Despite recent advances in optogenetics, it remains challenging to manipulate gene expression in specific populations of neurons. We present a dual-protein switch system, Cal-Light, that translates neuronal-activity-mediated calcium signaling into gene expression in a light-dependent manner. In cultured neurons and brain slices, we show that Cal-Light drives expression of the reporter EGFP with high spatiotemporal resolution only in the presence of both blue light and calcium. Delivery of the Cal-Light components to the motor cortex of mice by viral vectors labels a subset of excitatory and inhibitory neurons related to learned lever-pressing behavior. By using Cal-Light to drive expression of the inhibitory receptor halorhodopsin (eNpHR), which responds to yellow light, we temporarily inhibit the lever-pressing behavior, confirming that the labeled neurons mediate the behavior. Thus, Cal-Light enables dissection of neural circuits underlying complex mammalian behaviors with high spatiotemporal precision.
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We would like to thank B. Kuhlman (University of North Carolina, Chapel Hill) for iLID construct; K. Deisseroth (Stanford University) for an eNpHR-EYFP construct; C. Tucker (University of Colorado) for CRY2 and CIBN constructs; H. Zeng (Allen institute) for a TetO-EGFP construct; S-Y. Choi (Chonnam National University, Republic of Korea) for a P2A vector; W. Weber (University of Freiburg, Germany) for a pSAM200 vector. We thank K-S. Lee and D. Fitzpatrick for helping 3D reconstruction of neurons. We thank M.J. Yetman and H. Taniguchi for helping antibody staining and resource sharing. We thank Y. Chen and B.L. Sabatini for testing initial Cal-Light constructs. We thank all members of the laboratory for critical discussion and comments. This work was supported by funding from a Korea University Grant (to D.L.) and Max Planck Florida Institute for Neuroscience (to H.-B.K.). DNA plasmids used in this study have been deposited to Addgene (Deposit number: 74208).
The Max Planck Florida Institute for Neuroscience has filed a patent application that includes portions of the research described in this manuscript.
Integrated supplementary information
Supplementary Figure 1 Design of calcium and light sensitive Cre recombinase.
(a) DNA plasmids for testing an initial Cal-Light design. (b) Cre recombinase was split and each c-terminus and n-terminus are fused to light and calcium sensitive proteins, CRY2PHR and CaM, respectively. Blue light makes CRY2PHR associates with CIBN, but Cre-dependent gene expression will not occur because Cre-C is missing. Calcium influx makes CaM bind to M13, bringing Cre-C near Cre-N. Therefore, Cre recombinase regains functions only when light and calcium are present. (c) Representative image of calcium and light dependent gene expression. A few cells displayed EGFP expression suggesting successful gene expression, but the number of EGFP positive cells was too low. mCherry was a transfection marker and EGFP was a reporter gene product.
Supplementary Figure 2 Cal-Light design using EL222.
(a) DNA constructs for cell transfection. (b) Blue light induces dimerization of a bacterial LOV-HTH and makes them to bind gene promoter area. But gene transcription cannot be initiated due to the lack of transcription activator, VP16, which is recruited by calcium. (c) Representative images of calcium and light dependent gene expression in HEK293 cells. EGFP was a transfection marker and mCherry was a reporter. Note that there were more mCherry positive cells when light was on.
Supplementary Figure 3 Effects of light-exposure duration, cycle, and intensity on Cal-Light activity expressing reporter gene.
(a) To check the light effect of Cal-Light, we tested with various combinations of blue light illumination protocols (DARK, 30HF, 30MF, 120MF, 30LF, and 120LF; Number represents duration of illumination. HF, MF, and LF are short for High frequency, medium frequency, and low frequency, respectively). Summary graph shows light exposure duration- and cycle-dependent gene expression in rat hippocampal culture neurons (DARK = 0.111 ± 0.005, n=251 neurons; 30HF = 0.336 ± 0.049, n=182 neurons; 30MF = 0.361 ± 0.012, n=156 neurons; 120MF = 0.529 ± 0.034, n=217 neurons; 30LF = 0.264 ± 0.026, n=153 neurons; 120LF = 0.368 ± 0.020, n=151 neurons; mean ± s.e.m.; one-way ANOVA [F(5, 18) = 68.125, ***p<0.001]; Bonferroni post hoc test [DARK/30HF = ***p<0.001; DARK/30MF = ***p<0.001; DARK/120MF = ***p<0.001; DARK/30LF = ***p<0.001; DARK/120LF = ***p<0.001; 30HF/120MF = ***p<0.001; 30MF/120MF = ***p<0.001; 30MF/30LF = *p<0.05; 120MF/30LF = ***p<0.001; 120MF/120LF = ***p<0.001; 30LF/120LF = **p<0.01]). (b) Summary graph represents light intensity-dependent gene expression of Cal-Light in rat hippocampal culture neurons (DARK = 0.134 ± 0.009, n=326 neurons; 1.0 mW = 0.328 ± 0.042, n=338 neurons; 2.6 mW = 0.486 ± 0.023, n=229 neurons; 5.7 mW = 0.542 ± 0.072, n=205 neurons; mean ± s.e.m.; one-way ANOVA [F(3, 12) = 68.518, ***p<0.001]; Bonferroni post hoc test [DARK/1.0 mW = ***p<0.001; DARK/2.6 mW = ***p<0.001; DARK/5.7 mW = ***p<0.001; 1.0 mW/2.6 mW = **p<0.01; 1.0 mW/5.7 mW = ***p<0.001]). Small circles indicate the G/R value of individual neurons. (c) Representative confocal images in various extend of light level (1.0, 2.6, and 5.7mW). Small circles indicate the G/R value of individual neurons. Error bar represents s.e.m. Scale bar, 100 μm.
Supplementary Figure 4 The stoichiometric balance of two Cal-Light components is necessary for effective gene expression of reporter.
Summary graph shows the gene-expression level of Cal-Light in different ratio (TM-CaM-TevN-AsLOV2-tTA: M13-TevC = 8:1, 3:1, 1:3 or 1:8) of two Cal-Light components (8:1 = 0.217 ± 0.015, n=278 neurons; 3:1 = 0.486 ± 0.040, n=288 neurons; 1:3 = 0.531 ± 0.091, n=225 neurons; 1:8 = 0.238 ± 0.013, n=296 neurons; mean ± s.e.m.; one-way ANOVA [F(3, 12) = 40.678, ***p<0.001]; Bonferroni post hoc test [8:1/3:1 = ***p<0.001; 8:1/1:3 = ***p<0.001; 3:1/1:8 = ***p<0.001; 1:3/1:8 = ***p<0.001]). Small blue circles represent G/R value of individual neurons. Error bars indicate s.e.m.
Supplementary Figure 5 Calcium and light dependent gene expression with M13 deletion mutants.
(a) Schematic description of Cal-Light constructs. To determine the contribution of CaM-M13 interaction to gene expression, M13 domain was deleted (delM13). (b) Representative culture neuron images at different conditions. Blue light was illuminated (1 sec ON/9 sec OFF for 2 hours) at all conditions. When M13 deletion mutant (TEV-C only) was transfected, although blue light was illuminated and activity was increased by 30 μM bicuculline, high EGFP expressing neurons were not observed. On the other hand, when M13 containing construct was transfected, robust gene expression was induced by light and activity. (c) Both green and red fluorescence intensity from individual neurons were plotted. Neurons were divided into four groups (GREEN, YELLOW, RED, and BLACK) by the level of red and green fluorescence. Green and yellow fluorescence neurons were significantly increased by blue light and bicuculline only when normal M13 domain was transfected. Both bicuculline and blue light were not able to increase EGFP expression when M13 domain was deleted. Bar graph represents percentage of RED, GREEN, YELLOW, and BLACK groups. Red dot line indicates a division line between groups.
Supplementary Figure 6 Coincident detector of calcium and light.
