Abstract
Real-time activity measurements from multiple specific cell populations and projections are likely to be important for understanding the brain as a dynamical system. Here we developed frame-projected independent-fiber photometry (FIP), which we used to record fluorescence activity signals from many brain regions simultaneously in freely behaving mice. We explored the versatility of the FIP microscope by quantifying real-time activity relationships among many brain regions during social behavior, simultaneously recording activity along multiple axonal pathways during sensory experience, performing simultaneous two-color activity recording, and applying optical perturbation tuned to elicit dynamics that match naturally occurring patterns observed during behavior.
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Acknowledgements
We thank M. Kay (Stanford University, Stanford, California, USA) for providing the pRD-DJ plasmid used to produce AAV-DJ. We thank M. Wagner for generating videos of live Ca2+-imaging traces. This work is supported by the US National Science Foundation (graduate research fellowships to C.K.K. and I.K.), the US Department of Defense (National Defense Science and Engineering graduate fellowship to S.J.Y.), the US National Institute of Mental Health (National Research Service Award postdoctoral fellowship 1F32MH105053-01 to T.N.L.; additional funding to K.D.), the German Academic Exchange Service (DAAD; to A.B.), the Fidelity Foundation (to S.Y.L.), the Japan Agency for Medical Research and Development (a Core Research for Evolutional Science and Technology Program and a Brain/MINDS project, both to H.B.), the Japan Society for the Promotion of Science (KAKENHI Grants-in-Aid for Scientific Research 15K18372 to M.I. and 26115507 and 15H02358 to H.B.), the US National Institute on Drug Abuse (to K.D.), and the US Army Research Laboratory and Defense Advanced Research Projects Agency (Cooperative Agreement W911NF-14-2-0013 to K.D.).
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Contributions
C.K.K., S.J.Y. and K.D. designed the experiments and wrote the paper with comments from all other authors. C.K.K., S.J.Y. and I.K. designed the FIP microscope. C.K.K. and S.J.Y. built the FIP microscope. N.P.Y. and S.J.Y. wrote software for image acquisition. C.K.K. built the head-fixed behavioral apparatus and wrote software for stimulus delivery. C.K.K. and S.J.Y. performed simultaneous photoreceiver and sCMOS measurements. C.K.K., N.P. and J.H.J. performed surgeries and behavioral experiments. C.K.K., S.J.Y. and I.K. performed simultaneous stimulation and imaging experiments. T.N.L. performed the GECI isosbestic cell culture experiments. A.B., S.Y.L. and C.R. designed and characterized the bReaChES construct. C.R. designed and generated constructs for viruses and generated transfected cultured neurons. T.J.D. contributed to photoreceiver setup design and isosbestic control design. M.I. and H.B. provided the R-CaMP2 construct. C.K.K. analyzed all data. K.D. supervised all aspects of the project.
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C.K.K., S.J.Y., I.K. and K.D. have disclosed these methods to Stanford's Office of Technology Licensing; all reagents, protocols and software are distributed freely (http://web.stanford.edu/group/dlab/optogenetics/).
Integrated supplementary information
Supplementary Figure 1 Simultaneous camera and photoreceiver measurements of reward-related photometry signals in the VTA.
(a) Schematic of setup for simultaneous camera and photoreceiver measurements. Example images of the emission light from the fiber and the excitation spot are shown in the bottom left. Photoreceiver measurements were collected with a –3 dB rolloff frequency of 16 Hz, and camera measurements were collected at 32 Hz. The excitation LED was modulated at 448 Hz. (b) An example of a GCaMP6f Ca2+ transient measured from VTA-DA neurons in response to a single water reward is shown for both the camera and the photoreceiver (top). Six trials collected from the same animal are shown below for both the camera and photoreceiver. (c) The SNR of the camera measurements (14.03 ± 2.55) was significantly higher than that of the photoreceiver measurements (6.39 ± 0.70). Data plotted as mean ± s.e.m. Asterisks indicate p < 0.005, Wilcoxon’s signed-rank test, n = 11 trials from 2 mice. (d) There was no significant difference between the peak dF/F measured between the camera (8.98 ± 0.93 % dF/F) and photoreceiver (8.47 ± 0.79 % dF/F). Data plotted as mean ± s.e.m. p > 0.05, Wilcoxon’s signed-rank test, n = 11 trials from 2 mice.
