Focus on Neurotechniques

Targeting neurons and photons for optogenetics

Journal name:
Nature Neuroscience
Year published:
Published online


Optogenetic approaches promise to revolutionize neuroscience by using light to manipulate neural activity in genetically or functionally defined neurons with millisecond precision. Harnessing the full potential of optogenetic tools, however, requires light to be targeted to the right neurons at the right time. Here we discuss some barriers and potential solutions to this problem. We review methods for targeting the expression of light-activatable molecules to specific cell types, under genetic, viral or activity-dependent control. Next we explore new ways to target light to individual neurons to allow their precise activation and inactivation. These techniques provide a precision in the temporal and spatial activation of neurons that was not achievable in previous experiments. In combination with simultaneous recording and imaging techniques, these strategies will allow us to mimic the natural activity patterns of neurons in vivo, enabling previously impossible 'dream experiments'.

At a glance


  1. Intersectional strategies for targeting optogenetic manipulation.
    Figure 1: Intersectional strategies for targeting optogenetic manipulation.

    (a) Physical delivery of virus to a given anatomical location can exploit or uncover circuit connectivity patterns either by making use of axonal projections or by using viruses that are able to cross one or more synapses. (b) Cell types can be addressed if the cell type of interest has a known genetic identity. (c) Directing the illumination source to a given set of cells or even individual neurons and processes is useful when the targets of interest are separated in space relative to the spatial resolution of the technique used. (d) These three strategies can be combined, as shown in this example, in which axons of a particular cell class projecting to a subcellular domain of a neuron are photostimulated at different distances from the neuron.

  2. Viral targeting of optogenetic tools using knowledge of circuit connectivity.
    Figure 2: Viral targeting of optogenetic tools using knowledge of circuit connectivity.

    Schematic illustration of different strategies for targeting optogenetic tools to specific cell types based on their connectivity pattern. Neurons expressing an optogenetic tool are indicated in yellow, arrows next to cellular processes indicate the direction of viral spread, and the location of light stimulation is shown in blue. (a) Use of a retrograde virus with targeted virus injection to an axon projection region. (b) Use of an anterograde virus with targeted virus injection to the somatic region. (c) Use of a trans-synaptic retrograde virus starting from virus introduction (or infection) of a single postsynaptic cell, which leads to optogene expression in monosynaptically connected presynaptic partners. (d) Use of a trans-synaptic anterograde virus starting from virus injection in a given brain region to cause optogene expression in synaptically connected downstream neurons.

  3. Targeting optogene expression using single-cell electroporation.
    Figure 3: Targeting optogene expression using single-cell electroporation.

    (a) Schematic of the experimental setup for targeted single-cell electroporation in vivo. (b) Two-photon image of a small network of layer 2/3 parietal cortex neurons in vivo expressing channelrhodopsin-2 and enhanced green fluorescent protein (EGFP) 3 d after targeted electroporation of the respective plasmid DNA. Scale bar, 100 μm. (c) Targeted patch-clamp recording from a single layer 2/3 neuron (indicated with the red electrode in b) exhibiting spontaneous up and down states. Reliable and temporally precise spiking was triggered by illumination with brief pulses of blue light (5 ms; wavelength, 473 nm) to activate channelrhodopsin (ten consecutive traces are shown; 97% of pulses triggered a spike). Modified from ref. 31 with permission.

  4. Patterned illumination strategies.
    Figure 4: Patterned illumination strategies.

    (a) Top, pointing a single beam with galvanometer (galvo) mirrors is the most straightforward implementation of directing a focused beam of light onto different locations within a sample. Bottom, this approach is particularly useful for mapping studies91 in which independent activation of small, localized subsets of labeled neurons or axons is desired for readout by downstream neurons. (b) Top, pointing multiple beams with a digital micromirror device92. Bottom, this enables more complex patterns of activation across large areas of tissue, which has proven useful in studies of retinal circuitry63 and zebrafish behavior93. (c) Top, creating holographic patterns with a spatial light modulator combines the power of generating multiple individual beamlets with high efficiency in directing power into those beamlets. Bottom, this enables multi-site activation70, 76 when combined with two-photon excitation (see Fig. 5).