(a) Representative neuronal images at each condition. (b) Distribution of fluorescence level from individual neurons at different conditions. Four groups (GREEN, YELLOW, RED, and BLACK) of neurons were divided by the level of red and green fluorescence. Portion of GREEN and YELLOW groups were robustly increased only when activity and light were present simultaneously. Either light only or activity only did not cause increase of YELLOW group. When blue light exposure preceded by bicuculline, there was no noticeable changes in gene expression. When activity was increased and then blue light was illuminated, YELLOW group was slightly increased (~10 %). Red dot line indicates a division line between groups.
Supplementary Figure 7 No intrinsic property changes by Cal-Light constructs.
(a) Whole cell patch clamp recordings were made in Cal-Light positive neurons and neighboring control neurons in cortical pyramidal layer 2/3 pyramidal neurons. 200 pA current injection triggered the same number of APs in both fluorescent Cal-Light positive and negative neurons. (b) Summary graph of AP amplitude (Cal-Light (+), 77.7 ± 0.9 mV, n = 13; Cal-Light (−), 78.4 ± 1.5 mV, n = 13, p = 0.67) and threshold (Cal-Light (+), -34.9 ± 1.7 mV, n = 13; Cal-Light (−), -35.5 ± 1.5 mV, n = 13, p = 0.79).
Supplementary Figure 8 In vivo labeling of medium spiny neurons and striatal pathways by Cal-Light.
(a) A mixture of Cal-Light and ChR2 viruses (AAV1-hSYN-TM-CaM-TEV-N-AsLOV2-TEVseq-tTA: AAV1-hSYN-M13-TEV-C-P2A-TdTomato: AAV1-TRE-EGFP: AAV1-CMV-PI-Cre-rBG: dFlox-ChR2-mCherry = 1: 1: 2: 2: 4) were injected into striatum. 473 nm blue light was illuminated for 45 min (2 s ON/1 s OFF). (b) Image of sagittal section. (c-f) Individual medium spiny neurons and their long-range projection to globus pallidus (GP), subthalamic nucleus (STN), and substantia nigra (SNr) were visualized.
Supplementary Figure 9 Verification of Cal-Light system as a function of light duration in vivo.
Intermittent flash of blue light (1 sec ON/9 sec OFF) was shined to layer 2/3 of primary motor cortex of mice for 1, 2, or 4 hours. G/R was calculated from individual cells (Dark: 0.31 ± 0.01, n = 249 neurons; 1 hr Blue: 0.58 ± 0.02, n = 317 neurons, p < 0.005 compare to Dark; 2 hr Blue: 0.89 ± 0.04, n = 428 neurons, p < 0.005 compare to 1 hr Blue; 4 hr Blue: 1.28 ± 0.05, n = 428 neurons, p < 0.05 compare to 2 hr Blue / 4 independent animals for each condition. Boxes show the median, 25th and 75th percentiles and whiskers show min to max.
Supplementary Figure 10 Total blue light exposure time during training.
When mice press a lever, blue light was on for 5 sec. Open circles represent individual mouse used for the experiment. (CRF: 35.5 ± 2.7 sec, 11 mice; FR-2: 57.7 ± 2.6 sec, 11 mice, p < 0.005; FR-5: 96.8 ± 5.7 sec, 11 mice, p < 0.005; FR-8: 107.3 ± 4.7 sec, 11 mice; FR-10: 115.9 ± 6.0 sec, 11 mice; FR-12: 118.6 ± 3.5 sec, 11 mice, p > 0.05).
Supplementary Figure 11 Neuronal activity was diminished in an anesthetized state.
(a) GCaMP6s virus was injected into motor cortex layer 2/3 area. When mice were fully awake, neuronal activity was very high as monitored by GCaMP6s signals. (b) In the anesthetized condition, majority of neurons were quiet. Top image shows a representative in vivo GCaMP6s image. Bottom traces were Ca2+ transients from selected individual neurons.
Supplementary Figure 12 Both pyramidal neurons and interneurons were labelled by Cal-Light.
