Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Integration of optogenetics with complementary methodologies in systems neuroscience

Key Points

  • Modern optogenetics enables temporally precise, acute or chronic, excitatory or inhibitory modulation of neuronal activity with cell type and anatomical specificity that can be tuned to timing and magnitude of naturally occurring patterns within the same experimental subject.

  • Diverse opsin variants exhibit unique spectral and kinetic features that are specifically suited for distinct experimental requirements.

  • Optogenetics can be used in combination with electrophysiological or optical recordings, providing powerful approaches to simultaneously monitor and perturb neural function.

  • Activity-dependent labelling of opsins can be used to reactivate neural ensembles that encode particular behaviours or experiences.

  • New anatomical techniques (such as viral-tracing methods and hydrogel-embedding methods for optical access) enable molecular and anatomical profiling of the same cells that were active in vivo, providing integrative understanding of neural circuitry.

Abstract

Modern optogenetics can be tuned to evoke activity that corresponds to naturally occurring local or global activity in timing, magnitude or individual-cell patterning. This outcome has been facilitated not only by the development of core features of optogenetics over the past 10 years (microbial-opsin variants, opsin-targeting strategies and light-targeting devices) but also by the recent integration of optogenetics with complementary technologies, spanning electrophysiology, activity imaging and anatomical methods for structural and molecular analysis. This integrated approach now supports optogenetic identification of the native, necessary and sufficient causal underpinnings of physiology and behaviour on acute or chronic timescales and across cellular, circuit-level or brain-wide spatial scales.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Approaches to opsin targeting with anatomical and cell type specificity.
Figure 2: Integrating optogenetic control with in vivo electrophysiology.
Figure 3: Integrating optogenetic control with optical methods: matching naturally occurring activity patterns and linking to brain-wide projection activity.

Similar content being viewed by others

References

  1. Deisseroth, K. Optogenetics: 10 years of microbial opsins in neuroscience. Nat. Neurosci. 18, 1213–1225 (2015). This recent review covers the history and developments of optogenetics over the past 10 years and addresses potential limitations and standards of practice for application.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Grosenick, L., Marshel, J. H. & Deisseroth, K. Closed-loop and activity-guided optogenetic control. Neuron 86, 106–139 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zhang, F. et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat. Protoc. 5, 439–456 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Aravanis, A. M. et al. An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural Eng. 4, S143–S156 (2007).

    Article  PubMed  Google Scholar 

  5. Warden, M. R., Cardin, J. A. & Deisseroth, K. Optical neural interfaces. Annu. Rev. Biomed. Eng. 16, 103–129 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Montgomery, K. L. et al. Wirelessly powered, fully internal optogenetics for brain, spinal and peripheral circuits in mice. Nat. Methods 12, 969–974 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Park, S. I. et al. Soft, stretchable, fully implantable miniaturized optoelectronic systems for wireless optogenetics. Nat. Biotechnol. 33, 1280–1286 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lin, D. et al. Functional identification of an aggression locus in the mouse hypothalamus. Nature 470, 221–226 (2011). This study demonstrates the necessity of using cell type-specific optogenetic targeting as opposed to nonspecific electrical stimulation to delineate the hypothalamic neurons that are responsible for controlling aggression.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Young, N. P. & Deisseroth, K. Cognitive neuroscience: in search of lost time. Nature 542, 173–174 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Licata, A. et al. Posterior parietal cortex guides visual decisions in rats. Preprint at bioRxiv http://dx.doi.org/10.1101/066639 (2016).

    Google Scholar 

  11. Otchy, T. M. et al. Acute off-target effects of neural circuit manipulations. Nature 528, 358–363 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Goshen, I. et al. Dynamics of retrieval strategies for remote memories. Cell 147, 678–689 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gunaydin, L. A. et al. Natural neural projection dynamics underlying social behavior. Cell 157, 1535–1551 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fenno, L. E. et al. Targeting cells with single vectors using multiple-feature Boolean logic. Nat. Methods 11, 763–772 (2014). This paper reports a two-virus strategy for delivering a targeted recombinase virus alongside a recombinase-dependent (DIO) opsin-expressing virus; it also reports single viruses implementing Boolean logic on the presence of multiple recombinase types for refined multiple-feature cell type targeting.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012). This study demonstrates optogenetic reactivation of a population of neurons that were labelled by opsin expression during prior experience, using IEG-mediated expression methods.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ramirez, S. et al. Creating a false memory in the hippocampus. Science 341, 387–391 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Redondo, R. L. et al. Bidirectional switch of the valence associated with a hippocampal contextual memory engram. Nature 513, 426–430 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Root, C. M., Denny, C. A., Hen, R. & Axel, R. The participation of cortical amygdala in innate, odour-driven behaviour. Nature 515, 269–273 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ryan, T. J., Roy, D. S., Pignatelli, M., Arons, A. & Tonegawa, S. Engram cells retain memory under retrograde amnesia. Science 348, 1007–1013 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gore, F. et al. Neural representations of unconditioned stimuli in basolateral amygdala mediate innate and learned responses. Cell 162, 134–145 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ye, L. et al. Wiring and molecular features of prefrontal ensembles representing distinct experiences. Cell 165, 1776–1788 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hsiang, H. L. et al. Manipulating a “cocaine engram” in mice. J. Neurosci. 34, 14115–14127 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013). This paper provides the first demonstration of tissue–hydrogel hybrid creation to achieve high-resolution optical access by allowing full delipidation.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).

    Article  CAS  PubMed  Google Scholar 

  26. Yang, B. et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Tomer, R., Ye, L., Hsueh, B. & Deisseroth, K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat. Protoc. 9, 1682–1697 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sylwestrak, E. L., Rajasethupathy, P., Wright, M. A., Jaffe, A. & Deisseroth, K. Multiplexed intact-tissue transcriptional analysis at cellular resolution. Cell 164, 792–804 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Zemelman, B. V., Lee, G. A., Ng, M. & Miesenböck, G. Selective photostimulation of genetically chARGed neurons. Neuron 33, 15–22 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Zemelman, B. V., Nesnas, N., Lee, G. A. & Miesenböck, G. Photochemical gating of heterologous ion channels: remote control over genetically designated populations of neurons. Proc. Natl Acad. Sci. USA 100, 1352–1357 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Oesterhelt, D. & Stoeckenius, W. Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nature 233, 149–152 (1971).

    CAS  Google Scholar 

  32. 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). This paper offers the first demonstrations of optogenetics using microbial opsins.

    Article  CAS  PubMed  Google Scholar 

  33. Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Nagel, G. et al. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15, 2279–2284 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Li, X. et al. Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc. Natl Acad. Sci. USA 102, 17816–17821 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bi, A. et al. Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron 50, 23–33 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ishizuka, T., Kakuda, M., Araki, R. & Yawo, H. Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels. Neurosci. Res. 54, 85–94 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Schroll, C. et al. Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae. Curr. Biol. 16, 1741–1747 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Chow, B. Y. et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463, 98–102 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gradinaru, V., Thompson, K. R. & Deisseroth, K. eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol. 36, 129–139 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Zalocusky, K. A. et al. Nucleus accumbens D2R cells signal prior outcomes and control risky decision-making. Nature 531, 642–646 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Yizhar, O. et al. Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477, 171–178 (2011). This study describes the development of C1V1, the first red-light-activated excitatory opsin, which was suitable for integration with blue-light-excited GCaMPs; it also describes the excitatory stabilized step-function opsin SSFO.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Berndt, A., Lee, S. Y., Ramakrishnan, C. & Deisseroth, K. Structure-guided transformation of channelrhodopsin into a light-activated chloride channel. Science 344, 420–424 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wietek, J. et al. Conversion of channelrhodopsin into a light-gated chloride channel. Science 344, 409–412 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Govorunova, E. G., Sineshchekov, O. A., Janz, R., Liu, X. & Spudich, J. L. Natural light-gated anion channels: a family of microbial rhodopsins for advanced optogenetics. Science 349, 647–650 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wietek, J., Broser, M., Krause, B. S. & Hegemann, P. Identification of a natural green light absorbing chloride conducting channelrhodopsin from Proteomonas sulcata. J. Biol. Chem. 291, 4121–4127 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Govorunova, E. G., Cunha, S. R., Sineshchekov, O. A. & Spudich, J. L. Anion channelrhodopsins for inhibitory cardiac optogenetics. Sci. Rep. 6, 33530 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Berndt, A. et al. Structural foundations of optogenetics: determinants of channelrhodopsin ion selectivity. Proc. Natl Acad. Sci. USA 113, 822–829 (2015).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  49. Gunaydin, L. A. et al. Ultrafast optogenetic control. Nat. Neurosci. 13, 387–392 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Lin, J. Y., Lin, M. Z., Steinbach, P. & Tsien, R. Y. Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys. J. 96, 1803–1814 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Klapoetke, N. C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Berndt, A. et al. High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels. Proc. Natl Acad. Sci. USA 108, 7595–7600 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Huff, M. L., Miller, R. L., Deisseroth, K., Moorman, D. E. & LaLumiere, R. T. Posttraining optogenetic manipulations of basolateral amygdala activity modulate consolidation of inhibitory avoidance memory in rats. Proc. Natl Acad. Sci. USA 110, 3597–3602 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Berndt, A., Yizhar, O., Gunaydin, L. A., Hegemann, P. & Deisseroth, K. Bi-stable neural state switches. Nat. Neurosci. 12, 229–234 (2009).

    Article  CAS  PubMed  Google Scholar 

  55. Bamann, C., Gueta, R., Kleinlogel, S., Nagel, G. & Bamberg, E. Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond. Biochemistry 49, 267–278 (2010).

    Article  CAS  PubMed  Google Scholar 

  56. Yamamoto, K. et al. Chronic optogenetic activation augments Aβ pathology in a mouse model of Alzheimer disease. Cell Rep. 11, 859–865 (2015).

    Article  CAS  PubMed  Google Scholar 

  57. Lee, J. H. et al. Global and local fMRI signals driven by neurons defined optogenetically by type and wiring. Nature 465, 788–792 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Thanos, P. K. et al. Mapping brain metabolic connectivity in awake rats with μPET and optogenetic stimulation. J. Neurosci. 33, 6343–6349 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Kolodziej, A. et al. SPECT-imaging of activity-dependent changes in regional cerebral blood flow induced by electrical and optogenetic self-stimulation in mice. Neuroimage 103, 171–180 (2014).

    Article  PubMed  Google Scholar 

  60. Ferenczi, E. A. et al. Prefrontal cortical regulation of brainwide circuit dynamics and reward-related behavior. Science 351, aac9698 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Zhang, F. et al. Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri. Nat. Neurosci. 11, 631–633 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Lin, J. Y., Knutsen, P. M., Muller, A., Kleinfeld, D. & Tsien, R. Y. ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat. Neurosci. 16, 1499–1508 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Rajasethupathy, P. et al. Projections from neocortex mediate top-down control of memory retrieval. Nature 526, 653–659 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009). This study describes the development of GCaMP3-facilitated Ca2+ imaging of activity in awake, behaving mice, allowing for the eventual integration of activity imaging with optogenetics.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Szabo, V., Ventalon, C., De Sars, V., Bradley, J. & Emiliani, V. Spatially selective holographic photoactivation and functional fluorescence imaging in freely behaving mice with a fiberscope. Neuron 84, 1157–1169 (2014).

    Article  CAS  PubMed  Google Scholar 

  68. Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Rickgauer, J. P., Deisseroth, K. & Tank, D. W. Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields. Nat. Neurosci. 17, 1816–1824 (2014). This paper provides the first demonstration of all-optical manipulation at single-cell resolution that provided an activity readout from targeted neurons in an awake, behaving mammal.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Packer, A. M., Russell, L. E., Dalgleish, H. W. & Häusser, M. Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo. Nat. Methods 12, 140–146 (2015).

    Article  CAS  PubMed  Google Scholar 

  71. Rajasethupathy, P., Ferenczi, E. & Deisseroth, K. Targeting neural circuits. Cell 165, 524–534 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Kim, C. K. et al. Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain. Nat. Methods 13, 325–328 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M. & Deisseroth, K. Optogenetics in neural systems. Neuron 71, 9–34 (2011).

    Article  CAS  PubMed  Google Scholar 

  74. Adamantidis, A. R., Zhang, F., Aravanis, A. M., Deisseroth, K. & De Lecea, L. Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450, 420–424 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Knobloch, H. S. et al. Evoked axonal oxytocin release in the central amygdala attenuates fear response. Neuron 73, 553–566 (2012).

    Article  CAS  PubMed  Google Scholar 

  76. Mattis, J. et al. Frequency-dependent, cell type-divergent signaling in the hippocamposeptal projection. J. Neurosci. 34, 11769–11780 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Vandecasteele, M. et al. Optogenetic activation of septal cholinergic neurons suppresses sharp wave ripples and enhances theta oscillations in the hippocampus. Proc. Natl Acad. Sci. USA 111, 13535–13540 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Saunders, A., Johnson, C. & Sabatini, B. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons. Front. Neural Circ. 6, 47 (2012).

    CAS  Google Scholar 

  79. Saunders, A. et al. A direct GABAergic output from the basal ganglia to frontal cortex. Nature 521, 85–89 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Tye, K. M. et al. Amygdala circuitry mediating reversible and bidirectional control of anxiety. Nature 471, 358–362 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Jennings, J. H., Rizzi, G., Stamatakis, A. M., Ung, R. L. & Stuber, G. D. The inhibitory circuit architecture of the lateral hypothalamus orchestrates feeding. Science 341, 1517–1521 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Adhikari, A. et al. Basomedial amygdala mediates top-down control of anxiety and fear. Nature 527, 179–185 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Mahn, M., Prigge, M., Ron, S., Levy, R. & Yizhar, O. Biophysical constraints of optogenetic inhibition at presynaptic terminals. Nat. Neurosci. 19, 554–556 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Soudais, C., Laplace-Builhe, C., Kissa, K. & Kremer, E. J. Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo. FASEB J. 15, 2283–2285 (2001).

    Article  CAS  PubMed  Google Scholar 

  85. Salinas, S. et al. CAR-associated vesicular transport of an adenovirus in motor neuron axons. PLoS Pathog. 5, e1000442 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  86. Schwarz, L. A. et al. Viral-genetic tracing of the input–output organization of a central noradrenaline circuit. Nature 524, 88–92 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Lerner, T. N. et al. Intact-brain analyses reveal distinct information carried by SNc dopamine subcircuits. Cell 162, 635–647 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Beier, K. T. et al. Circuit architecture of VTA dopamine neurons revealed by systematic input-output mapping. Cell 162, 622–634 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Tervo, D. G. R. et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 92, 372–382 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Stamatakis, A. M. et al. A unique population of ventral tegmental area neurons inhibits the lateral habenula to promote reward. Neuron 80, 1039–1053 (2013).

    Article  CAS  PubMed  Google Scholar 

  91. Nieh, E. H. et al. Decoding neural circuits that control compulsive sucrose seeking. Cell 160, 528–541 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Lammel, S. et al. Input-specific control of reward and aversion in the ventral tegmental area. Nature 491, 212–217 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Kiritani, T., Wickersham, I. R., Seung, H. S. & Shepherd, G. M. Hierarchical connectivity and connection-specific dynamics in the corticospinal–corticostriatal microcircuit in mouse motor cortex. J. Neurosci. 32, 4992–5001 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Reardon, T. R. et al. Rabies virus CVS-N2c ΔG strain enhances retrograde synaptic transfer and neuronal viability. Neuron 89, 711–724 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Lerner, T. N., Ye, L. & Deisseroth, K. Communication in neural circuits: tools, opportunities, and challenges. Cell 164, 1136–1150 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. McGovern, A., Davis-Poynter, N., Farrell, M. & Mazzone, S. Transneuronal tracing of airways-related sensory circuitry using herpes simplex virus 1, strain H129. Neuroscience 207, 148–166 (2012).

    Article  CAS  PubMed  Google Scholar 

  98. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Marcinkiewcz, C. A. et al. Serotonin engages an anxiety and fear-promoting circuit in the extended amygdala. Nature 537, 97–101 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Tovote, P. et al. Midbrain circuits for defensive behaviour. Nature 534, 206–212 (2016).

    Article  CAS  PubMed  Google Scholar 

  101. Petreanu, L., Huber, D., Sobczyk, A. & Svoboda, K. Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat. Neurosci. 10, 663–668 (2007).

    Article  CAS  PubMed  Google Scholar 

  102. Root, D. H. et al. Single rodent mesohabenular axons release glutamate and GABA. Nat. Neurosci. 17, 1543–1551 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Matthews, G. A. et al. Dorsal raphe dopamine neurons represent the experience of social isolation. Cell 164, 617–631 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Wu, Z. et al. GABAergic projections from lateral hypothalamus to paraventricular hypothalamic nucleus promote feeding. J. Neurosci. 35, 3312–3318 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Kim, S.-Y. et al. Diverging neural pathways assemble a behavioural state from separable features in anxiety. Nature 496, 219–223 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Lee, H. et al. Scalable control of mounting and attack by Esr1+ neurons in the ventromedial hypothalamus. Nature 509, 627–632 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Stuber, G. D. et al. Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking. Nature 475, 377–380 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Spellman, T. et al. Hippocampal–prefrontal input supports spatial encoding in working memory. Nature 522, 309–314 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Padilla-Coreano, N. et al. Direct ventral hippocampal–prefrontal input is required for anxiety-related neural activity and behavior. Neuron 89, 857–866 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Lima, S. Q., Hromádka, 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  111. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Zhang, S.-J. et al. Optogenetic dissection of entorhinal-hippocampal functional connectivity. Science 340, 1232627 (2013).

    Article  PubMed  CAS  Google Scholar 

  113. 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). This is a thorough description of phototagging methods to identify specific cell types labelled with opsins in vivo using simultaneous optogenetics and electrophysiology.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Sohal, V. S., Zhang, F., Yizhar, O. & Deisseroth, K. Parvalbumin neurons and gamma rhythms enhance cortical circuit performance. Nature 459, 698–702 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Paz, J. T. et al. Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury. Nat. Neurosci. 16, 64–70 (2013). This paper demonstrates the use of real-time feedback from electrical readouts of cortical activity to trigger optogenetic intervention (in this case, inhibition) to silence both neural activity (seizure-like activity in the thalamus) and behaviour (toblock visible signs of seizures).

    Article  CAS  PubMed  Google Scholar 

  116. Krook-Magnuson, E., Szabo, G. G., Armstrong, C., Oijala, M. & Soltesz, I. Cerebellar directed optogenetic intervention inhibits spontaneous hippocampal seizures in a mouse model of temporal lobe epilepsy. eNeuro http://dx.doi.org/10.1523/ENEURO.0005-14.2014 (2014).

  117. Siegle, J. H. & Wilson, M. A. Enhancement of encoding and retrieval functions through theta phase-specific manipulation of hippocampus. eLife 3, e03061 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  118. Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L. & Tank, D. W. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat. Neurosci. 13, 1433–1440 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Harvey, C. D., Coen, P. & Tank, D. W. Choice-specific sequences in parietal cortex during a virtual-navigation decision task. Nature 484, 62–68 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Flusberg, B. A. et al. High-speed, miniaturized fluorescence microscopy in freely moving mice. Nat. Methods 5, 935–938 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Ziv, Y. et al. Long-term dynamics of CA1 hippocampal place codes. Nat. Neurosci. 16, 264–266 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Schulz, K. et al. Simultaneous BOLD fMRI and fiber-optic calcium recording in rat neocortex. Nat. Methods 9, 597–602 (2012).

    Article  CAS  PubMed  Google Scholar 

  123. Cui, G. et al. Concurrent activation of striatal direct and indirect pathways during action initiation. Nature 494, 238–242 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Mandelblat-Cerf, Y. et al. Arcuate hypothalamic AgRP and putative POMC neurons show opposite changes in spiking across multiple timescales. eLife 4, e07122 (2015).

    Article  PubMed Central  CAS  Google Scholar 

  125. Calipari, E. S. et al. In vivo imaging identifies temporal signature of D1 and D2 medium spiny neurons in cocaine reward. Proc. Natl Acad. Sci. USA 113, 2726–2731 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Prakash, R. et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat. Methods 9, 1171–1179 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. St-Pierre, F. et al. High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nat. Neurosci. 17, 884–889 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Gong, Y., Wagner, M. J., Li, J. Z. & Schnitzer, M. J. Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nat. Commun. 5, 3674 (2014).

    Article  PubMed  Google Scholar 

  130. Gong, Y. et al. High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor. Science 350, 1361–1366 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Vogt, N. Voltage sensors: challenging, but with potential. Nat. Methods 12, 921–924 (2015).

    Article  CAS  PubMed  Google Scholar 

  132. Lovett-Barron, M. et al. Dendritic inhibition in the hippocampus supports fear learning. Science 343, 857–863 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Chen, R., Romero, G., Christiansen, M. G., Mohr, A. & Anikeeva, P. Wireless magnetothermal deep brain stimulation. Science 347, 1477–1480 (2015).

    Article  CAS  PubMed  Google Scholar 

  134. Meister, M. Physical limits to magnetogenetics. eLife 5, e17210 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  135. Ibsen, S., Tong, A., Schutt, C., Esener, S. & Chalasani, S. H. Sonogenetics is a non-invasive approach to activating neurons in Caenorhabditis elegans. Nat. Commun. 6, 8264 (2015).

    Article  CAS  PubMed  Google Scholar 

  136. Garner, A. R. et al. Generation of a synthetic memory trace. Science 335, 1513–1516 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Guenthner, C. J., Miyamichi, K., Yang, H. H., Heller, H. C. & Luo, L. Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations. Neuron 78, 773–784 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Kawashima, T. et al. Functional labeling of neurons and their projections using the synthetic activity-dependent promoter E-SARE. Nat. Methods 10, 889–895 (2013).

    Article  CAS  PubMed  Google Scholar 

  139. Sheng, M. & Greenberg, M. E. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4, 477–485 (1990).

    Article  CAS  PubMed  Google Scholar 

  140. Kheirbek, M. A. et al. Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. Neuron 77, 955–968 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Rashid, A. J. et al. Competition between engrams influences fear memory formation and recall. Science 353, 383–387 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Deisseroth, K. A look inside the brain. Sci. Am. 315, 30–37 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Mattis, J. et al. Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat. Methods 9, 159–172 (2012).

    Article  CAS  Google Scholar 

  144. Felix-Ortiz, A. C. et al. BLA to vHPC inputs modulate anxiety-related behaviors. Neuron 79, 658–664 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Inoue, M. et al. Rational design of a high-affinity, fast, red calcium indicator R-CaMP2. Nat. Methods 12, 64–70 (2015).

    Article  CAS  PubMed  Google Scholar 

  146. Ziegler, T. & Möglich, A. Photoreceptor engineering. Front. Mol. Biosci. 2, 30 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  147. Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  148. Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34, 204–209 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Wang, H. et al. Molecular determinants differentiating photocurrent properties of two channelrhodopsins from chlamydomonas. J. Biol. Chem. 284, 5685–5696 (2009).

    Article  CAS  PubMed  Google Scholar 

  150. Carter, M. E. et al. Tuning arousal with optogenetic modulation of locus coeruleus neurons. Nat. Neurosci. 13, 1526–1533 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Han, S., Soleiman, M. T., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Elucidating an affective pain circuit that creates a threat memory. Cell 162, 363–374 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Kim, J.-M. et al. Light-driven activation of β2-adrenergic receptor signaling by a chimeric rhodopsin containing the β2-adrenergic receptor cytoplasmic loops. Biochemistry 44, 2284–2292 (2005).

    Article  CAS  PubMed  Google Scholar 

  153. Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H. & Deisseroth, K. Temporally precise in vivo control of intracellular signalling. Nature 458, 1025–1029 (2009).

    Article  CAS  PubMed  Google Scholar 

  154. Siuda, E. R. et al. Optodynamic simulation of β-adrenergic receptor signalling. Nat. Commun. 6, 8480 (2015).

    Article  CAS  PubMed  Google Scholar 

  155. van Wyk, M., Pielecka-Fortuna, J., Löwel, S. & Kleinlogel, S. Restoring the ON switch in blind retinas: opto-mGluR6, a next-generation, cell-tailored optogenetic tool. PLoS Biol. 13, e1002143 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  156. Brothers, S. P. & Wahlestedt, C. Therapeutic potential of neuropeptide Y (NPY) receptor ligands. EMBO Mol. Med. 2, 429–439 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank the members of the Deisseroth laboratory for helpful discussions; in particular, S.J.Y. and T.J.D. for helpful discussions about fibre photometry analysis. C.K.K. is supported by a National Research Service Award (NRSA) F31 award (NIDA F31DA041795). A.A. is supported by the Walter V. and Idun Berry award, a K99 award (NIMH K99MH106649), and a NARSAD Young Investigator fellowship. K.D. is supported by the National Institutes of Health (NIH), National Science Foundation (NSF), Defense Advanced Research Projects Agency (DARPA) and the Wiegers, Grosfeld, Snyder, Yu, and Woo Foundations.

Author information

Authors and Affiliations

Authors

Contributions

C.K.K., A.A. and K.D. wrote the paper; C.K.K. and A.A. contributed equally to this work.

Corresponding author

Correspondence to Karl Deisseroth.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information S1

Integration of optogenetics with complementary methodologies in systems neuroscience (PDF 644 kb)

PowerPoint slides

Glossary

Fibre-optic patch cord

A flexible and lightweight optical fibre that is used to connect a light source (such as a laser diode or a light-emitting diode (LED)) to a fibre-optic cannula implanted on an animal, allowing light delivery to target cell populations in freely moving animals.

Kinetic opsin variants

Opsin variants that have been engineered to have slower or faster deactivation kinetics, such as the stabilized step-function opsin or 'ChETA' (E123T mutation-containing channelrhodopsin) variants, respectively.

Step-function opsins

Opsin proteins with very slow deactivation kinetics, which can thus remain activated for tens of minutes following brief light delivery and can also be switched off in a temporally precise manner with a different wavelength of light.

Red-shifted excitatory opsins

Opsin proteins such as VChR1 and C1V1 that have been discovered and/or engineered to be excited by light of longer wavelengths (that is, red-shifted), in contrast to blue light-activated channelrhodopsins, making them useful for integrating optogenetic excitation with Ca2+ imaging through blue-light-excited GCaMP sensors.

Boolean logic

An algebraic framework in which the basic operations are “OR”, “NOT” and “AND”. These logical operators have been implemented for targeting cell types defined by the presence or absence of multiple features, such as through the use of multiple recombinases and INTRSECT viruses that allow expression of genes encoding opsins in neuronal populations that express Cre NOT Flp recombinase.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, C., Adhikari, A. & Deisseroth, K. Integration of optogenetics with complementary methodologies in systems neuroscience. Nat Rev Neurosci 18, 222–235 (2017). https://doi.org/10.1038/nrn.2017.15

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrn.2017.15

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing