotion and altered functional connectivity and spatiotemporal dynamics during locomotion compared to rest. CONCLUSIONS: This work establishes a new benchmark for awake rodent fMRI, enabling the direct investigation of naturalistic behaviors like locomotion and their whole-brain correlates without the confounding effects of anesthesia or excessive restraint.