Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators

Tian, Lin; Hires, Samuel Andrew; Mao, Tianyi; Huber, Daniel; Chiappe, M. Eugenia; Chalasani, Sreekanth H.; Petreanu, Leopoldo; Akerboom, Jasper; McKinney, Sean A.; Schreiter, Eric R.; Bargmann, Cornelia I.; Jayaraman, Vivek; Svoboda, Karel; Looger, Loren L.

doi:10.1038/nmeth.1398

Cited by 1,805 publications

(2,042 citation statements)

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“…[3][4][5][6][7][8] Nevertheless, though a useful correlation often exists between Ca 2+ dynamics and neuronal spiking across a range of spike frequencies, the slowkinetics and saturation of [Ca 2+ ]-related fluorescent signals constrain the utility of Ca 2+ imaging as a means of probing spiking dynamics in many neuron types. 4,9 Numerous voltage-sensitive indicators 1,[10][11][12][13][14][15][16][17][18] permit direct imaging of cellular membrane potentials. Organic voltage-sensitive dyes have allowed functional mapping studies in awake mammals 14 and studies of individual cells' dynamics in invertebrates 19 and mammalian brain slices.…”

Section: Introductionmentioning

confidence: 99%

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Marshall

Schnitzer

2013

ACS Nano

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Biophysicists have long sought optical methods capable of reporting the electrophysiological dynamics of large-scale neural networks with millisecond-scale temporal resolution. Existing fluorescent sensors of cell membrane voltage can report action potentials in individual cultured neurons, but limitations in brightness and dynamic range of both synthetic organic and genetically encoded voltage sensors have prevented concurrent monitoring of spiking activity across large populations of individual neurons. Here we propose a novel, inorganic class of fluorescent voltage sensors: semiconductor nanoparticles, such as ultrabright quantum dots (qdots). Our calculations revealed that transmembrane electric fields characteristic of neuronal spiking (~10 mV/nm) modulate a qdot's electronic structure and can induce ~5% changes in its fluorescence intensity and ~1 nm shifts in its emission wavelength, depending on the qdot's size, composition, and dielectric environment. Moreover, tailored qdot sensors composed of two different materials can exhibit substantial (~30%) changes in fluorescence intensity during neuronal spiking. Using signal detection theory, we show that conventional qdots should be capable of reporting voltage dynamics with millisecond precision across several tens or more individual neurons over a range of optical and neurophysiological conditions. These results unveil promising avenues for imaging spiking dynamics in neural networks and merit in-depth experimental investigation. Toward addressing these challenges, we studied whether tools from nanotechnology, specifically, bright, multifunctional semiconductor quantum dots (qdots), 22,23 might be useful for imaging neuronal membrane potentials. Qdots are known to possess voltagesensitive optical properties, including a red-shifting of the qdot's fluorescence emission peak, spectral broadening, and a decrease in the intensity of fluorescence emission ( Figure 1C,D). [24][25][26][27] Qdots also are highly resistant to photobleaching and exhibit large quantum yields and absorption cross sections for one-(σ 1 ) and two-photon (σ 2 ) excitation. For example, CdSe qdots with radius R ~ 3 nm exhibit cross sections of σ 1 > 1 nm 2 and σ 2 > 3 × 10 4 GM, as compared to σ 1 ~ 10 −2 nm 2 and σ 2 ~ 3 × 10 2 GM for green fluorescent protein. 28,29 Marshall and Schnitzer Page 2 ACS Nano. Author manuscript; available in PMC 2017 December 15. Graphical Abstract HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptTo evaluate the spike detection capabilities of qdot indicators quantitatively, we applied a theoretical approach that incorporates the physical, biophysical, and statistical components of the problem. We calculated the effect of applied electric fields on the qdot's electronic structure and examined how this modulates a qdot's fluorescence intensity and emission spectra. We also studied nonspherical, nonhomogenous qdots, which we refer to more generally as semiconductor nanocrystals, and found that these nanoparticles can demonstrate muc...

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Section: Introductionmentioning

confidence: 99%

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Marshall

Schnitzer

2013

ACS Nano

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“…Importantly, there was no pathological nuclear translocation of the sensor in hippocampal CA1 neurons of mice from age postnatal day 13 to 6-month-old animals. By contrast, studies on long-term expression of several GECIs by adeno-associated virus-mediated gene transfer recently reported progressing cytomorbidity and concomitant restricted localization of the sensor to the cell nucleus 11 . Thus, transgenic expression of calcium biosensors is likely to be more suitable for long-term expression of indicators.…”

Section: Discussionmentioning

confidence: 91%

“…In addition, we investigated whether long-term expression of TN-XXL could cause changes in cell morphology. In a previous study a pathological aggregation of several GECIs to the nucleus was reported after longterm adeno-associated virus vector-mediated expression in cortical neurons 11 . Here, we looked at 13 days, 36 days, 2-month-and 6-month-old brains from TN-XXL transgenic mice and performed confocal images of frozen sections of the CA1 and CA3 region of the hippocampus ( Supplementary Fig.…”

Section: Widespread Expression Of Tn-xxl In Transgenic Micementioning

confidence: 91%

“…The engineering of GECIs has followed two major trends: the development of single fluorophore sensors that modulate fluorescence intensity of a single FP through calcium binding or that of indicators based on Förster resonance energy transfer (FRET). Latest generation sensors include the single fluorophore sensors G-CaMP3 and HS-G-CaMP [11][12][13] , and the FRET sensors YC 3.6, D3cpV and TN-XXL [14][15][16] . Although most engineering efforts focused on signal strength and optimization of expression, the long-term effects of chronic GECI expression on the host organism have not been studied in detail.…”

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confidence: 99%

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Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

et al. 2012

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Engineering efforts of genetically encoded calcium indicators predominantly focused on enhancing fluorescence changes, but how indicator expression affects the physiology of host organisms is often overlooked. Here, we demonstrate biocompatibility and widespread functional expression of the genetically encoded calcium indicator Tn-XXL in a transgenic mouse model. To validate the model and characterize potential effects of indicator expression we assessed both indicator function and a variety of host parameters, such as anatomy, physiology, behaviour and gene expression profiles in these mice. We also demonstrate the usefulness of primary cells and organ explants prepared from these mice for imaging applications. Although we find mild signatures of indicator expression that may be further reduced in future sensor generations, the 'green' indicator mice generated provide a well-characterized resource of primary cells and tissues for in vitro and in vivo calcium imaging applications.

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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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Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators

Cited by 1,805 publications

References 45 publications

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

Calcium signaling in human pluripotent stem cells

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