Achieving temporally precise, noninvasive control over specific neural cell types in the deep brain would advance the study of nervous system function. Here we use the potent channelrhodopsin ChRmine to achieve transcranial photoactivation of defined neural circuits, including midbrain and brainstem structures, at unprecedented depths of up to 7 mm with millisecond precision. Using systemic viral delivery of ChRmine, we demonstrate behavioral modulation without surgery, enabling implant-free deep brain optogenetics.
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We thank C. Bedbrook, M. Lovett-Barron and L. Tan for feedback on the manuscript and the entire Deisseroth laboratory for advice and discussions. We also thank T. Gschwind for assistance with EEG analysis. This research was supported by grants from the NIH, NSF, Gatsby, Fresenius, Wiegers, Grosfeld and NOMIS Foundations (to K.D.); a Walter V. and Idun Berry Postdoctoral Fellowship, and grant K99 DA050662 (to F.G.); a NARSAD Young Investigator Grant (to R.C. and to F.G.); grant F32 NS106764 (to Q.A.N.); grants R01 NS112518 and R01 NS104071 and the University of Minnesota’s MnDRIVE (Minnesota’s Discovery, Research and Innovation Economy) initiative (to E.K.-M.); and grant NS94668 (to I.S.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Extracellular electrophysiological characterization of transcranial deep brain optogenetics at different photostimulation parameters in mice.
a, Confocal image of representative neurons in the VTA expressing either soma-localized (Kv2.1) or non-localized ChRmine-oScarlet. Scale bar: 100 µm. b, Representative peristimulus time histogram of transcranial light-evoked spikes at different pulse widths. Light-responsive single-unit neural recordings at different pulse widths plotted as: probability of one or more evoked spikes (c), number of spikes per pulse (d), and latency to the first spike (e) (n = 7 units from 2 mice; one-way ANOVA with Bonferroni post hoc tests: F(3,24) = 22.51, P = 0.0000004 (c); F(3,22)=2.10, P= 0.13 (d); and F(3,22) = 9.30, P = 0.0004 (e). Note two neurons exhibited no spike response at 1-ms pulse width and therefore do not have an associated latency or spike count at this pulse width). f, Representative peristimulus time histogram of transcranial light-evoked spikes at different frequencies. Light-responsive single-unit neural recordings at different frequencies plotted as: probability of one or more evoked spikes (g), number of spikes per pulse (h), and latency to the first spike (i) (n = 7 from 2 mice; one-way ANOVA with Bonferroni post hoc tests: F(3,24) = 6.31, P = 0.003 (g); F(3,24) = 2.82, P = 0.06 (h); and F(3,24)=0.52, P = 0.67 (i)). j, Representative raster plot and peristimulus time histogram of transcranial light-evoked spikes at low light power (40 mW mm-2, 100 ms duration). Light-responsive single-unit neural recordings at different irradiance (I) with an extended pulse width of 100 ms plotted as: probability of one or more evoked spikes (k), number of spikes per pulse (l), and latency to the first spike (m) (n = 12 units from 2 mice; one-way ANOVA with Bonferroni post hoc tests: (k) F(5,66) = 10.46, P = 0.0000002 (l) F(5,56) = 2.81, P = 0.02, and (m) F(5,56 ) = 14.08, P = 0.000000006. Note six (4 mW mm-2) and four (12 mW mm-2) neurons exhibited no spike response and therefore do not have an associated latency or spike count at these irradiances). n, Latency to first spike determined from patch recordings of cultured hippocampal neurons at low irradiance exhibiting increased time to fire action potentials with decreasing photon density (n = 5 neurons, one-way ANOVA with Bonferroni post hoc tests: F(2,12) = 36, P = 0.03). Light-responsive single-unit neural recordings at different irradiance for ChRmine-expressing neurons with (nonstriped) or without (striped) the Kv2.1 peptide tag:probability of one or more evoked spikes (o), number of spikes per pulse (p), and latency to the first spike (q) (n = 7 units from 2 mice (ChRmine) and n = 7 units from 2 mice (ChRmine-Kv2.1); two-way ANOVA with Bonferroni post hoc tests: ChRmine-Kv2.1 vs ChRmine (o) F(1,48)=0.02, P=0.88, (p) F(1,43)=0.08, P=0.78, and (q) F(1,43)=0.42, P=0.52). No differences in the ability to photoactivate neurons with or without the Kv2.1 tag was observed. In b–e, 635 nm light was delivered at 10 Hz and 800 mW mm-2 light power at different pulse width. In f–i, 635 nm light was delivered at 800 mW mm-2 mW light power with 5-ms pulse width at different frequencies. In j–m, 635 nm light was delivered at 1 Hz with 100 ms pulse at different irradiance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Data are mean ± sem.
