Reporting neural activity with genetically encoded calcium indicators

Hires, Samuel Andrew; Tian, Lin; Looger, Loren L.

doi:10.1007/s11068-008-9029-4

Cited by 123 publications

(117 citation statements)

References 79 publications

(135 reference statements)

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“…Two-photon Laser Scanning Microscopy of Neurons Expressing GCaMP2-To measure intracellular [GCaMP2], acute brain slices containing neurons expressing GCaMP2 were prepared and imaged as previously described (6,16). Purified GCaMP2 was diluted into pipette internal solution supplemented with 1 mM K 2 EGTA at 0.1, 1, and 10 M concentrations.…”

Section: Methodsmentioning

confidence: 99%

“…In the decade since their first publication, several formats have been explored for genetically encoded calcium indicator construction, including both two-fluorescent protein (FP) 2 sensors with a fluorescence resonance energy transfer signal change and single-FP intensity-modulated sensors (6). Genetically encoded calcium indicator-enabled calcium imaging has been used to detect single action potentials in awake mice (7,8).…”

mentioning

confidence: 99%

“…Incremental rounds of improvement by grafting of GFP-stabilizing mutations and random mutagenesis resulted in GCaMP2 (13)(14)(15), with significantly improved folding and fluorescence characteristics. This calcium indicator was extensively characterized in acute mouse cortical brain slice, with the conclusion that although useful, GCaMP2 suffers from low baseline fluorescence and cannot reproducibly detect single action potentials due to poor signalto-noise (6,16).…”

mentioning

confidence: 99%

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Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

Akerboom

Rivera

Guilbe

et al. 2009

Journal of Biological Chemistry

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242

252

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The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca 2؉ -induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

Akerboom

Rivera

Guilbe

et al. 2009

Journal of Biological Chemistry

Self Cite

242

252

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“…In addition, by using tissue specific promoters and selective cellular targeting modifications, these proteins became most useful for calcium signaling analysis within selected tissues, specific cell types or intracellular compartments (see Refs. [13][14][15][16][17]). In most cases, under proper expression conditions, GECIs have minimum effects on cellular activity, can be used in vivo as well, and therefore are also suitable for long-term studies (see Ref.…”

Section: Methods For Studying Calcium Signals In Human Ps Cellsmentioning

confidence: 99%

Calcium signaling in human pluripotent stem cells

2016

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a b s t r a c tHuman pluripotent stem cells provide new tools for developmental and pharmacological studies as well as for regenerative medicine applications. Calcium homeostasis and ligand-dependent calcium signaling are key components of major cellular responses, including cell proliferation, differentiation or apoptosis. Interestingly, these phenomena have not been characterized in detail as yet in pluripotent human cell sates. Here we review the methods applicable for studying both short-and long-term calcium responses, focusing on the expression of fluorescent calcium indicator proteins and imaging methods as applied in pluripotent human stem cells. We discuss the potential regulatory pathways involving calcium responses in hPS cells and compare these to the implicated pathways in mouse PS cells. A recent development in the stem cell field is the recognition of so called "naïve" states, resembling the earliest potential forms of stem cells during development, as well as the "fuzzy" stem cells, which may be alternative forms of pluripotent cell types, therefore we also discuss the potential role of calcium homeostasis in these PS cell types.

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“…Recently, calcium imaging has emerged as an approach for overcoming some of these liabilities. Because the concentration of calcium ions rises when a neuron is active, geneticallyencoded calcium indicators (GECIs) [76,77], which become more brightly fluorescent when calcium is high, report indirectly on a neuron's electrical activity. This technique is much slower and less sensitive [78] than electrophysiology, but comes with the great advantage that it can report on thousands of cells simultaneously [79][80][81][82].…”

Section: Fluorescent Proteins and The Development Of Gecismentioning

confidence: 99%

Imaging, manipulation and optogenetics in zebrafish

Favre-bulle¹

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This thesis investigates the advantages and limits of using laser light for illumination and micromanipulation of the nervous system deep in-vivo in zebrafish. Further interest in the study neuronal pathways and functions in zebrafish led to employ laser micro-manipulation for the stimulation of the systems of interest and the development of optogenetics system and light sheet microscopy for analyses of the processes. First I present the investigation of the interaction of light and brain tissue as we aim to quantify light scattered in zebrafish brain tissue with depth. This is of great importance for optogenetics as it uses light to control (turn on or off) neurons and the unwanted/scattered light out of the aimed region could significantly affect results of experiments. The measurements and model developed to predict light propagation through brain tissue and how much a focussed beam broadens and alters with depth are presented in details. Results show that illumination of individual neurons is possible at depth in the zebrafish brain, despite scattering that results from shallower neural tissue. This means that the approach presented allows for optogenetics manipulation of single neurons to be performed without optical correction for scattering. Secondly, I construct an optogenetics system where we employ advanced optics and novel algorithm to deliver versatile two dimensional illumination that can be used at any depth in the fish. I present an example of a problem that I have solved, and resulting optogenetics results that were obtained, using this new technology. In chapter 5, I was responsible for the improvements implemented on the optogenetics system and the different tests to show its performances. Lucy Heap has performed multiple experiments on that system and I present one example she performed and the results she got.In chapter 7, I performed the manipulation of free otolith in water. I with Alex Stilgoe performed scanning experiments of otolith on an optical trapping system that Alex Stilgoe built.We both performed the analysis of the experimental results. Alex Stilgoe performed the force modelisation with Ray optics methods and we compared it with experimental results. Statement of parts of the thesis submitted to qualify for the award of another degreeNone. I have special thanks to Shu, Swaantje and Lucy for our philosophical discussions, the sharing of our experiences in the good and bad moments of our PhDs and their support.Finally I have some friends that I would like to thank for their great support without necessarily realising it: Mari-wenn, Estelle and Arnaud.8 ABSTRACT

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Reporting neural activity with genetically encoded calcium indicators

Cited by 123 publications

References 79 publications

Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

Calcium signaling in human pluripotent stem cells

Imaging, manipulation and optogenetics in zebrafish

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