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Two-photon optogenetics of dendritic spines and neural circuits


We demonstrate a two-photon optogenetic method that generates action potentials in neurons with single-cell precision, using the red-shifted opsin C1V1T. We applied the method to optically map synaptic circuits in mouse neocortical brain slices and to activate small dendritic regions and individual spines. Using a spatial light modulator, we split the laser beam onto several neurons and performed simultaneous optogenetic activation of selected neurons in three dimensions.

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Figure 1: Two-photon activation of individual neurons with C1V1T in mouse brain slices.
Figure 2: Two-photon stimulation of individual dendrites and spines and optical mapping of connected neurons.
Figure 3: Two-photon 3D stimulation of two individual neurons with SLMs.


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We thank M. Agetsuma and Y. Shin for assistance with viral injections and surgeries, and other members of the laboratory for help and comments. R.Y., A.M.P., J.J.H. and D.S.P. are supported by the HHMI, Kavli Institute for Brain Science, National Eye Institute, Keck Foundation, Deutsche Forschungsgemeinschaft (DFG grant HI 1728/1-1 to J.J.H.) and Department of Defense Multidisciplinary University Research Initiative Program. R.P. is supported by the US National Institute of Mental Health. K.D. is supported by the HHMI, US National Institutes of Health, California Institute for Regenerative Medicine, Gatsby Foundation and Defense Advanced Research Projects Agency Reorganization and Plasticity to Accelerate Injury Recovery Program.

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A.M.P., D.S.P. and R.Y. designed and built the microscope software and hardware. A.M.P., D.S.P. and J.J.H. performed experiments, data analysis and quantification. R.P. and K.D. provided viral constructs, technical assistance, advice and opsin characterization. A.M.P., D.S.P., J.J.H. and R.Y. contributed to the writing of the manuscript.

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Correspondence to Rafael Yuste.

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The authors declare no competing financial interests.

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Supplementary Figures 1–10 and Supplementary Table 1 (PDF 4963 kb)

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Packer, A., Peterka, D., Hirtz, J. et al. Two-photon optogenetics of dendritic spines and neural circuits. Nat Methods 9, 1202–1205 (2012).

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