Abstract Many fundamental questions in neuroscience and neurological disorders, such as those associated with learning, memory, sleep, aging, and disease progression, would benefit greatly from a technology that enables brain-wide single-cell readout of signaling activities and gene expression changes over extended time periods (over weeks, months, to even years). Current single-cell physiology recording methods require external interfacing to live cells, either optically (via fluorescent probes plus microscopy) or electrically (via electrical probes). It has been a major challenge to perform long-term optical imaging in vivo beyond several hours, due to fast photobleaching of fluorescent probes. Electrical probes, on the other hand, often induce unwanted long- term immune response after implantation and do not support measurement of intracellular signaling and gene expression activities. In addition, these single-cell recording methods do not support large-scale readout across the whole brain, while modalities that can image whole brains, such as ultrasound, MRI, and CT, lack single-cell resolutions. We aim to bridge this technological gap by developing a genetically encoded, temporally scalable ‘protein ticker tape’ recording system, to enable long-term, brain-wide recording of single- cell physiology in vivo without any external interfacing to live cells. In our prior work, we found it is possible to record and store cell physiology histories over time, such as gene expression histories, along elongating protein self-assemblies in live cells (analogous to tree rings permanently storing wood’s growth conditions), for subsequent single-cell readout in situ via simple post-mortem tissue processing and imaging techniques. In this project, we will leverage our expertise in protein engineering and neurotechnology development to transform this protein ticker tape concept into a technological solution that enables brain-wide single-cell physiology recording over long (up to a year) and scalable periods of time, where the recording duration is externally defined by users. We will first engineer novel protein assemblies, via an AI-powered protein design approach, to boost the information storage capacity and recording precision of protein ticker tapes. Next, we will employ chemical and light dependent expression systems to externally control the elongation speed of the protein self-assembly, to enable various recording durations (from a day to a year) externally defined by users. Finally, we will couple protein ticker tapes to promoters and synthetic expression systems that respond to signaling events, transcription factor activities, circadian rhythms, and gene expression levels, to enable recording of these cell physiological events separately and in parallel. We will genetically deliver these protein ticker tapes in single cells across living mouse brains to characterize and validate their performance, fidelity, and safety to cells and tissue, followed by demonstrations of long-term, brain-wide, single-cell physiology recording in healthy and diseased mouse models. We envision this novel recording technology with both spatial and temporal scalabilities to have a broad impact in biology and medicine.

Project Narrative Many neurological disorders involve abnormalities in cell physiology that unfold and progress over prolonged durations, ranging from days to years. Understanding these cellular processes in healthy and diseased states, as well as under pharmaceutical interventions, requires long-term readout of cell physiology in the living brain. The proposed work aims to develop novel technologies to enable such a long-term readout across living brains and to make these tools widely accessible to both basic and translational research communities.