Functional Ultrasound Localization Microscopy on Freely Moving Rats

Brain-wide functional neuroimaging at single-vessel resolution in naturally behaving animals has been challenging to realize. Functional ultrasound localization microscopy (fULM) offers brain-wide hemodynamic imaging at microscopic resolution but has remained restricted to head-fixed preparations. Here we present a platform for fULM in freely moving rats. The system integrates a magnetic probe-clamping interface, a counterbalanced pulley-based tether, chronic jugular venous access for on-demand microbubble delivery, and a motion-correction processing pipeline tailored to freely moving ULM data. Using visual stimulation, we first show that the platform reliably captures functional hemodynamic responses and supports super-resolved vascular reconstruction across deep brain regions. We then demonstrate that freely moving fULM resolves stimulus-evoked vessel-specific changes in both diameter and flow velocity, revealing microvascular heterogeneity that cannot be recovered with conventional methods. Finally, we apply the platform to image brain responses induced by an experimental biased μ opioid agonist (SR-17018) in rats, revealing transient, region-dependent cerebrovascular responses, with flow-velocity changes of 20-30% that vary in magnitude and temporal profile across individual vessel segments in cortex and periaqueductal gray in free-moving rats. This work establishes freely moving fULM as a practical platform for whole-brain microvascular imaging under naturalistic behavioral conditions.