Erasable labeling of neuronal activity using a reversible calcium marker
- Howard Hughes Medical Institute, Janelia Research Campus, United States
Figures
Figure 1 with 9 supplements
Engineering and in vitro characterization of rsCaMPARI.
(a) Schematic of rsCaMPARI function. (b) Primary structure of rsCaMPARI relative to mEos3.1. (c) Off-switching time-course of rsCaMPARI under marking light illumination (200 mW/cm2) in the presence …
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Figure 1—source data 1
Off-switching time-course of rsCaMPARI under marking light illumination in the presence or absence of calcium.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig1-data1-v1.xlsx
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Figure 1—source data 2
On-switching time-course of rsCaMPARI under erasing light illumination in the presence or absence of calcium.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig1-data2-v1.xlsx
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Figure 1—source data 3
Relative fluorescence intensity and relative off-switching rate of rsCaMPARI as a function of free [Ca2+].
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig1-data3-v1.xlsx
Figure 1—figure supplement 1
Design and construction of libraries to engineer an erasable calcium activity marker.
(a) Outline of Gibson assembly strategy for constructing insertion libraries of calmodulin-binding domains into a reversibly photoswitchable variant of mEos3.1 (rs-mEos3.1). (b) Theoretical amino …
Figure 1—figure supplement 2
Outline of library screening to engineer rsCaMPARI.
Light intensities for 490 nm and 405 nm light illumination are 170 mW/cm2 and 200 mW/cm2, respectively. Initial concentration for Ca2+ and EGTA are 0.5 mM and 1 mM, respectively. Concentrations for …
Figure 1—figure supplement 3
In vitro characterization of variants selected from library screening.
Nineteen selected variants from calcium-binding domain insertion screen are shown. Relative brightness is green fluorescence normalized to mCherry red fluorescence and the template rs-Eos3.1. …
Figure 1—figure supplement 4
DNA and amino acid sequence of rsCaMPARI with sequence features annotated.
A nuclear export signal (NES) is added to the N-terminus for excluding rsCaMPARI from the nucleus when expressed in eukaryotic cells.
Figure 1—figure supplement 5
Amino acid sequence comparison of rsCaMPARI with mEos3.1.
Point mutations between rsCaMPARI and mEos3.1 outside of the calcium-binding domains are highlighted in yellow. The chromophore is underlined, the RS20 calmodulin-binding peptide is outlined with a …
Figure 1—figure supplement 6
rsCaMPARI secondary library screen showing relationship between photoswitching contrast and indicator behavior.
rsCaMPARI controls are shown in red and secondary library variants are shown in black. A dashed grey line illustrates a boundary beyond which no variants with high photoswitching contrast and low …
Figure 1—figure supplement 7
Two-photon action cross-section of rsCaMPARI in the presence and absence of Ca2+.
Figure 1—figure supplement 8
Optimization of blue light wavelengths for off-switching of rsCaMPARI.
(a) Spectra of bandpass filters tested. (b) Off-switching time-course of rsCaMPARI in the presence or absence of calcium under illumination of blue light with spectra shown in (a). Lines are …
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Figure 1—figure supplement 8—source data 1
Off-switching time-course of rsCaMPARI in the presence or absence of calcium under various wavelengths of light illumination.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig1-figsupp8-data1-v1.xlsx
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Figure 1—figure supplement 8—source data 2
Off-switching rate contrast and maximum fluorescence change (ΔF) as a function of wavelength.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig1-figsupp8-data2-v1.xlsx
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Figure 1—figure supplement 8—source data 3
On-switching time-course of rsCaMPARI from dim off-state under illumination with 460 nm light.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig1-figsupp8-data3-v1.xlsx
Figure 1—figure supplement 9
The relationship between rsCaMPARI off-switching rate and light power intensity.
Fitted lines are a simple linear regression to data. Error bars are standard deviation, n = 2 replicate measurements.
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Figure 1—figure supplement 9—source data 1
The relationship between rsCaMPARI off-switching rate and light power intensity.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig1-figsupp9-data1-v1.xlsx
Figure 2 with 4 supplements
rsCaMPARI reversibly and selectively marks activity in primary neurons.
(a) Fluorescence images of a representative primary rat hippocampal neuron undergoing multiple cycles of exposure to a 10 s window of marking light (224 mW/cm2) ± field stimulation (2 × 160 …
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Figure 2—source data 1
ΔF/F values of individual neurons ± field stimulations from 5 to 80 Hz during a 2-s pulse of marking light.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-data1-v1.xlsx
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Figure 2—source data 2
Fluorescence time-course of neurons undergoing one cycle of illumination ± stimulation (3 × 160 stims at 80 Hz).
