Functional ultrasound localization microscopy on freely moving rats

Functional ultrasound localization microscopy (fULM), which utilizes the neurovascular coupling effect, has become a critical tool for investigating neural activities in the rodent brain. This technique relies on intravenously injected microbubbles (MBs) as contrast agents, enabling exceptional spatial resolution. However, prior studies have predominantly applied fULM in anesthetized animals, a limitation that markedly reduces sensitivity to neural activity. Recent advancements in functional ultrasound imaging of freely moving rodents have demonstrated not only enhanced sensitivity but also an expanded application to behavioral studies, which is unattainable in anesthetized or head-fixed awake imaging setups. Nevertheless, the application of ULM in freely moving animals remains unexplored. In this study, we introduce a novel fULM imaging approach on freely moving rats. By utilizing indwelling jugular vein catheterization and a miniaturized ultrasound probe, this method achieves high spatial resolution and precise mapping of dynamic neural processes in vivo. We validate the system by visualizing cerebral blood flow and microvascular changes during visual stimulation, providing new insights into neurovascular coupling. This platform addresses the traditional limitations of immobilized and anesthetized imaging, offering a transformative tool for neuroscience research in naturalistic settings.