Characterizing the brain’s neural circuits would yield insight into the circuit’s functions in cognitive processes. However, shortcomings in current technologies and the brain’s inherent complexity have hindered progress in this area, demanding the development of novel strategies for marking neural circuitry. Here we exploit the intracellular Ca2+ increase that occurs during neural circuit activation to mark active neurons and reveal circuit connectivity. We describe a strategy to trace neural circuits by utilizing a novel, split Ca2+- sensing enzyme to generate a permanent signal in active neurons. Our strategy employs a split Tobacco Etch Virus (TEV) protease with each half fused to a dimerizing Ca2+ binding domain. In this strategy, the increase in Ca2+ in active neurons triggers association of the Ca2+ binding domains and assembly of the catalytically competent enzyme. Once assembled, the enzyme is poised to free a caged, genetically-encoded substrate, which activates expression of a desired product. Whereas previous tools for recording transient Ca2+ increases are limited by stringent imaging requirements and a solitary fluorescence readout, our system enables expression of assorted fluorophores, optogenetics tools to control neuron excitation or inhibition, or toxins to silence subsequent neural activity. We report the characterization of a library of split Ca2+-sensing TEV proteases and substrate modifications that will enable spatiotemporal control of neuronal recordings.

Development of a novel, neural-activity triggered split enzyme

253rd National Meeting (2017)

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