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-data2-v1.xlsx
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Figure 2—source data 3
Time-course of rsCaMPARI spontaneous recovery in the dark at 37°C following marking light illumination.
Previously stimulated neurons were stimulated (3 × 160 stims at 80 Hz) during 20 s marking light illumination. Previously non-stimulated neurons were not stimulated during 20 s marking light illumination.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-data3-v1.xlsx
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Figure 2—source data 4
Photofatigue of rsCaMPARI over successive cycles of marking light illumination with or without field stimulation.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-data4-v1.xlsx
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Figure 2—source data 5
Quantification of ΔF/F of individual neurons across three marking cycles for patched and non-patched cells.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-data5-v1.xlsx
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Figure 2—source data 6
Fluorescence time-course of rsCaMPARI in patched and non-patched cells during marking light illumination.
The patched cell was stimulated to fire action potentials using current injection as described in Materials and methods.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-data6-v1.xlsx
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Figure 2—source data 7
Quantification of ΔF/F of individual neurons across three marking cycles for +ChR and -ChR cells.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-data7-v1.xlsx
Figure 2—figure supplement 1
rsCaMPARI marks neurons stimulated by a field electrode (data from Figure 2a and c–e normalized using mRuby3).
(a) Fluorescence images of rsCaMPARI-mRuby3 in a representative neuron undergoing multiple cycles of exposure to a 10-s window of marking light (224 mW/cm2) ± stimulation (2 × 160 stims at 80 Hz). …
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Figure 2—figure supplement 1—source data 1
Fluorescence time-course of neurons undergoing one cycle of illumination ± stimulation (3 × 160 stims at 80 Hz).
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
Time-course of rsCaMPARI-mRuby3 spontaneous recovery in the dark at 37°C following marking light illumination.
Previously stimulated neurons were stimulated (3 × 160 stims at 80 Hz) during 20 s marking light illumination. Previously non-stimulated neurons were not stimulated during 20 s marking light illumination.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-figsupp1-data2-v1.xlsx
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Figure 2—figure supplement 1—source data 3
Photofatigue of rsCaMPARI-mRuby3 over successive cycles of marking light illumination with or without field stimulation.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-figsupp1-data3-v1.xlsx
Figure 2—figure supplement 2
Spontaneous recovery of rsCaMPARI.
(a) Same data as shown in Figure 2d except individual traces are shown. (b), Time-course of rsCaMPARI spontaneous recovery in the dark at 37°C following blue light illumination of previously …
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Figure 2—figure supplement 2—source data 1
Time-course of rsCaMPARI-mRuby3 spontaneous recovery in the dark at 37°C following marking light illumination.
No stimulations were delivered between each timepoint.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
rsCaMPARI selectively marks neurons stimulated by current injection through a patch pipette.
Fluorescence images of rsCaMPARI expressed in dissociated primary rat hippocampal neurons before and after 15 s of marking light illumination. A single cell, denoted by pipette drawing, is patched …
Figure 2—figure supplement 4
rsCaMPARI selectively marks neurons activated by a channelrhodopsin (data from Figure 2i–j normalized using mRuby3).
(a) Merged rsCaMPARI (green) and mRuby3 (magenta) fluorescence images pre- and post-marking light illumination (285 mW/cm2, 10 s) from three cycles. Neurons expressing ChrimsonR-HaloTag labeled with …
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Figure 2—figure supplement 4—source data 1
Logarithm values of the red-to-green fluorescence ratios of -ChR and +ChR neurons post-marking light illumination.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-figsupp4-data1-v1.xlsx
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Figure 2—figure supplement 4—source data 2
The relationship between log(red/green) values and ΔF/F.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig2-figsupp4-data2-v1.xlsx
Figure 3 with 5 supplements
rsCaMPARI reversibly marks patterns of elevated calcium in the freely moving larval zebrafish.
(a) rsCaMPARI expression in the Tg[elavl3:rsCaMPARI-mRuby3]jf93 zebrafish. Scale bar is 300 μm. (b) Cartoon schematic of experimental setup and image acquisition. (c) Maximum intensity Z projections …
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Figure 3—source data 1
Mean correlation matrix values of ΔF/F images across multiple cycles of rsCaMPARI marking in the same zebrafish pallium exposed to either cold or warm water.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Replicates of rsCaMPARI labeling in zebrafish brain after exposure to different stimuli (same experiment as Figure 3c, different fish).
Each image is a maximum intensity Z projection of the entire brain from zebrafish larvae (4 to 5 dpf). Top half of images are merged marked and reference erased images, pseudo-colored green and …
Figure 3—figure supplement 2
Comparison of rsCaMPARI and CaMPARI labeling in zebrafish brain after exposure to different stimuli.
