Chronic brain functional ultrasound imaging in freely moving rodents performing cognitive tasks

Functional ultrasound imaging (fUS) is an emerging imaging technique that indirectly measures neural activity via changes in blood volume. To date it has not been used to image chronically during cognitive tasks in freely moving animals. Performing those experiments faces a number of exceptional challenges: performing large durable craniotomies with chronic implants, designing behavioural experiments matching the hemodynamic timescale, stabilizing the ultrasound probe during freely moving behavior, accurately assessing motion artifacts and validating that the animal can perform cognitive tasks at high performance while tethered. In this study, we provide validated solutions for those technical challenges. In addition, we present standardized step-by-step repro-ducible protocols, procedures and data processing pipelines that open up the opportunity to perform fUS in freely moving rodents performing complex cognitive tasks. Moreover, we present proof-of-concept analysis of brain dynamics during a decision making task. We obtain stable recordings from which we can robustly decode task variables from fUS data over multiple months. Moreover, we find that brain wide imaging through hemodynamic response is nonlinearly related to cognitive variables, such as task difficulty, as compared to sensory responses previously explored.