(a) Several reconstructed images of interneurons. Brain slices were made from mice who acquired lever pressing behavior. Bright EGFP expressing neurons were reconstructed to visualize clear morphology of neurons. 3D reconstruction was performed using Neurolucida 360 (MBF Bioscience). (b) Sample image showing clear pyramidal neuron morphology and its firing pattern. (c) Putative interneuron image labelled by Cal-Light and its firing pattern.
Supplementary Figure 13 Learning-related Cal-Light positive interneuron population.
(a) Representative images showing PV (left), SOM (middle) and VIP (right) immunostaining from layer 2/3 mouse brain that underwent lever-pressing training (Green: Cal-Light (+), Magenta: PV, SOM or VIP; yellow arrow: Cal-Light (+) and PV or SOM or VIP (+) co-labelled cell). Scale bar, 50 μm. (b) Percentage of Cal-Light (+) cells co-labelled with PV, SOM and VIP antibodies (PV: 18.45 ± 2.14, n = 24 cells; SOM: 22.07 ± 4.88, n = 10 cells, p = 0.67 compare to PV; VIP: 42.64 ± 3.36, n = 17 cells, p < 0.005 compare to SOM). Boxes show the median, 25th and 75th percentiles and whiskers show min to max. (c) Percentage of relative brightness of green signal of Cal-Light (+) neurons which are co-labelled with PV or SOM or VIP immunostaining (PV: 95.93 ± 6.57, n = 24 cells; SOM: 153.99 ± 10.63, n = 10 cells, p < 0.005 compare to PV; VIP: 106.54 ± 6.23, n = 17 cells, p < 0.005 compare to SOM; zero dotted line indicates brightness of non-PV or –SOM or –VIP, respectively). Data are mean ± SEM, n = 3-4 animals per group.
Supplementary Figure 14 Distinct labeling profile of PV- and SOM-positive interneurons by Cal-Light.
(a) (Upper) Schematic protocol for current injection (from 50 to 200 pA, 50 pA increment) to the visually identified interneurons. The firing property of PV-IN (middle) and SOM-IN (bottom) by current injection. (b) (Upper) Schematic cartoon for optogenetic neuronal activation. The firing profiles of PV-IN (middle) and SOM-IN (bottom) by brief blue light activation. (c) The input-output curve from PV (black) and SOM-INs (magenta) by the step current injection. (d) Summary graph of firing profile of PV and SOM-INs evoked by blue light. (e) Average box plot chart of gene expression. G/R values from individual cells and a summary box plot chart are superimposed (WT: 0.63 ± 0.61, n = 284 cells / 3 independent cultures; PV: 0.78 ± 0.54, n = 180 cells / 4 cultures, p = 0.29; SOM: 1.13 ± 0.85, n = 209 cells / 5 cultures, p < 0.01). Boxes show the median, 25th and 75th percentiles and whiskers show min to max.
Supplementary Figure 15 Impaired learning behavior by 589 nm yellow light.
(a) When learning related neurons were inactivated by yellow light, inter-reward interval was prolonged (Blue label + 589 OFF: 0.5 ± 0.1 min, n = 7; Blue label + 589 ON: 6.0 ± 1.8 min, n = 5, Blue label + 589 OFF (1d after): 0.7 ± 0.3 min, n = 6, p < 0.005). (b) Summary of pressing-licking matching ratio. Well trained mice show almost one to one matching ratio between lever pressing and licking behaviors. When eNpHR was activated by 589 nm light, lever pressing and licking behaviors were dissociated to the significant level (Light−: 0.988 ± 0.013, n = 4; Light +: 0.761 ± 0.043, n = 4, p < 0.01; post 1 day: 0.975 ± 0.014, n = 4, p < 0.05). Boxes show the median, 25th and 75th percentiles and whiskers show min to max.
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Lee, D., Hyun, J., Jung, K. et al. A calcium- and light-gated switch to induce gene expression in activated neurons. Nat Biotechnol 35, 858–863 (2017). https://doi.org/10.1038/nbt.3902
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