Each image is an individual Z slice acquired at various depths from the brain of zebrafish larvae (4 to 5 dpf). Under each stimulus condition, images to the left are rsCaMPARI ΔF/F light sheet …
Figure 3—figure supplement 3
Replicate of multiple rsCaMPARI labeling cycles in the same zebrafish (same experiment as Figure 3d, different fish).
Top panels are individual Z slices from the pallium of the same fish (5 dpf) brain illustrating the same field of view from six marking cycles of alternating cold and warm water stimulus. Scale bar …
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Figure 3—figure supplement 3—source data 1
Mean correlation matrix values of ΔF/F images across multiple cycles of rsCaMPARI marking in the same zebrafish pallium exposed to either cold or warm water.
- https://cdn.elifesciences.org/articles/57249/elife-57249-fig3-figsupp3-data1-v1.xlsx
Figure 3—figure supplement 4
mRuby3 is poor for normalizing rsCaMPARI expression in larval zebrafish.
Two-photon image of rsCaMPARI-mRuby3 in five dpf zebrafish brain without prior exposure to marking light. Excitation wavelength is 960 nm with detection wavelength ranges of 490–534 nm and 570–695 …
Figure 3—figure supplement 5
Representative Z slices of rsCaMPARI labeling from different marking cycles in the same fish.
Left image panels are merged marked (green) and reference erased (pseudo-colored magenta) images from the same Z position in the zebrafish pallium. Center image panels are the corresponding ΔF/F …
Tables
Table 1
Photophysical properties of rsCaMPARI.
| Ca2+ | λabs (nm) | λex (nm) | ϵ (M−1cm−1) | ΦF | Brightness* | ↓ rate† (s−1) | ↑ rate‡ (s−1) | Kd (nM) | Hill coefficient | |
|---|---|---|---|---|---|---|---|---|---|---|
| rsCaMPARI (on) | - | 502 | 515 | 60304 | 0.54 | 32.6 | 0.028 ± 0.002 | - | 167 ± 6 | 3.96 |
| + | 500 | 515 | 38234 | 0.46 | 17.6 | 0.467 ± 0.066 | - | |||
| rsCaMPARI (off) | - | 392 | - | - | - | - | - | 4.37 ± 0.24 | - | - |
| + | 389 | - | - | - | - | - | 6.40 ± 0.28 |
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*Brightness = ϵ x ΦF / 1000.
†Measured during irradiation with 200 mW/cm2 of 484 nm light.
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‡Measured during irradiation with 200 mW/cm2 of 405 nm light.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Lobophyllia hemprichii) | mEos3.1 | FPbase | FPbase:73GT1 | |
| Recombinant DNA reagent | pRSET_His-rsCaMPARI-mRuby3 (plasmid) | This paper | RRID:Addgene_120804 | Plasmid available at Addgene |
| Recombinant DNA reagent | pAAV-hsyn_NES-His-rsCaMPARI-mRuby3 (plasmid) | This paper | RRID:Addgene_120805 | Plasmid available at Addgene |
| Recombinant DNA reagent | pAAV-hsyn_NLS-His-rsCaMPARI-mRuby3 (plasmid) | This paper | RRID:Addgene_122092 | Plasmid available at Addgene |
| Recombinant DNA reagent | pTol2-elavl3_NES-rsCaMPARI-mRuby3 (plasmid) | This paper | RRID:Addgene_122129 | Plasmid available at Addgene |
| Recombinant DNA reagent | pAAV-hsyn_ChrimsonR-HaloTag (plasmid) | PMID:24509633 and PMID:18533659 | ||
| Strain, strain background (Escherichia coli) | T7 express | New England Biolabs | C2566 | Competent cells |
| Biological sample (Rattus norvegicus) | Primary rat hippocampal neurons | Janelia Research Campus | Freshly isolated from Rattus norvegicus | |
| Genetic reagent (Danio rerio) | Tg[elavl3:rsCaMPARI-mRuby3]jf93 | This paper | jf93Tg/+ | ZFIN ID: ZDB-FISH-191008–1 |
| Chemical compound, drug | Synaptic blockers | Tocris; PMID:24155972 | Cat# 0190, 0247, 1262, 0337 | |
| Chemical compound, drug | Janelia Fluor 635 dye, HaloTag ligand | PMID:28924668 | Luke Lavis’ lab | |
| Chemical compound, drug | Tricaine methanesulfonate (MS-222) | Sigma | E10521 | |
| Chemical compound, drug | 4-Aminopyridine | Sigma | 275875 | |
| Software, algorithm | CMTK | PMID:12670015 | RRID:SCR_002234 |
Additional files
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Source code 1
Source code for all Fiji macro scripts used in image analysis are provided.
- https://cdn.elifesciences.org/articles/57249/elife-57249-code1-v1.docx.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/57249/elife-57249-transrepform-v1.docx