Supplementary Figure 2 GECI fluorescence emission to Ca2+-dependent and -independent wavelengths.
(a-b) Example images of a cultured neuron co-expressing GCaMP6m and R-CaMP2 taken with Ca2+-dependent (470 nm and 560 nm) and isosbestic wavelengths (405 nm). Inset in (a) shows a DIC image of the recording pipette attached to the cell. (c-d) Simultaneously collected voltage (top) and fluorescence (bottom) recordings in response to 30 APs elicited by current injections. Ca2+-dependent responses were measured with 470 and 560 nm light (left), while control responses were measured with 405 nm light (right). (e-f) Mean responses to 30 APs (dF/Fstimulus – dF/Fbaseline). For both GCaMP6m and R-CaMP2, Ca2+ responses measured with 470 nm or 560 nm light were significantly greater than control fluorescence changes measured with 405 nm light (GCaMP6m response: 54.03 ± 24 % dF/F, GCaMP6m control: –1.67 ± 0.43 % dF/F; R-CaMP2 response: 5.61 ± 2.12 % dF/F, R-CaMP2 control: –0.43 ± 0.17 % dF/F). Asterisk indicates p < 0.05, Wilcoxon’s rank-sum test, n = 5 cells for GCaMP6m and 4 cells for R-CaMP2.
Supplementary Figure 3 Correcting motion-related artifacts present in the 410-nm isosbestic wavelength.
Example of simultaneously recorded GCaMP6 signals using 470 nm and 410 nm excitation in vivo. The 410 nm signal has been scaled using least-squares regression to minimize the difference between the 410 and 470 nm signal. We then subtracted the scaled 410 nm trace from the 470 nm trace to generate the corrected 470 nm signal.
Supplementary Figure 4 Individual traces of simultaneous four-fiber recordings of VTA-DA cell bodies and projections during reward and tail shock.
(a-b) Raw GCaMP6f responses to reward or tail shock shown for 6 individual trials. Dark green traces correspond to Ca2+-dependent GCaMP6f emission, and light green traces correspond to control traces (GCaMP6f emission with 410 nm light). (c) Mean control responses to reward or shock (dF/Fstimulus – dF/Fbaseline). There were no significant responses in any brain region to reward or to shock (p > 0.05, Wilcoxon’s signed-rank test, n = 6 trials from 1 mouse).
Supplementary Figure 5 Confirmation of fiber location and virus expression for four-fiber surgeries.
(a-d) Top: 10× magnification images of slices containing PFC, NAc, BLA, or VTA. Blue stain is for DAPI, green stain is for GCaMP6f, and magenta stain is for TH. Dashed white rectangles indicate fiber location. Scale bar indicates 100 μm. Bottom: 63× magnification images of slices of same brain regions and staining. Bottom right image is a merge of all three channels. Scale bar indicates 25 μm. Note that we observed very sparse GCaMP6f fibers localized to the amygdala regions surrounding the BLA that could also be contributing to the signal.
Supplementary Figure 6 Microscope configuration used for dual-color imaging.
Schematic of setup for dual-color imaging. An image splitter was placed before the camera sensor, and an additional 560 nm LED was used to image R-CaMP2. The lower left diagram illustrates the time-division multiplexing strategy used to simultaneously image GCaMP6 and R-CaMP2 at both their Ca2+-dependent and -independent wavelengths.
Supplementary Figure 7 Confirmation of fiber location and virus specificity for dual-color imaging.
Top: 10× magnification images of a slice containing VTA. GCaMP6m fluorescence in VTA-non-DA neurons is shown in green, R-CaMP2 fluorescence in VTA-DA neurons is shown in magenta, and a TH stain is shown in white. Dashed white rectangle indicates fiber location. Scale bar indicates 100 μm. Bottom: 63× magnification images of VTA slice with the same staining. Bottom right image is a merge of all three channels. Scale bar indicates 25 μm.
Supplementary Figure 8 Characterization of novel bReaChES opsin.
