CaMPARI & Voltron

Type: Molecular / Cellular,

Keywords: Fluorescent protein-based integrator, Fluorescent voltage indicator, GENIE, Calcium activity, Neuronal activity, BRAIN Initiative

Fluorescent indicators for neuronal activity: CaMPARI and Voltron

CaMPARI (Calcium Modulated Photoactivatable Ratiometric Integrator) – a fluorescent protein-based integrator of calcium for permanent marking of neuronal activity, Voltron – a chemigenetic fluorescent voltage indicator for in vivo recording of electrical activity. Primary use cases are marking/monitoring of neuronal activity in vivo in model organisms. Goal is to disseminate as broadly as possible, primarily via publicly-accessible repositories, to enable new biology.

* CaMPARI – A photoconvertible protein for calcium activity history imaging
* Photoconvertible protein construct that enables imaging of the integrated calcium activity of large populations of cells over defined time windows
* New tool developed at HHMI’s Janelia Research Campus lets scientists permanently mark neurons that are active at a particular time
* A calcium indicator that can be rapidly photoconverted in active neurons to perform circuit mapping
* Based on EosFP, a fluorescent protein whose emission changes from green to red upon irradiation with UV-light (~400 nm)
* Bright green fluorescent protein that — via allosteric modulation of the chromophore — converts to a bright red fluorescent species upon illumination with violet light during high calcium availability
* Photoconverted CaMPARI neurons are labeled irreversibly, allowing for imaging of an active network long after the photoconversion snapshot of activity has been obtained
* A ratiometric integrator, where its red/green ratio indicates the level of their activity or, more precisely, the level of calcium influx
* Also a negative fluorescent indicator, meaning that it reports momentary calcium influx by a reduction in fluorescence intensity in both the unconverted green state and in the converted red state
* Led the development of CaMPARI, working as part of Janelia’s Genetically-Encoded Neuronal Indicator and Effector (GENIE) project team
* CaMPARI 2.0 – the second generation of CaMPARI molecules
* Improved green brightness by 50% and red brightness by 250%
* Recently generated monoclonal antibodies (mAbs) specific to the red form of the CaMPARI molecule
* Allow usage of immunocytochemistry to recover neurons that were marked in vivo
* System can be used in conjunction with IHC methods for fixed tissue analysis, in addition to facilitating the amplification of weaker red signals
* Voltron – Genetically-encoded fluorescent voltage indicator tracks neural activity over long time periods
* Combines a novel fluorescent dye with an engineered multi-part protein that alters the dye’s intensity when specific neurons are switched on
* Detect neural signals throughout the brain
* Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators has huge potential for monitoring neuronal activity with high spatial and temporal resolution
* Use Voltron with light-sheet microscopy and other light microscopes

* Validated in cultured rat hippocampal neurons, with observable photoconversion (PC) for 10 action potential trains and 2 s of PC exposure
* Ability to label active neural circuits in fruit flies, zebrafish, and mice
* Photoconversion or gene expression is triggered when light is applied to the cell culture, to the brain slice or through a cranial window onto the brain

* Calcium activity imaging across large cell populations and/or tissues
* Labeling of “active” cells within a tissue (such as brain) during stimulus or behavior in model organisms
* Tracing of neurons based on their calcium activity level
* Integration of subcellularly localized calcium activity when targeted to specific subcellular locations
* Can visualize neural activity during more complicated behaviors as it can be used while animals move freely

* In fruit flies, the team used CaMPARI to identify neurons that were activated in response to specific odors
* Demonstrated Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies
* In lab test, able to watch neurons light up in the spinal cord of developing zebrafish

* Can visualize neural activity across wide swaths of brain tissue
* Image total calcium activity during defined time windows (gated by photoconversion light)
* Not restricted to the field of view of a microscope, as during real-time calcium imaging with e.g. GCaMP
* Enables rapid network-wide, tunable, all-optical functional circuit mapping
* Converts from green to red when calcium floods a nerve cell after the cell fires
* Enables higher-throughput calcium assays with cultured cells
* CaMPARI 2.0 – faster kinetics, lower photoconversion rate in low calcium conditions

* Expression is sensitive to tissue fixation using formaldehyde-based solutions
* Not possible to make high quality images of the neurons post-fixation

Issued U.S. Patents 9,518,996 and 10,067,148

Fosque et al. 2015, Labeling of active neural circuits in vivo with designed calcium integrators, Science 347:755-60



Eric Schreiter, Group Leader





Ahmed Abdelfattah - postdoc



Karel Svoboda
Misha Ahrens
Glenn Turner