Rapid, biochemical tagging of cellular activity history in vivo

Intracellular calcium (Ca²⁺) is ubiquitous to cell signaling across biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca²⁺ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca²⁺ in vivo. Ca²⁺-activated split-TurboID (CaST) labels activated cells within 10 min with an exogenously delivered biotin molecule. The enzymatic signal increases with Ca²⁺ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca²⁺ activity. Furthermore, the CaST readout can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.