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

Background: Functional ultrasound imaging (fUS) is an emerging imaging technique that indirectly measures neural activity via changes in blood volume. Chronic fUS imaging during cognitive tasks in freely moving animals faces multiple exceptional challenges: performing large durable craniotomies with chronic implants, designing behavioral 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 while tethered. New method: We provide validated solutions for those technical challenges. In addition, we present standardized step-by-step reproducible protocols, procedures, and data processing pipelines. Finally, we present proof-of-concept analysis of brain dynamics during a decision making task. Results: 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. Comparison with existing methods: Computational pipelines in fUS are nascent and we present an initial development of a full processing pathway to correct and segment fUS data. Conclusions: Our methods provide stable imaging and analysis of behavior with fUS that will enable new experimental paradigms in understanding brain-wide dynamics in naturalistic behaviors.