  5. One-photon versus two-photon activation strategies: from spines to circuits.
    Figure 5: One-photon versus two-photon activation strategies: from spines to circuits.

    (a) In one-photon excitation (left), opsin molecules illuminated above and below the focal plane of interest are excited. In two-photon excitation (right), generally only opsin molecules in the focal plane are excited (but see ref. 68), leading to optical sectioning that allows activation to be restricted to the particular neurons of interest. (b) Spatiotemporal patterns for illuminating neurons with two-photon beams require different power budgets and yield different spatial and temporal resolutions (see Table 1). (c) Two-photon point stimulation of a dendritic spine on a neuron expressing C1V1 (top panel) generates current detectable at the soma (bottom trace). (d) Two-photon raster-scanning of neuron 2 (top panel, red box) during electrophysiological recording from neuron 1 (white circle, top panel and bottom trace) indicates that neuron 2 is monosynaptically connected to neuron 1. (e) Simultaneous action potential generation in two neurons in three dimensions using a spatial light modulator to generate separate laser beamlets over each neuron. Data in panels ce adapted from ref. 76.

  6. Using targeted optogenetics to enable 'dream experiments'.
    Figure 6: Using targeted optogenetics to enable 'dream experiments'.

    A schematic illustration of how 'targeted optogenetics' can be used to probe the neural code in a cortical circuit. The figure highlights the close interplay that is necessary between behavioral experiments, optical readout of patterns of activity and replay of the same patterns in the 'right' neurons using optogenetics. Targeted optogenetics allows the precision of temporal patterns and the precise membership of the neuronal ensemble to be tested directly to investigate their importance for the neural code driving the behavior.


  1. Miesenböck, G. The optogenetic catechism. Science 326, 395399 (2009).
  2. Bamann, C., Nagel, G. & Bamberg, E. Microbial rhodopsins in the spotlight. Curr. Opin. Neurobiol. 20, 610616 (2010).
  3. Yizhar, O., Fenno, L.E., Davidson, T.J., Mogri, M. & Deisseroth, K. Optogenetics in neural systems. Neuron 71, 934 (2011).
  4. Madisen, L. et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat. Neurosci. 15, 793802 (2012).
  5. Kienle, E. et al. Engineering and evolution of synthetic adeno-associated virus (AAV) gene therapy vectors via DNA family shuffling. J. Vis. Exp. 62, 3819 (2012).
  6. Cronin, J., Zhang, X.Y. & Reiser, J. Altering the tropism of lentiviral vectors through pseudotyping. Curr. Gene Ther. 5, 387398 (2005).
  7. Wall, N.R., Wickersham, I.R., Cetin, A., De La Parra, M. & Callaway, E.M. Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus. Proc. Natl. Acad. Sci. USA 107, 2184821853 (2010).
  8. Tripodi, M., Stepien, A.E. & Arber, S. Motor antagonism exposed by spatial segregation and timing of neurogenesis. Nature 479, 6166 (2011).
  9. Yonehara, K. et al. Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit. Nature 469, 407410 (2011).
  10. Calame, M. et al. Retinal degeneration progression changes lentiviral vector cell targeting in the retina. PLoS ONE 6, e23782 (2011).
  11. Atasoy, D., Aponte, Y., Su, H.H. & Sternson, S.M.A. FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J. Neurosci. 28, 70257030 (2008).
  12. Farrow, K. et al. Ambient illumination toggles a neuronal circuit switch in the retina and visual perception at cone threshold. Neuron, doi:10.1016/j.neuron.2013.02.014 (2013).
  13. Dymecki, S.M., Ray, R.S. & Kim, J.C. Mapping cell fate and function using recombinase-based intersectional strategies. Methods Enzymol. 477, 183213 (2010).
  14. Busskamp, V. et al. Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa. Science 329, 413417 (2010).
  15. Busskamp, V., Picaud, S., Sahel, J.A. & Roska, B. Optogenetic therapy for retinitis pigmentosa. Gene Ther. 19, 169175 (2012).
  16. Tye, K.M. & Deisseroth, K. Optogenetic investigation of neural circuits underlying brain disease in animal models. Nat. Rev. Neurosci. 13, 251266 (2012).
  17. Arlotta, P. et al. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45, 207221 (2005).
  18. Fried, S.I., Munch, T.A. & Werblin, F.S. Mechanisms and circuitry underlying directional selectivity in the retina. Nature 420, 411414 (2002).
  19. Enquist, L.W. & Card, J.P. Recent advances in the use of neurotropic viruses for circuit analysis. Curr. Opin. Neurobiol. 13, 603606 (2003).
  20. Petreanu, L., Mao, T., Sternson, S.M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 11421145 (2009).
  21. Osakada, F. et al. New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron 71, 617631 (2011).
  22. Wickersham, I.R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639647 (2007).
  23. Beier, K.T. et al. Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors. Proc. Natl. Acad. Sci. USA 108, 1541415419 (2011).
  24. Beier, K.T. et al. Transsynaptic tracing with vesicular stomatitis virus reveals novel retinal circuitry. J. Neurosci. 33, 3551 (2013).
  25. Marshel, J.H., Mori, T., Nielsen, K.J. & Callaway, E.M. Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron 67, 562574 (2010).
  26. Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154165 (2010).
  27. Xu, W. & Sudhof, T.C. A neural circuit for memory specificity and generalization. Science 339, 12901295 (2013).
  28. Lo, L. & Anderson, D.J.A. Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons. Neuron 72, 938950 (2011).
  29. Miyashita, T., Shao, Y.R., Chung, J., Pourzia, O. & Feldman, D.E. Long-term channelrhodopsin-2 (ChR2) expression can induce abnormal axonal morphology and targeting in cerebral cortex. Front. Neural Circuits 7, 8 (2013).
  30. Kitamura, K., Judkewitz, B., Kano, M., Denk, W. & Haüsser, M. Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo. Nat. Methods 5, 6167 (2008).
  31. Judkewitz, B., Rizzi, M., Kitamura, K. & Haüsser, M. Targeted single-cell electroporation of mammalian neurons in vivo. Nat. Protoc. 4, 862869 (2009).
  32. Reijmers, L. & Mayford, M. Genetic control of active neural circuits. Front. Mol. Neurosci. 2, 27 (2009).
  33. Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381385 (2012).
  34. Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266269 (2012).
  35. Han, X. et al. Millisecond-timescale optical control of neural dynamics in the nonhuman primate brain. Neuron 62, 191198 (2009).
  36. Diester, I. et al. An optogenetic toolbox designed for primates. Nat. Neurosci. 14, 387397 (2011).
  37. Han, X. et al. A high-light sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex. Front. Syst. Neurosci. 5, 18 (2011).
  38. Gerits, A. et al. Optogenetically induced behavioral and functional network changes in primates. Curr. Biol. 22, 17221726 (2012).
  39. Jazayeri, M., Lindbloom-Brown, Z. & Horwitz, G.D. Saccadic eye movements evoked by optogenetic activation of primate V1. Nat. Neurosci. 15, 13681370 (2012).
  40. Cavanaugh, J. et al. Optogenetic inactivation modifies monkey visuomotor behavior. Neuron 76, 901907 (2012).
  41. Mattis, J. et al. Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat. Methods 9, 159172 (2012).
  42. Schoenenberger, P., Scharer, Y.P. & Oertner, T.G. Channelrhodopsin as a tool to investigate synaptic transmission and plasticity. Exp. Physiol. 96, 3439 (2011).
  43. Hirase, H., Nikolenko, V., Goldberg, J.H. & Yuste, R. Multiphoton stimulation of neurons. J. Neurobiol. 51, 237247 (2002).
  44. Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 1394013945 (2003).
  45. Cardin, J.A. et al. Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2. Nat. Protoc. 5, 247254 (2010).
  46. LeChasseur, Y. et al. A microprobe for parallel optical and electrical recordings from single neurons in vivo. Nat. Methods 8, 319325 (2011).
  47. Iwai, Y., Honda, S., Ozeki, H., Hashimoto, M. & Hirase, H. A simple head-mountable LED device for chronic stimulation of optogenetic molecules in freely moving mice. Neurosci. Res. 70, 124127 (2011).
  48. Flusberg, B.A. et al. High-speed, miniaturized fluorescence microscopy in freely moving mice. Nat. Methods 5, 935938 (2008).
  49. Helmchen, F., Fee, M.S., Tank, D.W. & Denk, W. A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals. Neuron 31, 903912 (2001).
  50. Foutz, T.J., Arlow, R.L. & McIntyre, C.C. Theoretical principles underlying optical stimulation of a channelrhodopsin-2 positive pyramidal neuron. J. Neurophysiol. 107, 32353245 (2012).
  51. Scanziani, M. & Haüsser, M. Electrophysiology in the age of light. Nature 461, 930939 (2009).
  52. Lee, S.H. et al. Activation of specific interneurons improves V1 feature selectivity and visual perception. Nature 488, 379383.
  53. Anikeeva, P. et al. Optetrode: a multichannel readout for optogenetic control in freely moving mice. Nat. Neurosci. 15, 163170 (2012).
  54. Royer, S. et al. Multi-array silicon probes with integrated optical fibers: light-assisted perturbation and recording of local neural circuits in the behaving animal. Eur. J. Neurosci. 31, 22792291 (2010).
  55. Lim, D.H. et al. In vivo large-scale cortical mapping using channelrhodopsin-2 stimulation in transgenic mice reveals asymmetric and reciprocal relationships between cortical areas. Front. Neural Circuits 6, 11 (2012).
  56. Guo, Z.V., Hart, A.C. & Ramanathan, S. Optical interrogation of neural circuits. in Caenorhabditis elegans. Nat. Methods 6, 891896 (2009).
  57. Stroh, A. et al. Making waves: initiation and propagation of corticothalamic Ca2+ waves in vivo. Neuron 77, 11361150 (2013).
  58. Wilson, N.R., Runyan, C.A., Wang, F.L. & Sur, M. Division and subtraction by distinct cortical inhibitory networks in vivo. Nature 488, 343348 (2012).
  59. Little, J.P. & Carter, A.G. Subcellular synaptic connectivity of layer 2 pyramidal neurons in the medial prefrontal cortex. J. Neurosci. 32, 1280812819 (2012).
  60. Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).
  61. Callaway, E.M. & Katz, L.C. Photostimulation using caged glutamate reveals functional circuitry in living brain slices. Proc. Natl. Acad. Sci. USA 90, 76617665 (1993).
  62. Shepherd, G.M., Pologruto, T.A. & Svoboda, K. Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex. Neuron 38, 277289 (2003).
  63. Münch, T.A. et al. Approach sensitivity in the retina processed by a multifunctional neural circuit. Nat. Neurosci. 12, 13081316 (2009).
  64. Leifer, A.M., Fang-Yen, C., Gershow, M., Alkema, M.J. & Samuel, A.D. Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans. Nat. Methods 8, 147152 (2011).
  65. Stirman, J.N. et al. Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans. Nat. Methods 8, 153158 (2011).
  66. Nikolenko, V. et al. SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators. Front. Neural Circuits 2, 5 (2008).
  67. Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 7376 (1990).
  68. Rickgauer, J.P. & Tank, D.W. Two-photon excitation of channelrhodopsin-2 at saturation. Proc. Natl. Acad. Sci. USA 106, 1502515030 (2009).
  69. Andrasfalvy, B.K., Zemelman, B.V., Tang, J. & Vaziri, A. Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc. Natl. Acad. Sci. USA 107, 1198111986 (2010).
  70. Papagiakoumou, E. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848854 (2010).
  71. Peron, S. & Svoboda, K. From cudgel to scalpel: toward precise neural control with optogenetics. Nat. Methods 8, 3034 (2011).
  72. Theer, P. & Denk, W. On the fundamental imaging-depth limit in two-photon microscopy. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 31393149 (2006).
  73. Ji, N., Sato, T.R. & Betzig, E. Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex. Proc. Natl. Acad. Sci. USA 109, 2227 (2012).
  74. Papagiakoumou, E. et al. Functional patterned multiphoton excitation deep inside scattering tissue. Nat. Photonics 7, 274278 (2013).
  75. Prakash, R. et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat. Methods 9, 11711179 (2012).
  76. Packer, A.M. et al. Two-photon optogenetics of dendritic spines and neural circuits. Nat. Methods 9, 12021205 (2012).
  77. Duemani Reddy, G., Kelleher, K., Fink, R. & Saggau, P. Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat. Neurosci. 11, 713720 (2008).
  78. Katona, G. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat. Methods 9, 201208 (2012).
  79. Kirkby, P.A., Srinivas Nadella, K.M. & Silver, R.A. A compact acousto-optic lens for 2D and 3D femtosecond based 2-photon microscopy. Opt. Express 18, 1372113745 (2010).
  80. Grewe, B.F., Langer, D., Kasper, H., Kampa, B.M. & Helmchen, F. High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat. Methods 7, 399405 (2010).
  81. DeFelipe, J. et al. New insights into the classification and nomenclature of cortical GABAergic interneurons. Nat. Rev. Neurosci. (2013).
  82. Toledo-Rodriguez, M. et al. Correlation maps allow neuronal electrical properties to be predicted from single-cell gene expression profiles in rat neocortex. Cereb. Cortex 14, 13101327 (2004).
  83. Siegert, S. et al. Transcriptional code and disease map for adult retinal cell types. Nat Neurosci. 15, 487495 (2012).
  84. Lima, S.Q., Hromadka, T., Znamenskiy, P. & Zador, A.M. PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording. PLoS ONE 4, e6099 (2009).
  85. Royer, S. et al. Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition. Nat. Neurosci. 15, 769775 (2012).
  86. Cardin, J.A. et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459, 663667 (2009).
  87. Cohen, J.Y., Haesler, S., Vong, L., Lowell, B.B. & Uchida, N. Neuron-type-specific signals for reward and punishment in the ventral tegmental area. Nature 482, 8588 (2012).
  88. Kravitz, A.V. et al. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature 466, 622626 (2010).
  89. Huber, D. et al. Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice. Nature 451, 6164 (2008).
  90. Houweling, A.R. & Brecht, M. Behavioural report of single neuron stimulation in somatosensory cortex. Nature 451, 6568 (2008).
  91. Petreanu, L., Huber, D., Sobczyk, A. & Svoboda, K. Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat. Neurosci. 10, 663668 (2007).
  92. Jerome, J., Foehring, R.C., Armstrong, W.E., Spain, W.J. & Heck, D.H. Parallel optical control of spatiotemporal neuronal spike activity using high-speed digital light processing. Front. Syst. Neurosci. 5, 70 (2011).
  93. Blumhagen, F. et al. Neuronal filtering of multiplexed odour representations. Nature 479, 493498 (2011).
  94. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 12631268 (2005).
  95. Zhang, F. et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat. Protoc. 5, 439456 (2010).
  96. Wang, H. et al. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 81438148 (2007).
  97. Zhu, P., Fajardo, O., Shum, J., Zhang Scharer, Y.P. & Friedrich, R.W. High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device. Nat. Protoc. 7, 14101425 (2012).
  98. Nikolenko, V., Peterka, D.S. & Yuste, R. A portable laser photostimulation and imaging microscope. J. Neural Eng. 7, 045001 (2010).
  99. Reutsky-Gefen, I. et al. Holographic optogenetic stimulation of patterned neuronal activity for vision restoration. Nat. Commun. 4, 1509 (2013).
  100. Zhu, P. et al. Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the tet system. Front. Neural Circuits 3, 21 (2009).

Download references

Author information


  1. Wolfson Institute for Biomedical Research and Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.

    • Adam M Packer &
    • Michael Häusser
  2. Neural Circuit Laboratories, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

    • Botond Roska

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Additional data