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Nat Commun. 2018 Oct 25;9(1):4440. doi: 10.1038/s41467-018-06935-2.

Improved methods for marking active neuron populations.

Author information

1
Howard Hughes Medical Institute, Janelia Research Campus, 19700 Helix Drive, Ashburn, VA, 20147, USA.
2
Program in Computational Biology and Bioinformatics, Duke University, Durham, NC, 27708, USA.
3
Neuronal Ensembles in Addiction Section, Behavioral Neuroscience Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, 21224, USA.
4
Institute for Synaptic Physiology, University Medical Center Hamburg-Eppendorf, 20251, Hamburg, Germany.
5
Department of Biology, Brandeis University, Waltham, MA, 02454, USA.
6
Institute of Biochemistry, Charité - Universitätsmedizin Berlin, 10117, Berlin, Germany.
7
NeuroCure Cluster of Excellence, Department of Biology, Humboldt University, 10117, Berlin, Germany.
8
Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195, USA.
9
Howard Hughes Medical Institute, Janelia Research Campus, 19700 Helix Drive, Ashburn, VA, 20147, USA. schreitere@janelia.hhmi.org.

Abstract

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

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PMID:
30361563
PMCID:
PMC6202339
DOI:
10.1038/s41467-018-06935-2
[Indexed for MEDLINE]
Free PMC Article

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