Studying complex neurological function and disease requires imaging technologies that can provide a comprehensive view of the mammalian brain with high spatiotemporal resolution. Ideally, these technologies should be scalable from small brain regions in rodents to whole-brain imaging in larger organisms such as non- human primates. They should provide access to endogenous signals such as hemodynamics, and be able to image neuron-specific signals such as calcium and activity-dependent gene expression using targeted reporters. They should also be compatible with freely moving subjects to enable behavioral studies, and should allow simultaneous electrophysiology and targeted modulation. Finally, they should be non-invasive or minimally invasive to enable the possibility of future clinical translation. No existing technology fulfills these criteria because optical and electrophysiological techniques face inherent limitations in scaling, while functional magnetic resonance imaging has relatively low spatiotemporal resolution and severely constrains behavioral paradigms. Recently, functional ultrasound was introduced as a revolutionary technique that can image neural activity non-invasively with more than an order of magnitude improved spatiotemporal resolution compared to fMRI (<100µm and <10ms), using transducers that can be mounted on freely moving animals. In rodents, fUS has been used to image activity patterns associated with epileptic seizures, whisker stimulation, olfactory perception and resting state connectivity, and studies are underway to demonstrate the use of fUS in larger animals and pediatric patients. In its present form, fUS tracks changes in blood flow arising from neurovascular coupling. While this already provides unprecedented brain imaging performance, it is limited by the relatively low strength of intrinsic hemodynamic signals and the lack of reporters to connect ultrasound more directly to neural activity. Here, we propose to take the next major leap in ultrasonic functional imaging of the brain by developing biomolecular imaging agents that enhance fUS signals by more than two orders of magnitude and enable direct imaging of activity-dependent gene expression and calcium signaling in genetically targeted neurons. As part of this work, we will also demonstrate the ability of fUS to image brain activity in non-human primates in combination with electrical recording and pharmacological modulation. If we are successful in these goals, the resulting technology will provide neuroscience researchers with truly revolutionary capabilities, especially for the study of larger animal models. Furthermore, the potential merger of of this technology with ultrasonic neuromodulation (which uses a different regime of frequencies and intensities) will open the possibility of all-acoustic interfaces to simultaneously image and control neural activity in freely moving subjects.

Studying complex neurological function and disease requires imaging technologies that can provide a comprehensive view of the mammalian brain with high spatiotemporal resolution. Functional ultrasound (fUS) is a new technology that offers unprecedented ability to image deep brain areas with high spatial and temporal resolution. In this project, we will dramatically expand the capabilities of fUS by combining it with the first genetically engineered and encoded neural imaging agents for ultrasound.