Abstract
A central goal in neuroscience is to determine how the brain’s neuronal circuits generate perception, cognition and emotions and how these lead to appropriate behavioural actions. A methodological platform based on genetically encoded voltage indicators (GEVIs) that enables the monitoring of large-scale circuit dynamics has brought us closer to this ambitious goal. This Review provides an update on the current state of the art and the prospects of emerging optical GEVI imaging technologies.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
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
Similar content being viewed by others
References
Markram, H. et al. Reconstruction and simulation of neocortical microcircuitry. Cell 163, 456–492 (2015).
Seeman, S. C. et al. Sparse recurrent excitatory connectivity in the microcircuit of the adult mouse and human cortex. eLife 7, e37349 (2018).
Knöpfel, T. Genetically encoded optical indicators for the analysis of neuronal circuits. Nat. Rev. Neurosci. 13, 687–700 (2012).
Knöpfel, T., Diez-Garcia, J. & Akemann, W. Optical probing of neuronal circuit dynamics: genetically encoded versus classical fluorescent sensors. Trends Neurosci. 29, 160–166 (2006). This study is an early account of the potential of genetically encoded indicators, with arguments that have become common sense over the past few years.
Scanziani, M. & Hausser, M. Electrophysiology in the age of light. Nature 461, 930–939 (2009). This article provides an appraisal of then-emerging optical methods.
Weisenburger, S. et al. Volumetric Ca2+ imaging in the mouse brain using hybrid multiplexed sculpted light microscopy. Cell 177, 1050–1066 (2019).
Grundemann, J. et al. Amygdala ensembles encode behavioral states. Science 364, eaav8736 (2019).
Liang, B. et al. Distinct and dynamic on and off neural ensembles in the prefrontal cortex code social exploration. Neuron 100, 700–714 (2018).
Inoue, M. et al. Rational engineering of xcamps, a multicolor geci suite for in vivo imaging of complex brain circuit dynamics. Cell 177, 1346–1360 (2019).
Akemann, W., Lundby, A., Mutoh, H. & Knöpfel, T. Effect of voltage sensitive fluorescent proteins on neuronal excitability. Biophys. J. 96, 3959–3976 (2009).
Sakai, R., Repunte-Canonigo, V., Raj, C. D. & Knöpfel, T. Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein. Eur. J. Neurosci. 13, 2314–2318 (2001).
Siegel, M. S. & Isacoff, E. Y. A genetically encoded optical probe of membrane voltage. Neuron 19, 735–741 (1997). This early report describes a genetically encoded probe of membrane voltage in which a GFP was attached to the channel-forming domain of a potassium channel; although lack of function in mammalian cells turned out to be a major setback in the development of modern GEVIs, this work is often cited as the invention of the first GEVI.
Dimitrov, D. et al. Engineering and characterization of an enhanced fluorescent protein voltage sensor. PLOS ONE 2, e440 (2007). This study reports the first GEVI that reliably monitored voltage transients in mammalian cells; the described approach set the standard for much of the subsequent work in the field.
Kang, B. E., Lee, S. & Baker, B. J. Optical consequences of a genetically-encoded voltage indicator with a pH sensitive fluorescent protein. Neurosci. Res. 146, 13–21 (2019).
Bando, Y., Sakamoto, M., Kim, S., Ayzenshtat, I. & Yuste, R. Comparative evaluation of genetically encoded voltage indicators. Cell Rep. 26, 802–813 (2019).
Akemann, W., Mutoh, H., Perron, A., Rossier, J. & Knöpfel, T. Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins. Nat. Methods 7, 643–649 (2010).
Daigle, T. L. et al. A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 174, 465–480 (2018).
Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).
Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).
Meng, G. et al. High-throughput synapse-resolving two-photon fluorescence microendoscopy for deep-brain volumetric imaging in vivo. eLife 8, e40805 (2019).
Hillman, E. M. et al. High-speed 3D imaging of cellular activity in the brain using axially-extended beams and light sheets. Curr. Opin. Neurobiol. 50, 190–200 (2018).
Skocek, O. et al. High-speed volumetric imaging of neuronal activity in freely moving rodents. Nat. Methods 15, 429–432 (2018).
Nobauer, T. et al. Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy. Nat. Methods 14, 811–818 (2017).
Tang, Q. et al. In vivo voltage-sensitive dye imaging of subcortical brain function. Sci. Rep. 5, 17325 (2015).
Marshall, J. D. et al. Cell-type-specific optical recording of membrane voltage dynamics in freely moving mice. Cell 167, 1650–1662 (2016).
Chamberland, S. et al. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. eLife 6, e25690 (2017).
Miyazawa, H. et al. Optical interrogation of neuronal circuitry in zebrafish using genetically encoded voltage indicators. Sci. Rep. 8, 6048 (2018).
Aimon, S. et al. Fast near-whole-brain imaging in adult Drosophila during responses to stimuli and behavior. PLOS Biol. 17, e2006732 (2019).
Xu, Y., Zou, P. & Cohen, A. E. Voltage imaging with genetically encoded indicators. Curr. Opin. Chem. Biol. 39, 1–10 (2017).
Lin, M. Z. & Schnitzer, M. J. Genetically encoded indicators of neuronal activity. Nat. Neurosci. 19, 1142–1153 (2016).
Sepehri Rad, M. et al. Voltage and calcium imaging of brain activity. Biophys. J. 113, 2160–2167 (2017).
Song, C., Barnes, S. & Knöpfel, T. Mammalian cortical voltage imaging using genetically encoded voltage indicators: a review honoring professor Amiram Grinvald. Neurophotonics 4, 031214 (2017).
Grinvald, A. & Hildesheim, R. VSDI: a new era in functional imaging of cortical dynamics. Nat. Rev. Neurosci. 5, 874–885 (2004).
Grinvald, A. & Petersen, C. C. Imaging the dynamics of neocortical population activity in behaving and freely moving mammals. Adv. Exp. Med. Biol. 859, 273–296 (2015).
Akemann, W. et al. Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. J. Neurophysiol. 108, 2323–2337 (2012).
Carandini, M. et al. Imaging the awake visual cortex with a genetically encoded voltage indicator. J. Neurosci. 35, 53–63 (2015).
Song, C., Piscopo, D. M., Niell, C. M. & Knöpfel, T. Cortical signatures of wakeful somatosensory processing. Sci. Rep. 8, 11977 (2018).
Adam, Y. et al. Voltage imaging and optogenetics reveal behaviour-dependent changes in hippocampal dynamics. Nature 569, 413–417 (2019).
Piatkevich, K. D. et al. A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat. Chem. Biol. 14, 352–360 (2018).
Song, M., Kang, M., Lee, H., Jeong, Y. & Paik, S. B. Classification of spatiotemporal neural activity patterns in brain imaging data. Sci. Rep. 8, 8231 (2018).
Maatuf, Y., Stern, E. A. & Slovin, H. Abnormal population responses in the somatosensory cortex of Alzheimer’s disease model mice. Sci. Rep. 6, 24560 (2016).
Abdelfattah, A. S. et al. Bright and photostable chemigenetic indicators for extended in vivo voltage imaging. Science 365, 699–704 (2019). This study provides a description of a recent breakthrough in the development of hybrid GEVIs.
Kim, T. H. et al. Long-term optical access to an estimated one million neurons in the live mouse cortex. Cell Rep. 17, 3385–3394 (2016).
Antic, S. D., Empson, R. M. & Knöpfel, T. Voltage imaging to understand connections and functions of neuronal circuits. J. Neurophysiol. 116, 135–152 (2016).
Buzsaki, G., Anastassiou, C. A. & Koch, C. The origin of extracellular fields and currents—EEG, ECoG, LFP and spikes. Nat. Rev. Neurosci. 13, 407–420 (2012).
Shimaoka, D., Harris, K. D. & Carandini, M. Effects of arousal on mouse sensory cortex depend on modality. Cell Rep. 25, 3230 (2018).
Perin, R. & Markram, H. A computer-assisted multi-electrode patch-clamp system. J. Vis. Exp. 18, e50630 (2013).
Zou, P. et al. Bright and fast multicoloured voltage reporters via electrochromic FRET. Nat. Commun. 5, 4625 (2014).
Abdelfattah, A. S. et al. A bright and fast red fluorescent protein voltage indicator that reports neuronal activity in organotypic brain slices. J. Neurosci. 36, 2458–2472 (2016).
Werley, C. A. et al. All-optical electrophysiology for disease modeling and pharmacological characterization of neurons. Curr. Protoc. Pharmacol. 78, 11.20.1–11.20.24 (2017).
Kannan, M. et al. Fast, in vivo voltage imaging using a red fluorescent indicator. Nat. Methods 15, 1108–1116 (2018).
Hochbaum, D. R. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014).
Piatkevich, K. D. et al. Population imaging of neural activity in awake behaving mice. Nature 574, 413–417 (2019).
Stuart, G., Schiller, J. & Sakmann, B. Action potential initiation and propagation in rat neocortical pyramidal neurons. J. Physiol. 505, 617–632 (1997).
Short, S. M. et al. The stochastic nature of action potential backpropagation in apical tuft dendrites. J. Neurophysiol. 118, 1394–1414 (2017).
Pan-Vazquez, A., Wefelmeyer, W., Gonzalez Sabater, V. & Burrone, J. Homeostatic plasticity rules control the wiring of axo-axonic synapses at the axon initial segment. Preprint at https://doi.org/10.1101/453753 (2019).
Antic, S. D., Hines, M. & Lytton, W. W. Embedded ensemble encoding hypothesis: the role of the ‘prepared’ cell. J. Neurosci. Res. 96, 1543–1559 (2018).
Bittner, K. C. et al. Conjunctive input processing drives feature selectivity in hippocampal CA1 neurons. Nat. Neurosci. 18, 1133–1142 (2015).
Gambino, F. et al. Sensory-evoked LTP driven by dendritic plateau potentials in vivo. Nature 515, 116–119 (2014).
Xu, N. L. et al. Nonlinear dendritic integration of sensory and motor input during an active sensing task. Nature 492, 247–251 (2012).
Volgushev, M., Chauvette, S., Mukovski, M. & Timofeev, I. Precise long-range synchronization of activity and silence in neocortical neurons during slow-wave sleep. J. Neurosci. 26, 5665–5672 (2006).
Diekelmann, S. & Born, J. The memory function of sleep. Nat. Rev. Neurosci. 11, 114–126 (2010).
Steriade, M., Timofeev, I. & Grenier, F. Natural waking and sleep states: a view from inside neocortical neurons. J. Neurophysiol. 85, 1969–1985 (2001).
Barttfeld, P. et al. Organization of brain networks governed by long-range connections index autistic traits in the general population. J. Neurodev. Disord. 5, 16 (2013).
Kern, J. K. et al. Shared brain connectivity issues, symptoms, and comorbidities in autism spectrum disorder, attention deficit/hyperactivity disorder, and Tourette syndrome. Brain Connect. 5, 321–335 (2015).
Bassett, D. S., Xia, C. H. & Satterthwaite, T. D. Understanding the emergence of neuropsychiatric disorders with network neuroscience. Biol. Psychiatry Cogn. Neurosci. Neuroimaging 3, 742–753 (2018).
Lundby, A., Akemann, W. & Knöpfel, T. Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP. Eur. Biophys. J. 39, 1625–1635 (2010).
Platisa, J. & Pieribone, V. A. Genetically encoded fluorescent voltage indicators: are we there yet? Curr. Opin. Neurobiol. 50, 146–153 (2018).
Lam, A. J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).
Tsutsui, H., Karasawa, S., Okamura, Y. & Miyawaki, A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat. Methods 5, 683–685 (2008).
Mishina, Y., Mutoh, H., Song, C. & Knöpfel, T. Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain. Front. Mol. Neurosci. 7, 78 (2014).
Sung, U. et al. Developing fast fluorescent protein voltage sensors by optimizing fret interactions. PLOS ONE 10, e0141585 (2015).
Gautam, S. G., Perron, A., Mutoh, H. & Knöpfel, T. Exploration of fluorescent protein voltage probes based on circularly permuted fluorescent proteins. Front. Neuroeng. 2, 14 (2009).
Kost, L. A. et al. Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor. PLOS ONE 12, e0184225 (2017).
Jin, L. et al. Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe. Neuron 75, 779–785 (2012).
Lee, S. et al. Improving a genetically encoded voltage indicator by modifying the cytoplasmic charge composition. Sci. Rep. 7, 8286 (2017).
