SUMMARY A major goal of the BRAIN initiative is to develop neural imaging technologies enabling whole-brain imaging of specific molecular signals such as neuronal calcium. Currently, fluorescent genetically encoded calcium indicators (GEGIs) combined with advanced microscopy techniques enable single-neuron Ca2+ imaging in volumes smaller than 1 mm3, typically at depths shallower than 1 mm. Alternatively, GECIs combined with implanted fiber photometry enable point-measurements of the aggregate activity of genetically defined neuronal populations in deep brain regions with dimensions on the order of 200 µm. While both of these optical approaches have proven their great value in enabling neuroscience discoveries, they fall short of providing simultaneous whole-brain access to neural signals. If a technology could be developed for whole-brain Ca2+ imaging it would have a transformative impact on neuroscience research. In this project, we will address this ambitious goal by developing ultrasonic genetically encoded calcium indicators (UGECIs) and showing that we can use them to image brain-wide calcium in mice. Ultrasound has unique advantages as a modality for neural imaging due to its ability to penetrate much deeper than light (several cm) while providing relatively high spatial (tens of µm) and temporal (ms) resolution. This potential has been demonstrated by hemodynamic functional ultrasound (fUS), which uses ultrafast Doppler imaging of blood flow to visualize neural activity with 100 µm and 100 ms resolution. fUS has shown that ultrasound imaging of neural activity is possible in species ranging from mice to humans and is compatible with awake, behaving, freely moving animals. However, hemodynamics provide only an indirect measure of neural activity. In contrast, the measurement of calcium with GECIs provides access to a molecular signal integral to neuronal excitation, and allows the probing of specific cellular populations. This project will combine the whole-brain coverage of ultrasound with the molecular and genetic specificity of GECIs by developing the ultrasound versions of these tools. Our proposal to develop UGECIs arises from a long-standing research program in our lab to develop the first genetically encoded reporters and sensors for ultrasound. These constructs are based on gas vesicles (GVs), a unique class of genetically encoded air-filled protein nanostructures derived from buoyant bacteria, which we discovered are capable of scattering sound waves and thereby producing ultrasound contrast. We recently showed that GVs can be engineered to incorporate molecular binding domains allowing their protein shells to change their mechanical properties and resulting ultrasound contrast in response to molecules such as calcium. Building on these advances, we will develop UGECIs, express them in the mouse brain and use them to image brain-wide calcium signals using new ultrasound techniques allowing rapid 2-dimensional and 3-dimensional molecular imaging. The resulting technology will provide neuroscience researchers with revolutionary capabilities for whole-brain molecular neural imaging.

NARRATIVE Imaging neural activity on a brain-wide scale is a major technology goal of the BRAIN Initiative. To achieve this goal, we will develop ultrasonic genetically encoded calcium indicators, which will allow the activity of genetically defined neuronal populations to be imaged with ultrasound – a deeply penetrant imaging modality with high resolution and wide coverage. This will enable the study of healthy and diseased neural circuits in model organisms, and facilitate the development of more selective treatments for neurological and neuropsychiatric disease.