Super-resolution ultrasound imaging of visual stimulus-evoked activity in freely-moving rats

Introduction: : High-resolution imaging of deep, brain-wide tissue in awake and unrestrained animals is critical for studying brain activity under naturalistic conditions. However, achieving this goal remains technically challenging for existing neuroimaging modalities. Ultrasound localization microscopy (ULM) has emerged as a promising technique that enables super-resolution vascular imaging with deep tissue penetration. More recently, functional ULM (fULM) has shown the potential to capture hemodynamic responses based on microbubble (MB) fluctuations. However, current fULM implementations remain limited to anesthetized and head-fixed animals, which restrict their application in behaviorally relevant neuroscience studies. This work aims to establish fULM imaging in freely-moving rats to enable naturalistic functional brain imaging with microvascular spatial resolution.

Materials and

Methods: : We developed an imaging platform that supports non-head-fixed and awake fULM imaging in rats. The setup incorporates a tethering system for suspending a lightweight ultrasound transducer, which is adapted from an existing study [El Hady, A. et al. J. Neurosci. Methods (2024)]. Additionally, continuous MB infusion was achieved via a chronic jugular catheter linked to a Vessel Access Button (Instech Laboratories, PA, USA). Both the transducer cable and catheter are supported by a pulley-counterweight system. It allows continuous data acquisition for tens of minutes without manual intervention, which shows the potential of incorporating fULM into behaviorally driven neuroscience experiments. Functional imaging was performed under visual stimulation using a 3 Hz flashing white LED array (20 s ON / 40 s OFF, 5 cycles) in a dark environment.

Results, Conclusions, and Discussions:: Fig.(a) illustrates the freely-moving imaging setup with the integration of the tethered transducer and catheter system. Fig.(b) presents a ULM directional flow density map (at Bregma -6.2 mm) with a pixel size of 10 µm. The minimal motion artifacts observed during ULM imaging indicate the stability of the imaging system. Functional responses obtained without (Fig. c) and with MB (Fig. d) demonstrate the activation of superior colliculus. fULM shows enhanced sensitivity in non-head-fixed awake, freely-moving animals, detecting activation with shorter stimulation durations (20 s ON, 5 cycles), compared to longer protocols (30 s ON, 10–20 cycles) reported in previous anesthetized fULM studies. These results validate the feasibility of fULM imaging in freely-moving animals and demonstrate its potential to support high-resolution, behavior-compatible functional brain imaging.

Acknowledgements and/or References (Optional):: Reference:
El Hady, Ahmed, et al. "Chronic brain functional ultrasound imaging in freely moving rodents performing cognitive tasks." Journal of neuroscience methods 403 (2024): 110033.