Labeling of active neural circuits in vivo with designed calcium integrators

TLDR

Identifying active neurons and circuits in vivo is a key challenge, with genetically encoded calcium indicators providing high‑resolution monitoring but limited field of view, while immediate early gene staining covers whole brains but lacks temporal precision. The study aimed to create CaMPARI, a fluorescent sensor that merges GECI targetability with permanent IEG‑like labeling to capture temporally precise activity snapshots across large tissue volumes. CaMPARI irreversibly switches from green to red fluorescence only when high intracellular Ca²⁺ coincides with controlled illumination, enabling permanent labeling of active neurons. The sensor was successfully applied in freely moving zebrafish and fly larvae, as well as in head‑fixed mice and adult flies, demonstrating its broad applicability.

Abstract

The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.