Extended Data Fig. 2 ChRmine photoactivatable up to 7 mm from the skull.
a, Schematic of experiment for extracellular recording in anesthetized rats. b, Confocal image of a coronal slice from rat depicting DAPI-stained cells (blue) and soma-localized ChRmine-oScarlet-Kv2.1 (red) expression in neurons at 6 mm (left) and 7 mm (right). Inset: expanded view of neurons expressing ChRmine. Scale bar: 1 mm, (inset) 100 µm. c, d, Example voltage trace of light-evoked activity in response to 5-ms pulse width of light delivered at 10 Hz with a 635 nm laser at 800 mW mm-2. Light-responsive single-unit neural recordings at 6 mm plotted as: probability of one or more evoked spikes (e), number of spikes per pulse (f), and latency to the first spike (g) (n = 15 units from 2 rats). Light-responsive single-unit neural recordings at 7 mm plotted as: probability of one or more evoked spikes (h), number of spikes per pulse (i), and latency to the first spike (j) (n = 6 units from 2 rats). In e–g and h–j, 635 nm light was delivered at 1 Hz with a pulse width of 100 ms at different irradiance (I). At 400 mW mm-2, these conditions resulted in a spike probability of 0.96 with an average spike latency of 27 ± 6 ms and a spike count of 3.7 ± 0.6 recorded at 6 mm in depth, and a spike probability of 0.97 with an average spike latency of 39 ± 7 ms and a spike count of 3 ± 0.6 at 7 mm in depth. At the same conditions, no light-evoked activity was recorded at 8 mm deep across 2 rats. Data are mean ± sem.
Extended Data Fig. 3 ChRmine enabled transcranial deep brain optogenetics at light powers that elicit minimal tissue heating.
a, Light transmission profile through brain tissue from a 400-µm 0.39 NA optical fiber calculated from Monte Carlo simulations of photon propagation. b, Calculated maximum temperature change associated with a 635 nm laser delivered at various light powers and frequencies with a pulse width of 5 ms. Note conditions used for transcranial optogenetics in mice (800 mW mm-2 and up to 10% duty cycle) resulted in minimal tissue heating at steady state (~0.3 °C, gray dashed line). c, Calculated maximum temperature change associated with a 635 nm laser delivered at various light powers as a function of continuous irradiation time. Note conditions used for transcranial optogenetics in rats (400 mW mm-2 with 100-ms pulse width at 1 Hz) resulted in minimal tissue heating at steady state (~0.4 °C, gray dashed line). d, Modeled temperature distribution in brain tissue at steady state for 635 nm light delivered at 800 mW mm-2, 20 Hz, 5-ms pulse width. e, Calculated maximum temperature change for various laser parameters used for transcranial optogenetics applied for 10 s (see Supplementary Table 2 for summary of parameters). Note stimulation conditions for ChRmine (red) heat tissue less than range reported to have nonspecific temperature effects on behavior (green, 532 nm 222 mW mm-2). Percentage of Iba+ microglia (f) and GFAP+ astrocytes (g) among DAPI-labeled cells within 200 µm of fiber optic source on the photostimulated (ipsilateral, red) or non-photostimulated (contralateral, gray) side. 635 nm light was delivered at 20 Hz with 5 ms pulse at different light powers (800, 3200, and 6400 mW mm-2) for 1 hour. Animals were perfused 24 hours after photostimulation. No glial accumulation was evident at 800 mW mm-2 and 10% duty cycle conditions (n = 3 mice; two-sided paired t-test, *P = 0.03; **P = 0.005; NS, not significant). h, Representative confocal image depicting a coronal slice treated at 6400 mW mm-2 exhibiting tissue lesioning on the ipsilateral side. i, Representative confocal images with indicated side of photostimulation and irradiance. Scale bar: 100 µm. Data are mean ± sem.
