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ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation


Channelrhodopsins (ChRs) are used to optogenetically depolarize neurons. We engineered a variant of ChR, denoted red-activatable ChR (ReaChR), that is optimally excited with orange to red light (λ 590–630 nm) and offers improved membrane trafficking, higher photocurrents and faster kinetics compared to existing red-shifted ChRs. Red light is less scattered by tissue and is absorbed less by blood than the blue to green wavelengths that are required by other ChR variants. We used ReaChR expressed in the vibrissa motor cortex to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illumination with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window or implant optical fibers.

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Figure 1: Basic properties of ReaChR compared to those of C-VChR1 (VChR1 with the ChIEF N terminus) and C1V1E122T.
Figure 2: Kinetics of C-VChR1, C1V1E122T and ReaChR.
Figure 3: Characterization and comparison of different ChR variants in primary cultured hippocampal neurons.
Figure 4: The responses of neurons expressing the different ChR variants to a 10 Hz pulsed-light stimulation train.
Figure 5: In vivo expression and utilization of ReaChR for cortical control.
Figure 6: In vivo expression and utilization of ReaChR for brainstem activation.

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J.Y.L. was funded by the Foundation of Research, Science and Technology New Zealand, and P.M.K. was supported by a long-term fellowship from the Human Frontier Science Program (HFSP). The project was supported by grants to R.Y.T. from the US National Institutes of Health (NS027177) and the Howard Hughes Medical Institute and grants to D.K. from the US National Institutes of Health (DA029706, OD006831 and NS058668). We thank P. Tsai for suggesting the name ReaChR. We thank K. Deisseroth (Stanford University) for the hChR2H134R-eYFP construct, K. Svoboda (Howard Hughes Medical Institute Janelia Farm Research Campus) for the CAG promoter vector, E. Boyden (Massachusetts Institute of Technology) for the lentiviral vector, L. Tian (Howard Hughes Medical Institute Janelia Farm Research Campus) for the AAV2 vector and D. Trono (École Polytechnique Fédérale De Lausane) for the psPAX2 and pMD2.G lentivirus packaging vectors. AAV2-ReaChR-Citrine and pLenti-ReaChR-Citrine constructs can be requested from

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Authors and Affiliations



J.Y.L. designed and developed ReaChR and conducted and analyzed the experiments in HEK293 and neuron cultures. P.M.K. and A.M. conducted and analyzed the in vivo experiments. D.K. and R.Y.T. contributed to the design and analysis of the experiments. All authors contributed to the writing and discussions of the manuscripts.

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Correspondence to John Y Lin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-10 (PDF 3215 kb)

Supplementary Data Set

Supplementary Data Set 1 (XLS 172 kb)

Supplementary Video 1

Example of whisker protraction evoked by inter-aural stimulation of a mouse expressing ReaChR in FN motoneurons. Movements were captured with high-speed video (800 fps), during isoflurane anesthesia, and tracked offline. FN was stimulated by placing a 617 nm LED at the opening of the external auditory canal (100 mW light output; red vertical bars; see Methods). Upon photo-stimulation the whiskers promptly protracted and remained protracted for the duration of the light pulse (100 msec). The whiskers then retracted to their initial reference position. (MOV 1115 kb)

Supplementary Video 2

Example of whisker retraction evoked by inter-aural stimulation of a mouse expressing ReaChR in FN motoneurons. Movements were captured with high-speed video (800 fps), and whisker position and angle tracked offline. As the mouse recovered from isoflurane anesthesia, the whiskers slowly protracted and remained in a protracted position for approximately one minute. During this time window, ReaChR expressing neurons in FN were stimulated by inter-aural illumination with a 617 nm LED (100 mW light output; red vertical bars). Upon photo-activation, the whiskers promptly retracted and remained retracted for the duration of the light pulse (100 msec). As the light turned off, the whiskers then returned to their initial protracted position. (MOV 1556 kb)

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Lin, J., Knutsen, P., Muller, A. et al. ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat Neurosci 16, 1499–1508 (2013).

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