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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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.).
The authors declare no competing financial interests.
Supplementary Figures 1–20, Supplementary Tables 1 and 2 and Supplementary Note (PDF 8774 kb)
Dissociated hippocampal neuron, transfected with short-GCaMP3-WT, displaying spontaneous activity. (MOV 1703 kb)
Sequential photoactivation of sPA-GCaMP3 transfected in HeLa cells. (MOV 3059 kb)
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)
Dissociated neuron transfected with sPA-GCaMP6f undergoing highlighting (color coded). (MOV 3851 kb)
Ca2+ activity observed in dendrites and spines, following highlighting with sPA-GCaMP6f (MOV 6625 kb)
Simultaneous functional highlighting of many dissociated hippocampal neurons, expressing sPA-GCaMP6m, in which spontaneous activity can be seen detected. (MOV 18498 kb)
Sequential photoactivation of closely situated dissociated neurons, enable tracing of the neurons' processes. (MOV 15599 kb)
sPA-GCaMP6m highlights the cell and enables imaging Ca2+ signals from the cell's soma, dendrites and spines. (AVI 16979 kb)
Color-coded Highlighting of individual somata, expressing sPA-GCaMP6f, in the ventral nerve cord of a transgenic Drosophila larvae. (AVI 45241 kb)
In vivo imaging of Ca2+ activity in many photoactivated Müller glia in Zebrafish larvae, transiently expressing sPA-GCaMP6f (color coded). (AVI 7211 kb)
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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