ABSTRACT
Astrocytes are the most abundant cells in the brain. They support neurons, adjust synaptic strength, and modulate neuronal signaling, yet the full extent of their functions is obscured by the dearth of methods for their visualization and analysis. Here, we report a chemical reporter that targets small molecules specifically to astrocytes both in vitro and in vivo. Fluorescent versions of this tag are imported through an organic cation transporter to label glia across species. The structural modularity of this approach will enable wide-ranging applications for understanding astrocyte biology.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , Fluorescent Dyes/chemistry , Animals , Fluorescence , HEK293 Cells , Humans , Mice , Microscopy, Confocal/methods , Pyridinium Compounds/chemistry , Rats , Rhodamines/chemistry , Spinal Cord/metabolism , ZebrafishABSTRACT
Technologies for measuring the transient Ca2+ spikes that accompany neural signaling have revolutionized our understanding of the brain. Nevertheless, microscopic visualization of Ca2+ spikes on the time scale of neural activity across large brain regions or in thick specimens remains a significant challenge. The recent development of stable integrators of Ca2+, instead of transient reporters, provides an avenue to investigate neural signaling in otherwise challenging systems. Here, we describe an engineered Ca2+-sensing enzyme consisting of a split Tobacco Etch Virus (TEV) protease with each half tethered to a calmodulin or M13 Ca2+ binding domain. This Split TEV, Ca2+ Activated Neuron Recorder (SCANR) remains separate and catalytically incompetent until a spike in cellular Ca2+ triggers its reconstitution and the subsequent turnover of a caged, genetically encoded reporter substrate. We report the identification of a successful Ca2+-sensing split TEV from a library of chimeras and deployment of the enzyme in primary rat hippocampal neurons.