Perron, A., Mutoh, H., Launey, T. & Knöpfel, T. Red-shifted voltage-sensitive fluorescent proteins. Chem. Biol. 16, 1268–1277 (2009).
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). This article provides a description of a major breakthrough in the development of voltage-sensing domain-based GEVIs for action potential monitoring.
Kralj, J. M., Douglass, A. D., Hochbaum, D. R., Maclaurin, D. & Cohen, A. E. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat. Methods 9, 90–95 (2011).
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).
Gong, Y., Wagner, M. J., Zhong Li, J. & Schnitzer, M. J. Imaging neural spiking in brain tissue using FRET–opsin protein voltage sensors. Nat. Commun. 5, 3674 (2014). This study provides a description of a major breakthrough in the development of opsin FRET GEVIs for action potential monitoring.
Chanda, B. et al. A hybrid approach to measuring electrical activity in genetically specified neurons. Nat. Neurosci. 8, 1619–1626 (2005).
Bayguinov, P. O., Ma, Y., Gao, Y., Zhao, X. & Jackson, M. B. Imaging voltage in genetically defined neuronal subpopulations with a Cre recombinase-targeted hybrid voltage sensor. J. Neurosci. 37, 9305–9319 (2017).
Grenier, V., Daws, B. R., Liu, P. & Miller, E. W. Spying on neuronal membrane potential with genetically targetable voltage indicators. J. Am. Chem. Soc. 141, 1349–1358 (2019).
Song, C., Do, Q. B., Antic, S. D. & Knöpfel, T. Transgenic strategies for sparse but strong expression of genetically encoded voltage and calcium indicators. Int. J. Mol. Sci. 18, E1461 (2017).
Acknowledgements
The authors thank S. Antic for suggestions and a set of figures for an earlier version of this article. Work in our laboratory is supported by grants from the BRAIN initiative (US National Institutes of Health grants U01MH109091 and U01NS099573).
Peer reviewer information
Nature Reviews Neuroscience thanks M. Hoppa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Author information
Authors and Affiliations
Contributions
Both authors contributed equally to all aspects of the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Glossary
- Quenched
-
Submitted to a process that (reversibly) deactivates fluorescence emission.
- 2p cross-section
-
A measure describing how well a fluorescent dye is excited by light of a given intensity; similar to the one-photon absorption extinction coefficient, but two-photon absorption increases with the square of the light intensity.
- Signal-to-noise ratio
-
(SNR). A measure that compares the level of a desired signal (for example, voltage-dependent change in fluorescence) to the level of background noise (in this case, random fluctuations of measured fluorescence). The SNR is defined as the ratio of signal power to noise power.
- Pixel
-
A term standing for ‘picture element’; the light detected by one pixel of the detector may come from anywhere within the corresponding area in the object plane.
- Bessel beam
-
A laser beam with a profile shaped in the form of a Bessel function that can be used to generate an axially elongated excitation volume.
- Light sheet illumination
-
A method in which a thin slice (usually from a few hundred nanometres to a few micrometres) of a sample is illuminated. Compared with conventional epifluorescence microscopy, light sheet illumination produces reduced out-of-focus background fluorescence.
- Light field deconvolution
-
A technique for high-speed volumetric imaging. Using an array of lenses, the object is imaged at different angles, providing 3D information about a sample. The 3D structure is reconstructed by mathematical operations termed deconvolution. This technique allows for imaging in three dimensions simultaneously with a 2D detector.
Rights and permissions
About this article
Cite this article
Knöpfel, T., Song, C. Optical voltage imaging in neurons: moving from technology development to practical tool. Nat Rev Neurosci 20, 719–727 (2019). https://doi.org/10.1038/s41583-019-0231-4
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41583-019-0231-4
This article is cited by
-
Mesoscale volumetric light-field (MesoLF) imaging of neuroactivity across cortical areas at 18 Hz
Nature Methods (2023)
-
110 μm thin endo-microscope for deep-brain in vivo observations of neuronal connectivity, activity and blood flow dynamics
Nature Communications (2023)
-
All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals
Nature Communications (2023)
-
QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins
Nature Communications (2022)
-
Emerging strategies for the genetic dissection of gene functions, cell types, and neural circuits in the mammalian brain
Molecular Psychiatry (2022)