There is a clear need for imaging protocols in awake animals. Yet, there are only a few approaches in mice, despite their usefulness as translational research subjects. We introduce the first protocol for longitudinal simultaneous fMRI and Ca2+ imaging in awake mice. The same animals are imaged four times (from 4 to 12 months of age) – twice under anesthesia, and twice awake. Our framework includes two acclimation protocols: an initial intensive training followed by a ‘refresher' course. We find a marked improvement in motion between the two awake sessions. Implications for future dual-imaging experiments in awake mice are considered.
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