1. Volume 26, Issue 4, Pages 1033-1043.e5 (January 2019)
Large Timescale Interrogation of Neuronal Function by Fiberless Optogenetics Using Lanthanide Micro-particles 
Toh Miyazaki, Srikanta Chowdhury, Takayuki Yamashita, Takanori Matsubara, Hiromu Yawo, Hideya Yuasa, Akihiro Yamanaka 
Cell Reports 
Volume 26, Issue 4, Pages e5 (January 2019)
DOI: /j.celrep
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2. Cell Reports 2019 26, 1033-1043.e5DOI: (10.1016/j.celrep.2019.01.001)
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3. Figure 1 Characteristics of Lanthanide Micro-particles
(A) Blue-, green-, and red-emitting lanthanide micro-particles (LMPs) with (right) or without (left) NIR illumination at a wavelength of 976 nm.
(B) Scanning electron microscopy (SEM) images of blue-, green-, and red-emitting LMPs.
(C) Distribution histograms for LMPs dissolved in Ringer’s solution measured by dynamic light scattering. Qi is the particle size distribution normalized to the peak of the distribution.
(D) Luminescence spectra of LMPs in Ringer’s solution.
(E) Experimental setup for measurement of up-conversion efficiency.
(F) Relationship between input NIR power (0, 360, 570, or 800 mW) and detected output power.
See also Figure S1.
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4. Figure 2 In Vitro Up-conversion Optogenetics Using HEK293 Cells
(A) Experimental setup. LS, LMP-containing silicone sheet.
(B) Fluorescence images of HEK293 cells expressing ChR2 (ET/TC)-EYFP, C1V1-Venus, or ChrimsonR-GFP.
(C) Light-induced currents recorded from HEK293 cells expressing ChR2 (ET/TC), C1V1, or ChrimsonR in the presence or absence of LS. LED light in blue (475 ± 17.5 nm, 1.0 mW/mm2), green (549 ± 7.5 nm, 0.9 mW/mm2), or red (660 nm, 0.9 mW/mm2) and NIR (976 nm, 18.6 mW/mm2) were applied through the objective lens. In the NIR+LS experiments, the color of the trace indicates the color of up-conversion luminescence.
(D) Summary of photocurrent data. The amplitudes of light-induced sustained currents were quantified. The numbers under the bars indicate the number of experiments. Data are shown as mean ± SEM. Significant differences (p < 0.05, one-way ANOVA followed by post hoc Bonferroni test): ∗ChR2 (ET/TC) versus C1V1, #ChR2 (ET/TC) versus ChrimsonR, and ΦC1V1 versus ChrimsonR.
See also Figure S2.
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5. Figure 3
Control of Locomotor Activity of Mice by Up-conversion-Mediated Optogenetics
(A) Experimental setup.
(B) NIR light intensity at the floor.
(C) Five centimeters above the floor (estimated position of mouse head).
(D) Sites of AAV/LMP injections (top). Epifluorescence images indicating expression of C1V1-Venus in the mPFC and dorsal striatum (dSTR; middle) and up-conversion luminescence from injected LMPs in fixed slices with thicknesses of 100 μm (bottom, illuminated by NIR at a wavelength of 976 nm under a stereomicroscope).
(E) Immuno-staining against GFAP (green, top) and IBA-1 (red, top) or NeuN (green, bottom) in coronal sections obtained from mice injected with vehicle (left) or LMPs (right) into the dSTR (blue: DAPI). See also Figures S3 and S4.
(F) Locomotor activity of mice upon irradiation with NIR light pulses of different durations from 1 to 20 ms at 10 Hz (left), upon irradiation with 10 ms NIR light pulses at different frequencies ranging from 1 to 20 Hz (middle), and under repeated irradiation with 10 ms NIR light pulses at 10 Hz. Red bars indicate the timing of NIR irradiation (right). Top: injection into the mPFC; bottom: injection into the dSTR.
Data are shown as mean ± SEM; ∗p < 0.05 and ∗∗∗p < 0.001, unpaired Student’s t test. One-way ANOVA followed by post hoc Tukey test, comparing LMP-injected mice and control mice.
See also Figures S3 and S5.
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6. Figure 4
In Vivo Activation of Neuronal Population in the dSTR and wM1 by Up-conversion Luminescence
(A) Experimental setup for LFP recording (top). Epifluorescence image of a recording site (bottom, red: DiI; blue: DAPI). LFP recordings (a recording glass pipette illustrated by dashed lines in the bottom image) were obtained in the dSTR of anesthetized mice, where AAV-hSyn-C1V1-Venus and LMPs were injected.
(B) Representative LFP responses evoked by up-conversion luminescence. NIR was illuminated at different intensities from 0.1 to 0.4 W/mm2 through a collimator lens (upper red trace, 10 Hz, 10 ms).
(C) Representative average LFP responses (top, superimposed). Summary plot of LFP amplitudes evoked by NIR irradiation (bottom) with LMP (red, n = 6) or without LMP (black, n = 6) in the AAV injection site. Lightly colored lines correspond to individual recordings. Bold lines and filled circles with error bars show mean ± SEM.
