Optogenetic and chemogenetic control of an enzymatic reporter of neural activity

Abstract

The brain's control of our thoughts, feelings, and behaviors stems from neural circuits, which perform logical operations based on the temporal patterns of neural activity and the connectivity of the neurons as the circuit traverses the brain. Recent studies have produced strategies for visualizing and controlling a circuit's neural activity. In some cases, specialized microscopy systems have enabled imaging of entire brain volumes on the timescale of neural activity and during behavior, enabling the reconstruction of neural circuitry at cellular resolution. However, recording neural activity from whole brain volumes to reconstruct neural circuitry is a significant challenge when working with larger brains, less specialized microscopes, or stimuli and behaviors that are not conducive to concurrent imaging. In these situations, strategies that enable permanent recording of neural activity during defined time windows for later readout would be highly beneficial. Here, we describe an approach for permanent recording of neural activity using calcium-dependent enzymatic activation of genetically encoded substrates. Our efforts focus on an engineered Tobacco Etch Virus (TEV) protease, which does not have endogenous substrates in vertebrate systems. We describe our strategy for engineering a calcium-dependent TEV protease that involves a split version of the enzyme with each half attached to the calcium-binding partners calmodulin and M13. These TEV fragments are designed so that, in the absence of neural activity, they will remain separate and inactive, but in the presence of neural activity and the resulting high calcium concentrations, the association of calmodulin and M13 will permit TEV reconstitution and enzyme activity. Once active, the split TEV protease can cleave a variety of genetically encoded substrates that link TEV protease activity with transcription of a desired gene or direct creation of a fluorophore. Finally, we will discuss controlling the split TEV protease and substrate association using opto- and chemogenetics approaches to provide precise spatial and temporal control over the time period of neural recording. This strategy will enable visualization and control of neurons activated by a sensory stimulus or behavior, permitting detailed analysis of neural circuitry over distant brain regions.