title: Optimization of a GCaMP calcium indicator for neural activity imaging.
abstract: Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia 20147, USA.
authors.forename: Jasper
authors.initials: J
authors.lastname: Akerboom
authors.forename: Tsai-Wen
authors.initials: TW
authors.lastname: Chen
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Wardill
authors.forename: Lin
authors.initials: L
authors.lastname: Tian
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Marvin
authors.forename: Sevinç
authors.initials: S
authors.lastname: Mutlu
authors.forename: Nicole Carreras
authors.initials: NC
authors.lastname: Calderón
authors.forename: Federico
authors.initials: F
authors.lastname: Esposti
authors.forename: Bart G
authors.initials: BG
authors.lastname: Borghuis
authors.forename: Xiaonan Richard
authors.initials: XR
authors.lastname: Sun
authors.forename: Andrew
authors.initials: A
authors.lastname: Gordus
authors.forename: Michael B
authors.initials: MB
authors.lastname: Orger
authors.forename: Ruben
authors.initials: R
authors.lastname: Portugues
authors.forename: Florian
authors.initials: F
authors.lastname: Engert
authors.forename: John J
authors.initials: JJ
authors.lastname: Macklin
authors.forename: Alessandro
authors.initials: A
authors.lastname: Filosa
authors.forename: Aman
authors.initials: A
authors.lastname: Aggarwal
authors.forename: Rex A
authors.initials: RA
authors.lastname: Kerr
authors.forename: Ryousuke
authors.initials: R
authors.lastname: Takagi
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kracun
authors.forename: Eiji
authors.initials: E
authors.lastname: Shigetomi
authors.forename: Baljit S
authors.initials: BS
authors.lastname: Khakh
authors.forename: Herwig
authors.initials: H
authors.lastname: Baier
authors.forename: Leon
authors.initials: L
authors.lastname: Lagnado
authors.forename: Samuel S-H
authors.initials: SS
authors.lastname: Wang
authors.forename: Cornelia I
authors.initials: CI
authors.lastname: Bargmann
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Kimmel
authors.forename: Vivek
authors.initials: V
authors.lastname: Jayaraman
authors.forename: Karel
authors.initials: K
authors.lastname: Svoboda
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Kim
authors.forename: Eric R
authors.initials: ER
authors.lastname: Schreiter
authors.forename: Loren L
authors.initials: LL
authors.lastname: Looger
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000648:ultrastructure; D017173:Caenorhabditis elegans; D020013:Calcium Signaling; D018360:Crystallography, X-Ray; D004331:Drosophila melanogaster / Q000254:growth & development; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods*; D005813:Genes, Synthetic; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D057809:HEK293 Cells / Q000737:chemistry / Q000648:ultrastructure; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology; D006801:Humans; D007814:Larva; D007834:Lasers; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D059906:Neuroimaging / Q000379:methods*; D009469:Neuromuscular Junction / Q000737:chemistry / Q000648:ultrastructure; D009474:Neurons / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure; D019581:Neuropil / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D018034:Olfactory Receptor Neurons / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D010455:Peptides / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D010775:Photic Stimulation; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D051245:Retinal Bipolar Cells / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D009435:Synaptic Transmission; D015027:Zebrafish / Q000254:growth & development
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins