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'.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Scientific Reports Open Access 11 April 2023
Molecular Brain Open Access 23 February 2022
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Miesenböck, G. The optogenetic catechism. Science 326, 395–399 (2009).
Bamann, C., Nagel, G. & Bamberg, E. Microbial rhodopsins in the spotlight. Curr. Opin. Neurobiol. 20, 610–616 (2010).
Yizhar, O., Fenno, L.E., Davidson, T.J., Mogri, M. & Deisseroth, K. Optogenetics in neural systems. Neuron 71, 9–34 (2011).
Madisen, L. et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat. Neurosci. 15, 793–802 (2012).
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).
Cronin, J., Zhang, X.Y. & Reiser, J. Altering the tropism of lentiviral vectors through pseudotyping. Curr. Gene Ther. 5, 387–398 (2005).
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, 21848–21853 (2010).
Tripodi, M., Stepien, A.E. & Arber, S. Motor antagonism exposed by spatial segregation and timing of neurogenesis. Nature 479, 61–66 (2011).
Yonehara, K. et al. Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit. Nature 469, 407–410 (2011).
Calame, M. et al. Retinal degeneration progression changes lentiviral vector cell targeting in the retina. PLoS ONE 6, e23782 (2011).
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, 7025–7030 (2008).
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).
Dymecki, S.M., Ray, R.S. & Kim, J.C. Mapping cell fate and function using recombinase-based intersectional strategies. Methods Enzymol. 477, 183–213 (2010).
Busskamp, V. et al. Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa. Science 329, 413–417 (2010).
Busskamp, V., Picaud, S., Sahel, J.A. & Roska, B. Optogenetic therapy for retinitis pigmentosa. Gene Ther. 19, 169–175 (2012).
Tye, K.M. & Deisseroth, K. Optogenetic investigation of neural circuits underlying brain disease in animal models. Nat. Rev. Neurosci. 13, 251–266 (2012).
Arlotta, P. et al. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45, 207–221 (2005).
Fried, S.I., Munch, T.A. & Werblin, F.S. Mechanisms and circuitry underlying directional selectivity in the retina. Nature 420, 411–414 (2002).
Enquist, L.W. & Card, J.P. Recent advances in the use of neurotropic viruses for circuit analysis. Curr. Opin. Neurobiol. 13, 603–606 (2003).
Petreanu, L., Mao, T., Sternson, S.M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).
Osakada, F. et al. New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron 71, 617–631 (2011).
Wickersham, I.R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).
Beier, K.T. et al. Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors. Proc. Natl. Acad. Sci. USA 108, 15414–15419 (2011).
Beier, K.T. et al. Transsynaptic tracing with vesicular stomatitis virus reveals novel retinal circuitry. J. Neurosci. 33, 35–51 (2013).
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, 562–574 (2010).
Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).
Xu, W. & Sudhof, T.C. A neural circuit for memory specificity and generalization. Science 339, 1290–1295 (2013).
Lo, L. & Anderson, D.J.A. Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons. Neuron 72, 938–950 (2011).
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).
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, 61–67 (2008).
Judkewitz, B., Rizzi, M., Kitamura, K. & Haüsser, M. Targeted single-cell electroporation of mammalian neurons in vivo. Nat. Protoc. 4, 862–869 (2009).
Reijmers, L. & Mayford, M. Genetic control of active neural circuits. Front. Mol. Neurosci. 2, 27 (2009).
Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012).
Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266–269 (2012).
Han, X. et al. Millisecond-timescale optical control of neural dynamics in the nonhuman primate brain. Neuron 62, 191–198 (2009).
Diester, I. et al. An optogenetic toolbox designed for primates. Nat. Neurosci. 14, 387–397 (2011).
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).
Gerits, A. et al. Optogenetically induced behavioral and functional network changes in primates. Curr. Biol. 22, 1722–1726 (2012).
Jazayeri, M., Lindbloom-Brown, Z. & Horwitz, G.D. Saccadic eye movements evoked by optogenetic activation of primate V1. Nat. Neurosci. 15, 1368–1370 (2012).
Cavanaugh, J. et al. Optogenetic inactivation modifies monkey visuomotor behavior. Neuron 76, 901–907 (2012).
Mattis, J. et al. Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat. Methods 9, 159–172 (2012).
Schoenenberger, P., Scharer, Y.P. & Oertner, T.G. Channelrhodopsin as a tool to investigate synaptic transmission and plasticity. Exp. Physiol. 96, 34–39 (2011).
Hirase, H., Nikolenko, V., Goldberg, J.H. & Yuste, R. Multiphoton stimulation of neurons. J. Neurobiol. 51, 237–247 (2002).
Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 13940–13945 (2003).
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, 247–254 (2010).
LeChasseur, Y. et al. A microprobe for parallel optical and electrical recordings from single neurons in vivo. Nat. Methods 8, 319–325 (2011).
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, 124–127 (2011).
Flusberg, B.A. et al. High-speed, miniaturized fluorescence microscopy in freely moving mice. Nat. Methods 5, 935–938 (2008).
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, 903–912 (2001).
Foutz, T.J., Arlow, R.L. & McIntyre, C.C. Theoretical principles underlying optical stimulation of a channelrhodopsin-2 positive pyramidal neuron. J. Neurophysiol. 107, 3235–3245 (2012).
Scanziani, M. & Haüsser, M. Electrophysiology in the age of light. Nature 461, 930–939 (2009).
Lee, S.H. et al. Activation of specific interneurons improves V1 feature selectivity and visual perception. Nature 488, 379–383.
Anikeeva, P. et al. Optetrode: a multichannel readout for optogenetic control in freely moving mice. Nat. Neurosci. 15, 163–170 (2012).
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, 2279–2291 (2010).
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).
Guo, Z.V., Hart, A.C. & Ramanathan, S. Optical interrogation of neural circuits. in Caenorhabditis elegans. Nat. Methods 6, 891–896 (2009).
Stroh, A. et al. Making waves: initiation and propagation of corticothalamic Ca2+ waves in vivo. Neuron 77, 1136–1150 (2013).
Wilson, N.R., Runyan, C.A., Wang, F.L. & Sur, M. Division and subtraction by distinct cortical inhibitory networks in vivo. Nature 488, 343–348 (2012).
Little, J.P. & Carter, A.G. Subcellular synaptic connectivity of layer 2 pyramidal neurons in the medial prefrontal cortex. J. Neurosci. 32, 12808–12819 (2012).
Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).
Callaway, E.M. & Katz, L.C. Photostimulation using caged glutamate reveals functional circuitry in living brain slices. Proc. Natl. Acad. Sci. USA 90, 7661–7665 (1993).
Shepherd, G.M., Pologruto, T.A. & Svoboda, K. Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex. Neuron 38, 277–289 (2003).
Münch, T.A. et al. Approach sensitivity in the retina processed by a multifunctional neural circuit. Nat. Neurosci. 12, 1308–1316 (2009).
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, 147–152 (2011).
Stirman, J.N. et al. Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans. Nat. Methods 8, 153–158 (2011).
Nikolenko, V. et al. SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators. Front. Neural Circuits 2, 5 (2008).
Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Rickgauer, J.P. & Tank, D.W. Two-photon excitation of channelrhodopsin-2 at saturation. Proc. Natl. Acad. Sci. USA 106, 15025–15030 (2009).
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, 11981–11986 (2010).
Papagiakoumou, E. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848–854 (2010).
Peron, S. & Svoboda, K. From cudgel to scalpel: toward precise neural control with optogenetics. Nat. Methods 8, 30–34 (2011).
Theer, P. & Denk, W. On the fundamental imaging-depth limit in two-photon microscopy. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 3139–3149 (2006).
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, 22–27 (2012).
Papagiakoumou, E. et al. Functional patterned multiphoton excitation deep inside scattering tissue. Nat. Photonics 7, 274–278 (2013).
Prakash, R. et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat. Methods 9, 1171–1179 (2012).
Packer, A.M. et al. Two-photon optogenetics of dendritic spines and neural circuits. Nat. Methods 9, 1202–1205 (2012).
Duemani Reddy, G., Kelleher, K., Fink, R. & Saggau, P. Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat. Neurosci. 11, 713–720 (2008).
Katona, G. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat. Methods 9, 201–208 (2012).
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, 13721–13745 (2010).
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, 399–405 (2010).
DeFelipe, J. et al. New insights into the classification and nomenclature of cortical GABAergic interneurons. Nat. Rev. Neurosci. (2013).
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, 1310–1327 (2004).
Siegert, S. et al. Transcriptional code and disease map for adult retinal cell types. Nat Neurosci. 15, 487–495 (2012).
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).
Royer, S. et al. Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition. Nat. Neurosci. 15, 769–775 (2012).
Cardin, J.A. et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459, 663–667 (2009).
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, 85–88 (2012).
Kravitz, A.V. et al. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature 466, 622–626 (2010).
Huber, D. et al. Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice. Nature 451, 61–64 (2008).
Houweling, A.R. & Brecht, M. Behavioural report of single neuron stimulation in somatosensory cortex. Nature 451, 65–68 (2008).
Petreanu, L., Huber, D., Sobczyk, A. & Svoboda, K. Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat. Neurosci. 10, 663–668 (2007).
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).
Blumhagen, F. et al. Neuronal filtering of multiplexed odour representations. Nature 479, 493–498 (2011).
Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).
Zhang, F. et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat. Protoc. 5, 439–456 (2010).
Wang, H. et al. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 8143–8148 (2007).
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, 1410–1425 (2012).
Nikolenko, V., Peterka, D.S. & Yuste, R. A portable laser photostimulation and imaging microscope. J. Neural Eng. 7, 045001 (2010).
Reutsky-Gefen, I. et al. Holographic optogenetic stimulation of patterned neuronal activity for vision restoration. Nat. Commun. 4, 1509 (2013).
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).
We are grateful to B. Clark, B. Judkewitz, D. Peterka, A. Roth, T. Sato, S. Smith, D. Sosulski, C. Wilms, K. Yonehara, R. Yuste and F. Zhang for discussions and for comments on the manuscript. We thank B. Judkewitz for help with Figure 4. This work was supported by an EMBO Long-Term Fellowship (A.M.P.) and by grants from the Friedrich Miescher Institute, Alcon, European Research Council and the European Union (B.R.) and from the Wellcome Trust, ERC and Gatsby Charitable Foundation (M.H.).
The authors declare no competing financial interests.
About this article
Cite this article
Packer, A., Roska, B. & Häusser, M. Targeting neurons and photons for optogenetics. Nat Neurosci 16, 805–815 (2013). https://doi.org/10.1038/nn.3427
This article is cited by
Nature Reviews Bioengineering (2023)
Scientific Reports (2023)
Applied Physics A (2023)
Molecular Brain (2022)
Nature Protocols (2022)