PROJECT SUMMARY/ABSTRACT Protein ticker-tapes for brain-wide neural recordings Behavior emerges from the interacting activity of widely distributed ensembles of neurons; but all existing tools for measuring brain activity sample only a small subset of these dynamics. Here we propose a protein-based approach to record brain-wide dynamics of two key measures of neural activity: immediate early gene (IEG) expression and Ca2+ concentration. This proposal focuses on in vitro proof of concept; follow-up efforts will focus on in vivo application if warranted. Tree rings and ticker tapes. A core taken in a tree today can reveal climate events from centuries past. The present proposal adapts this idea to record the history of neural activation. The idea is to form slowly growing intracellular protein filaments whose position-dependent color reflects the history of IEG activity or Ca2+ dynamics. High-resolution imaging ex vivo reads this history. The steps to achieve this goal are: 1) Engineer protein fibers as molecular ticker-tapes. The HaloTag receptor (HT) will be fused to filament-forming proteins (FFPs) that form linearly growing structures in cells. Sequential addition of different- colored brain-permeant HaloTag dyes will create colored stripes in the growing filaments, mapping fiber growth to wall-clock time. Protein engineering will be used to optimize the nucleation, growth, labeling and mechanical properties of these fibers. 2) Recording IEG activity on protein fibers. A HT-FFP gene will be expressed under a constitutive promoter and eGFP-FFP under an IEG promoter. IEG activation will lead to green stripes in the fiber, whose timing will be determined by reference to the different-colored HaloTag stripes. Promoters and protein stability will be optimized to achieve high-resolution and long time-base IEG recordings. 3) Record Ca2+ dynamics on protein fibers. The HT receptor will be engineered to contain a TEV protease recognition motif (termed HT*) which inactivates the HT when cleaved. A fusion protein, HT*-FFP, and a Ca2+-dependent protease, CaTEV, will be co-expressed in neurons, both under constitutive promoters. Elevated Ca2+ will drive proteolytic cleavage of HT*, leading to incorporation of dark bands in the filament (HT* labels within the filament will be protected from proteolysis by the surrounding crystal). The outcome of this work will be an in vitro proof of concept of a new approach to brain-wide neural recording which does not require electrodes or optical access to the live tissue.

PROJECT NARRATIVE Behavior emerges from the interacting activity of widely distributed ensembles of neurons; but all existing tools for measuring brain activity sample only a small subset of these dynamics. We propose to develop a tool for recording brain-wide patterns of neural activity into protein microstructures within each cell. This tool will give new insights into brain function in health and disease.