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Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging

Abstract

Circuit mapping requires knowledge of both structural and functional connectivity between cells. Although optical tools have been made to assess either the morphology and projections of neurons or their activity and functional connections, few probes integrate this information. We have generated a family of photoactivatable genetically encoded Ca2+ indicators that combines attributes of high-contrast photolabeling with high-sensitivity Ca2+ detection in a single-color protein sensor. We demonstrated in cultured neurons and in fruit fly and zebrafish larvae how single cells could be selected out of dense populations for visualization of morphology and high signal-to-noise measurements of activity, synaptic transmission and connectivity. Our design strategy is transferrable to other sensors based on circularly permutated GFP (cpGFP).

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Figure 1: Structural highlighting with sPA-GCaMP6f.
Figure 2: Activity imaging with sPA-GCaMP6s and sPA-GCaMP6f.
Figure 3: Functional highlighting of connected cultured rat hippocampal neurons.
Figure 4: In vivo highlighting and imaging of neural activity in Drosophila.
Figure 5: In vivo highlighting and imaging neural and glial activity in larval zebrafish.
Figure 6: Transferability of the engineering strategy to other GECIs.

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Acknowledgements

We thank C. Stanley and Z. Fu for help with molecular biology, H. Aaron for technical help with microscopy and C. Chang for fluorimeter use. We also thank R.Y. Tsien (University of California, San Diego) for the pRSETB vector, J.L. Brusés (University of Kansas) for the generous gift of the mnx1-GAL4 construct and D. Friedmann for generating the mnx1-GAL4 transgenic zebrafish line. The work was supported by US National Science Foundation (NSF) Graduate Research Fellowship (1106400; Z.L.N.), NSF Major Research Instrumentation (1041078; E.Y.I.), US National Institute of General Medical Sciences (R01 GM068552; J.C.L.) and US National Institutes of Health Nanomedicine Development Center for the Optical Control of Biological Function (2PN2EY01824; E.Y.I.).

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Authors and Affiliations

Authors

Contributions

S.B., E.C.C. and E.Y.I. conceived of the project. S.B. designed and constructed all the clones with help from H.O.O. S.B. performed all experiments in cultured cells and patching experiments. Z.L.N. maintained transgenic Drosophila lines and conducted larval Drosophila experiments with help from S.B. and E.C.C. B.K. conducted adult Drosophila experiments with help from S.B., E.C.C. and Z.L.N. E.C.C. conducted zebrafish experiments with help from C.M.Q. and S.B. S.S.M. purified proteins with supervision of J.C.L. and N.C.R. E.C.C. and S.B. characterized the purified proteins. S.B., E.C.C. and E.Y.I. wrote the manuscript.

Corresponding author

Correspondence to Ehud Y Isacoff.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–20, Supplementary Tables 1 and 2 and Supplementary Note (PDF 8774 kb)

short-GCaMP3-WT

Dissociated hippocampal neuron, transfected with short-GCaMP3-WT, displaying spontaneous activity. (MOV 1703 kb)

Highlighting of HeLa cells

Sequential photoactivation of sPA-GCaMP3 transfected in HeLa cells. (MOV 3059 kb)

Highlighting of a hippocampal neuron by ssPA-GCaMP6m

Repetitive photoactivation bouts targeted at the soma of a dissociated hippocampal neuron, transfected with ssPA-GCaMP6m, yield progressive highlighting of the neuron and its processes. (MOV 8968 kb)

Highlighting a hippocampal neuron by sPA-GCaMP6f

Dissociated neuron transfected with sPA-GCaMP6f undergoing highlighting (color coded). (MOV 3851 kb)

Ca2+ activity in dendrites and spines

Ca2+ activity observed in dendrites and spines, following highlighting with sPA-GCaMP6f (MOV 6625 kb)

Simultaneous photoactivation of individual cells

Simultaneous functional highlighting of many dissociated hippocampal neurons, expressing sPA-GCaMP6m, in which spontaneous activity can be seen detected. (MOV 18498 kb)

Sequential single cell photoactivation

Sequential photoactivation of closely situated dissociated neurons, enable tracing of the neurons' processes. (MOV 15599 kb)

Connected cells

sPA-GCaMP6m highlights the cell and enables imaging Ca2+ signals from the cell's soma, dendrites and spines. (AVI 16979 kb)

Simultaneous photoactivation of individual motor neuron somata in the VNC

Color-coded Highlighting of individual somata, expressing sPA-GCaMP6f, in the ventral nerve cord of a transgenic Drosophila larvae. (AVI 45241 kb)

Zebrafish glia displaying Ca2+ activity, in vivo

In vivo imaging of Ca2+ activity in many photoactivated Müller glia in Zebrafish larvae, transiently expressing sPA-GCaMP6f (color coded). (AVI 7211 kb)

Confined Ca2+ activity in glia cells, in vivo

In vivo imaging of Ca2+ activity in a single Müller glia in Zebrafish larvae, transiently expressing sPA-GCaMP6f. The cell displays distinct patters of Ca2+ activity in different regions of the cell. (AVI 3935 kb)

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Berlin, S., Carroll, E., Newman, Z. et al. Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging. Nat Methods 12, 852–858 (2015). https://doi.org/10.1038/nmeth.3480

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