(a) Example of internal current elicited by a 4 s pulse of 590 nm light (orange) for neurons expressing ReaChR or bReaChES. (b) Voltage recordings showing 4 APs in response to 4, 5 ms pulses of 590 nm light (orange) delivered at 1 Hz to neurons expressing ReaChR or bReaChES. (c) Voltage recordings showing APs in response to 80, 5 ms pulses of 590 nm light (orange) delivered at 20 Hz to neurons expressing ReaChR or bReaChES. (d) The mean tau-off for bReaChES was significantly smaller than that of ReaChR (ReaChR: 531.83 ± 40.29 ms; bReaChES: 39.33 ± 3.69 ms). Asterisks indicate p < 0.005, Wilcoxon’s rank-sum test, n = 6 cells. (e) There was no significant difference between steady-state current between ReaChR and bReaChES (ReaChR: 946.00 ± 121.97 pA; bReaChES: 941.17 ± 169.30 pA). p > 0.05, Wilcoxon’s rank-sum test, n = 6 cells. (f) Percentage of APs successfully elicited by a 4 s train of 590 nm light pulses (5 ms pulse width) delivered at 1, 2, 5, 10, and 20 Hz to neurons expressing ReaChR or bReaChES. At 10 and 20 Hz, bReaChES stimulation elicited a significantly higher percentage of successful APs than ReaChR (ReaChR: 12.08 ± 9.58 % at 10 Hz, 2.29 ± 1.04 % at 20 Hz; bReaChES: 100 ± 0 % at 10 and 20 Hz). Asterisks indicate p < 0.005, Wilcoxon’s rank-sum test, n = 6 cells.
Supplementary Figure 9 Microscope configuration used for experiments involving simultaneous imaging and perturbation.
Schematic of setup for simultaneous imaging and perturbation experiments. The 560 nm LED was replaced with a 594 nm laser for optogenetic stimulation. For cross-stimulation measurements, the 594 nm laser was replaced with an additional 470 nm LED, and the dichroic combining the 470 nm and 594 nm light was replaced with a 50:50 beamsplitter.
Supplementary Figure 10 Controls for experiments involving simultaneous imaging and perturbation.
(a) Example GCaMP6f fluorescence traces in response to bReaChES cross-stimulation with 470 nm light (light to dark blue represents 10 μW, 50 μW, and 220 μW of power). (b) Summary of the mean GCaMP6f responses to bReaChES cross-stimulation with 470 nm light (dF/Fstimulus – dF/Fbaseline). (c) Top: Example GCaMP6f fluorescence trace taken from a control mouse expressing mCherry instead of bReaChES to demonstrate that there is functional GCaMP6f present. Bottom: GCaMP6f fluorescence traces in response to 0.5 mW 594 nm stimulation pulses (orange), and to 20 or 50 μW 470 nm stimulation pulses (blue). (d) Mean GCaMP6f responses to light (dF/Fstimulus – dF/Fbaseline) in the mCherry control mouse. There were no increases in GCaMP6f fluorescence with 470 nm or 594 nm stimulation light.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–10, Supplementary Table 1 and Supplementary Notes 1–6 (PDF 6482 kb)
Freely moving mouse with seven fibers implanted into the brain
The first part of the video shows the mouse exploring the cage alone. The second part of the video shows the mouse interacting with a novel mouse. (MOV 40092 kb)
GCaMP6f fiber emission during administration of a reward
Video of fibers containing GCaMP6 emission from VTA-DA cell bodies or axonal projections in four different brain regions. For visualization, the minimum pixel value across the entire movie has been subtracted from each frame. The computed dF/F trace for each brain region is shown to the right in synchronization with the video frames. A 0.5 s water reward is administered at 2 s. Vertical scale bar indicates 10 % dF/F for VTA-DA cell bodies, and 1 % dF/F for VTA-DA projections in other regions. Horizontal scale bar indicates 1 s. (MOV 319 kb)
GCaMP6f fiber emission during administration of a tail shock
Video of fibers containing GCaMP6 emission from VTA-DA cell bodies or axonal projections from four different brain regions. For visualization, the minimum pixel value across the entire movie has been subtracted from each frame. The computed dF/F trace for each brain region is shown to the right in synchronization with the video frames. A 2 s tail shock (450 ms pulses at 0.5 Hz) is administered at 2 s. Vertical scale bar indicates 10 % dF/F for VTA-DA cell bodies, and 1 % dF/F for VTA-DA projections in other regions. Horizontal scale bar indicates 1 s. (MOV 317 kb)
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Kim, C., Yang, S., Pichamoorthy, N. et al. Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain. Nat Methods 13, 325–328 (2016). https://doi.org/10.1038/nmeth.3770
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DOI: https://doi.org/10.1038/nmeth.3770
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