Biphasic acclimation for simultaneous wide-field fluorescent Ca²⁺ imaging and fMRI of 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 fMRI 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 while awake. Recent years have seen a push toward the development of acclimation protocols for performing fMRI in 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-Ca²⁺) imaging. Our group works with a unique co-implementation of WF-Ca²⁺ and fMRI which enables the collection of complementary measures of brain function, but-until now-has necessitated the use of anesthesia. We present the first longitudinal protocol for simultaneous WF-Ca²⁺ and fMRI in awake mice. Our approach comprised a two-phase (biphasic) acclimation protocol that results in high-quality multimodal data. Comparisons of cortex-wide neuronal activity (WF-Ca²⁺ imaging) and blood-oxygen-level dependent fMRI signals between isoflurane-anesthetized and awake mice reveal both convergent and divergent patterns in brain function between modalities and across time.