(D) Representative traces of the angle of the C2 whisker from an awake mouse upon irradiation with green LED (top), NIR of 0.1 W/mm2 (middle), or NIR of 0.2 W/mm2 (bottom) on the surface of wM1 with C1V1 expression and LMP injection. Cumulative whisker movement (superimposed) was quantified by integrating the absolute value of the angular velocity (bin size = 50 ms; see STAR Methods). Timing of photostimulation (duration 50 ms, 10 Hz, 20 pulses) is indicated as a bar.
(E) Summary data for cumulative whisker movement evoked by photostimulation with LMPs (red, n = 6) or without LMPs (black, n = 6) in the AAV injection site. Individual data plots are also shown. The image in the inset is a minimum intensity projection of the frames taken over 10 s upon irradiation with green LED (left), NIR of 0.1 W/mm2 (middle), or NIR of 0.2 W/mm2 (right) from the mouse shown in (D).
(F) Summary data for cumulative whisker movement with the data from each mouse normalized to the value during green LED illumination. Data are shown as mean ± SEM.
(G) Example raster plot (left top) and corresponding peri-stimulus time histogram (PSTH; left bottom) aligned to the onset of NIR stimulation (duration 50 ms, 10 Hz, 20 pulses), obtained from wM1 of an anesthetized mouse with AAV/LMP injection. Average traces for extracellular spikes of the same unit detected during NIR stimulus periods (red) and those at other timings (black) are also shown (right, superimposed).
(H) Averaged data of Z scored PSTHs obtained from all recorded units (red, C1V1 [+] LMP [+], 82 units in 3 mice; black, C1V1 [+] LMP [−], 108 units in three mice). A red bar indicates the timing of NIR irradiation (duration 50 ms, 10 Hz, 20 pulses). Error bars are shown as shadows. Error bars (± SEM) are shown as shadows.
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7. Figure 5
In Vitro Up-conversion Optogenetics Using HEK293 Cells Expressing Inhibitory Opsins
(A) Sample voltage-clamp recordings of ACR1-, ACR2-, or ArchT-mediated currents in the presence or absence of the LS. Blue (475 ± 17.5 nm, 1.0 mW/mm2), green (549 ± 7.5 nm, 0.9 mW/mm2), red (660 nm, 0.9 mW/mm2), or NIR (976 nm, 18.6 mW/mm2) light pulses were applied for 1 s through the objective lens. In the NIR+LS experiments, the color of the trace indicates the color of the up-conversion luminescence.
(B) Summary bar graphs of sustained photo-induced currents. The numbers above the bars indicate the number of experiments.
(C) Fluorescence images of HEK293 cells expressing ACR1-Venus, ACR2-mCherry, and ArchT-tdTomato. Data are shown as mean ± SEM. Significant differences (p < 0.05, one-way ANOVA followed by post hoc Bonferroni test): ∗ACR1 versus ACR2, #ACR1 versus ArchT, and ΦACR2 versus ArchT.
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8. Figure 6
In Vivo Up-conversion-Mediated Optogenetics for Neural Inhibition
(A) AAV and LMP injection sites (left). Epifluorescence images indicating expression of ACR1-Venus (top right) and location of LMPs (bottom right).
(B) Experimental protocol of the motor coordination test.
(C) Retention ratio for 360 s with irradiation of 10 ms NIR light pulses applied at 10 Hz (right, n = 8 mice) and without NIR-irradiation (left, n = 8 mice).
(D) Latency to fall from the onset of NIR pulse irradiation. The numbers above the bars indicate the number of mice. Data are shown as mean ± SEM. ∗∗∗p < (unpaired t test).
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9. Figure 7
Analysis of the Distribution of Injected LMPs in the Intact Brain Using MRI
(A) LMPs at different concentrations were injected into an agarose gel (top) that was scanned by an MRI scanner (lower). Note that higher concentrations of LMPs caused lower signal intensities in the T2-weighted MRI.
(B) MB and LMPs were bilaterally injected into the dSTR (left). MRIs from vehicle injections (right top, Ringer 600 nL, mineral oil 600 nL) and MB (1.8 μL) and LMP (600 nL) injections (right bottom).
(C) Sizes of low-signal voxels in MRIs from the brain injected with different doses of LMPs.
(D) MRIs of the brain at different timings after MB and LMP injections.
(E) Summary of low-signal voxel sizes in the brain after MB and LMP injections (n = 7).
(F) Locomotor activity upon 10 min NIR irradiation after LMP or vehicle injection into the mPFC (n = 8).
(G) Trajectory of mouse movement during NIR irradiation for 10 min at 4 weeks after vehicle (black) or LMP (red) injection in the mPFC (upper insets). Maximum (Max) and mean speeds during NIR irradiation from five mice were extracted from the trajectories.
(H) Same as in (F), but AAV and LMPs were injected into the dSTR (n = 8).
(I) Same as in (G), but AAV and LMPs were injected into the dSTR (n = 3).
In (G) and (I), ∗p < 0.05 (unpaired t test). In (E), (F), and (H), ∗p < 0.05 and ∗∗p < 0.01 (two-way ANOVA followed by post hoc Tukey test, comparing LMP-injected mice and control mice). Data are shown as mean ± SEM.
See also Figure S7.
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