Extended Data Fig. 4 Evaluation of long-term expression of ChRmine at 7 months.
a, DAT-Cre mice following 7 months of ChRmine-oScarlet expression were subjected to a real-time place preference test with stimulation parameters at 800 mW mm-2, 500 ms ON/OFF at 20 Hz with 5-ms pulse width (5% duty cycle) (n = 5 mice; two-sided paired t-test, P = 0.003). b, Representative images of cFos expression with (bottom) or without (top) stimulation. Scale bar: 100 µm. c, Percentage of cFos+ cells among DAPI-labeled cells in the VTA. Mice were sacrificed 90 minutes following 10 minutes of transcranial photostimulation at 0 or 800 mW mm-2 laser delivered at 20 Hz and 5-ms pulse width (n = 3 per group; two-sided unpaired t-test, P = 0.001). d, Representative confocal image of neurons in the VTA expressing ChRmine-oScarlet (red fluorescent protein (FP)), stained with DAPI (blue, separate channel not shown), Iba1+ microglia (magenta), and GFAP+ astrocytes (white) used to assess glia distribution within the injection (ipsilateral) and contralateral side. Scale bar: 1 mm and 100 µm. Percentage of astrocytes (e) and microglia (f) among DAPI-labeled cells. No statistical difference in glial accumulation was observed in the VTA with (ipsilateral) or without (contralateral) local ChRmine expression (n = 3 per group; two-sided unpaired t-test, P = 0.38 (e and f)). **P < 0.01; NS, not significant. Data are mean ± sem.
Extended Data Fig. 5 Transcranial photoactivation of ChRmine enabled functional control of dopaminergic neurons.
DAT-Cre mice with ChRmine-oScarlet expression in dopaminergic neurons were subjected to a real-time place preference test. Percent of time spent on the stimulation side receiving transcranial photostimulation with the following parameters: a, tonic (1 Hz) or phasic (20 Hz) stimulation delivered at 800 mW mm-2 and 5-ms pulse width; b, with and without stimulation at 40 mW mm-2 delivered at 5 Hz with 100-ms pulse width (n=5 mice; two-sided paired t-test, P = 0.018 (a); P = 0.023 (b)). c, DAT-Cre mice expressing bReaChES did not exhibit place preference even at irradiance (I) of 3200 mW mm-2, 500 ms ON/OFF, 20 Hz and 5-ms pulse width (n = 4 mice (bReaChES), n = 6 mice (ChRmine); one-way repeated-measure ANOVA: F(4,12)=1.15, P = 0.38 (bReaChES) and two-sided paired t-test (ChRmine), P = 0.02). d, Representative confocal images of neurons in the VTA expressing red fluorescent protein and/or the indicated opsin (red) stained with DAPI (blue) and cFos (white). Scale bar: 100 µm. e, Percentage of cFos+ cells among DAPI-labeled cells in the VTA following 10 minutes of transcranial photostimulation at 20 Hz and 800 mWmm-2 with 5-ms pulse width. Animals were sacrificed after 90 minutes (n = 4 per group; one-way ANOVA with Bonferroni post hoc tests: F(3,12) = 14.24, P = 0.0003). *P < 0.05; **P < 0.01; NS, not significant. Data are mean ± sem.
Extended Data Fig. 6 Evaluation of cell type specificity for seizure inhibition.
Representative confocal image of the hippocampus depicting ChRmine-YFP (white) neurons co-stained with DAPI (blue), the vesicular GABA transporter VGAT (cyan), and parvalbumin PV (magenta) regulated by either the E2 enhancer derived from the Scna1 gene (a) or the Dlx5/6 enhancer (b). Scale bar: 1 mm and (expanded view) 100 µm. White arrows point to YFP+/VGAT+/PV+ neurons while proximal ‘*’ indicate YFP+/VGAT+/PV- neurons. Pie chart depicts quantification of YFP+ cells regulated by the E2 (a) or Dlx5/6 (b) enhancer that are either eYFP+/VGAT+/PV+ (magenta), eYFP+/VGAT+/PV- (cyan), or eYFP+/VGAT-/PV- (grey). Note all PV+ neurons were also VGAT+ and that a subset of YFP+ neurons (3% (E2) and 10% (Dlx5/6)) were not labeled by the VGAT GABAergic marker. A total of 302 and 227 neurons were counted from 3 (E2) and 4 (Dlx5/6) mice respectively. c, Comparison of mean seizure distribution without light stimulation showed comparable seizure duration across cohorts (n = 4 per group; one-way ANOVA: F(2,9)=0.56, P = 0.59). Data are mean ± sem. Box plot of each animal from E2::ChRmine-eYFP (d), Dlx5/6::ChRmine-eYFP (e) and Dlx5/6::eYFP (f) cohorts depicting seizure duration from trigger with (filled) and without (not filled) light treatment (n = 4 mice per group; two-sided Mann-Whitney U Test). Each box denotes lower (25%) and upper (75%) quartile with median (line) and mean (square) and whiskers depict the 10-90% range for all measured seizures. *P < 0.05; **P < 0.01; ***P < 0.01; ****P < 0.0001; NS, not significant.
