Labeling of active neural circuits in vivo with designed calcium integrators

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.

Learning to Fish with Genetics: A Primer on the Vertebrate ModelDanio rerio

“…However, studies in zebrafish can go beyond a wiring diagram to a dissection of the in vivo functions of connections. Using multiphoton calcium imaging in fish carrying calcium sensors, it is possible to record neuronal activity simultaneously throughout the brain (Friedrich et al 2013;Renninger and Orger 2013;Fosque et al 2015). Neural networks involved in visual activity, hunting behavior, and navigation have already been defined (Ahrens et al 2013;Muto et al 2013;Bianco and Engert 2015;Romano et al 2015).…”

Design and Synthesis of a Calcium‐Sensitive Photocage

“…This new class of small-molecule Ca 2+ indicator yields ap ermanent fluorescent signal that could enable simpler assays for high-throughput screening or the post hoc mapping of active cells in genetically intractable organisms.A dditionally,our modular synthetic strategy should facilitate improvements to this probe,such as modulating the Ca 2+ affinity, [2d, 15] altering the properties of the released fluorophore, [16] increasing the two-photon cross-section of the photocage by using the indoline scaffold, [9a,17] and changing selectivity to other ions that are similarly unable to elicit chemical reactions on their own (e.g.,N a + ). [2c, 18] Finally,u nlike the genetically encoded systems, [5,6] the flexibility of this small-molecule scaffold opens the door for pharmacological agents or chemical gene inducers to be photocaged and released using a" functional photochemistry" approach, allowing sophisticated biological experiments where active cells are selectively targeted for chemical manipulation.…”

“…In particular, caging af luorophore with such an ion-sensitive photocage would yield af luorescent ion "snapshot indicator" or "integrator" that would permanently record increased ion concentration during an illumination-defined time period. Integrator systems based on fluorescent proteins [5] and rhodopsins [6] have been described recently,b ut small-molecule ion integrators remain unknown. This currently limits the use of such probes to biological systems that are amenable to genetic manipulation.…”

In vivo calcium imaging and Parkinson’s disease

“…CaMPARI has the characteristics of permanent, large-scale labeling of immediate early genes, which enables a temporally precise "activity snapshot" of a large tissue volume. Professor Schreiter (Fosque et al, 2015) showed that when intracellular calcium is elevated, CaMPARI undergoes green-to-red conversion efficiently and irreversibly in response to a violet light shower in zebrafish, flies, head-fixed mice, and adult flies. Thus, the development of CaMPARI made it possible to efficiently study behavioral neuronal circuits in the entire brain.…”

“…Therefore, the introduction of a new, permanent calcium molecular reporter that is able to record neural activity across the whole brain would be transformative, enabling focus on only marker neurons active during behavioral processes. Fortuitously, in 2015, Professor Schreiter's group (Fosque et al, 2015) designed a fluorescent sensor, the calcium-modulated photoactivatable ratiometric integrator (CaMPARI). CaMPARI has the characteristics of permanent, large-scale labeling of immediate early genes, which enables a temporally precise "activity snapshot" of a large tissue volume.…”