Longitudinal, simultaneous wide-field fluorescent Ca ²⁺ imaging and fMRI in awake mice

Functional magnetic resonance imaging (fMRI) can be applied in mice and humans making it a key technology in translational neuroimaging research. Yet, most neuroimaging studies in rodents use anesthesia to limit subject motion and stress. This puts a hard boundary on the range of brain and behavioral states that can be studied in animals, and deviates from the near-universal practice of imaging people whilst awake. Recent years have seen a push towards the development of acclimation protocols for imaging mice without anesthesia, but the results have been mixed. In parallel, imaging mice without anesthesia has become routine for complementary neuroimaging methods including wide-field fluorescence calcium (WF-Ca ²⁺ ) imaging. We present the first longitudinal protocol for simultaneous WF-Ca ²⁺ and fMRI in awake mice. Our approach is comprised of a two-phase (biphasic) acclimation protocol that results in high-quality multimodal data, enabling direct comparison of neuronal mesoscopic and hemodynamic signals across brain states and time. Data from awake mice are compared with data from isoflurane-anesthetized imaging sessions to unlock state-dependent differences in functional connectivity, which show both convergent and divergent patterns between imaging modalities. Together, these results establish a framework for longitudinal awake multimodal imaging and uncover new insights into the neural and vascular functional organization of the mouse brain across states and time.