Extended Data Fig. 7 Effects of brain-noninvasive transcranial activation of 5-HT neurons on novelty preference, anxiety-related behaviors, and induction of the neural activity marker cFos.
a, Mice were assessed for novelty preference by quantifying ratio of time spent in the chamber containing a novel object relative to time spent in the empty chamber with and without photostimulation for YFP (gray) and ChRmine-eYFP (red) mice. Stimulation does not alter novel object interaction (n = 8 mice; two-sided paired t-test). b, Mice were assessed for anxiety-related behavior with a 15 min open field test, where the first and last 5 min block were not paired with light stimulation (OFF), while the middle 5 min was paired with 635 nm stimulation (800 mW mm-2, 20 Hz, 5-ms pulse width repeated in 10 s intervals). Stimulation does not alter anxiety-behavior based on time spent in the center of the arena. (n = 8 mice; repeated-measure one-way ANOVA: F(2,14) = 2.23, P = 0.15 (eYFP) and F(2,14) = 0.05, P = 0.83 (ChRmine-eYFP)). c, Example path-tracing of a 5-HT ChRmine-YFP mouse during the 3 5-min blocks of the 15 min long open field test. Tracks are color coded for velocity (v). d, Representative confocal image of ChRmine-YFP neurons (white) in the raphe stained for cFos (magenta) by in situ hybridization and DAPI (blue). White arrows point to example YFP+/cFos+ neurons. Scale bar: 100 µm and (expanded view) 10 µm. e, Percentage of cFos+ cells among DAPI-labeled cells in the raphe following 10 min of transcranial photostimulation at 20 Hz and 800 mW mm-2 with 5-ms pulse width. (n = 4 per group; one-way ANOVA: F(1,6)=16, P = 0.007). **P < 0.01; NS, not significant. Data are mean ± sem.
Extended Data Fig. 8 Comparison of expression level and photoactivity of ChRmine-expressing neurons targeted by intracranial or retro-orbital delivery.
a, Confocal images of Purkinje neurons expressing eYFP (white) co-labeled with in situ hybridization for ChRmine mRNA (magenta). Neurons were targeted by AAVPHP.eB-L7::ChRmine-p2a-eYFP by direct or retro-orbital injection. Scale bar: 100 µm. Box plot of mean intensity signal from individual neurons for (b) eYFP and (c) ChRmine mRNA (n = 2 mice, 60 neurons per group; two-sided unpaired t-test, P = 6.7e-10 (eYFP), 5.2e-10 (ChRmine mRNA)). The mean signal of neurons targeted by retro-orbital injection was 42% (eYFP) and 34% (ChRmine mRNA) relative to the signal from direct injection, indicative of the lower multiplicity of infection. Each box denotes lower (25%) and upper (75%) quartile with median (line) and mean (square) and whiskers depict the 10-90% range for neurons quantified. In vivo extracellular recordings at 3 mm from skull following transcranial stimulation from neurons expressing ChRmine targeted by intracranial (not filled) or retro-orbital (gray) injections plotted as: probability of one or more evoked spikes (d); number of spikes per pulse (e); and latency to the first spike (f) across the population of neurons as a function of irradiance (I) delivered at 10 Hz with 5-ms pulse width. No statistically significant difference was observed in single-unit response between the viral delivery methods (n = 9 units from 2 mice (intracranial) and n = 10 units from 2 mice (retro-orbital); two-way ANOVA with Bonferroni post hoc tests: intracranial vs. retro-orbital F(1,85) = 0.002, P = 0.96 (d); F(1,83) = 0.05, P = 0.82 (e); and F(1,83) = 0.65, P = 0.42 (f). Note one neuron in each group exhibited no spike response at 80 mW mm-2 and as such has no corresponding spike count or latency at this irradiance). ****P < 0.0001; NS, not significant. Data are mean ± sem.
Supplementary Note 1 and Tables 1 and 2.
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Chen, R., Gore, F., Nguyen, QA. et al. Deep brain optogenetics without intracranial surgery. Nat Biotechnol 39, 161–164 (2021). https://doi.org/10.1038/s41587-020-0679-9
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