To understand how the brain relates to behavior, it is essential to record neural activity in awake, behaving animals. To achieve this goal, a large variety of genetically encoded sensors have been developed to monitor and record the series of events following neuronal firing, including action potentials, intracellular calcium rise, neurotransmitter release and immediate early gene expression. In this Review, we discuss the existing genetically encoded tools for detecting and integrating neuronal activity in animals and highlight the remaining challenges and future opportunities for molecular biologists.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Communications Biology Open Access 29 July 2021
Genetically encoded sensors enable micro- and nano-scopic decoding of transmission in healthy and diseased brains
Molecular Psychiatry Open Access 04 December 2020
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
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 is the first optogenetic paper using channelrhodopsin demonstrating robust light-stimulated neuronal activation.
Gradinaru, V., Thompson, K. R. & Deisseroth, K. eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol. 36, 129–139 (2008).
Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl. Acad. Sci. USA 104, 5163–5168 (2007).
Burt, R. S. A note on social capital and network content. Soc. Networks 19, 355–373 (1997).
Ziv, Y. et al. Long-term dynamics of CA1 hippocampal place codes. Nat. Neurosci. 16, 264–266 (2013).
Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M. & Keller, P. J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013). This is the first report of whole-brain functional imaging of larval zebrafish at cellular resolution.
Chhetri, R. K. et al. Whole-animal functional and developmental imaging with isotropic spatial resolution. Nat. Methods 12, 1171–1178 (2015).
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).
Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012).
Kitamura, T. et al. Engrams and circuits crucial for systems consolidation of a memory. Science 356, 73–78 (2017).
Ye, L. et al. Wiring and molecular features of prefrontal ensembles representing distinct experiences. Cell 165, 1776–1788 (2016).
Kim, C. K. et al. Molecular and circuit-dynamical identification of top-down neural mechanisms for restraint of reward seeking. Cell 170, 1013–1027.e14 (2017).
Allen, W. E. et al. Thirst-associated preoptic neurons encode an aversive motivational drive. Science 357, 1149–1155 (2017).
Moser, E. I., Kropff, E. & Moser, M.-B. Place cells, grid cells, and the brain’s spatial representation system. Annu. Rev. Neurosci. 31, 69–89 (2008).
Tovote, P., Fadok, J. P. & Lüthi, A. Neuronal circuits for fear and anxiety. Nat. Rev. Neurosci. 16, 317–331 (2015).
Guzowski, J. F., McNaughton, B. L., Barnes, C. A. & Worley, P. F. Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles. Nat. Neurosci. 2, 1120–1124 (1999).
Helmchen, F., Borst, J. G. & Sakmann, B. Calcium dynamics associated with a single action potential in a CNS presynaptic terminal. Biophys. J. 72, 1458–1471 (1997).
Kuhn, B., Denk, W. & Bruno, R. M. In vivo two-photon voltage-sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulness. Proc. Natl. Acad. Sci. USA 105, 7588–7593 (2008).
Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).
Akemann, W., Song, C., Mutoh, H. & Knöpfel, T. Route to genetically targeted optical electrophysiology: development and applications of voltage-sensitive fluorescent proteins. Neurophotonics 2, 021008 (2015).
St-Pierre, F., Chavarha, M. & Lin, M. Z. Designs and sensing mechanisms of genetically encoded fluorescent voltage indicators. Curr. Opin. Chem. Biol. 27, 31–38 (2015).
Hamel, E. J. O., Grewe, B. F., Parker, J. G. & Schnitzer, M. J. Cellular level brain imaging in behaving mammals: an engineering approach. Neuron 86, 140–159 (2015).
Xu, Y., Zou, P. & Cohen, A. E. Voltage imaging with genetically encoded indicators. Curr. Opin. Chem. Biol. 39, 1–10 (2017).
Akemann, W. et al. Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. J. Neurophysiol. 108, 2323–2337 (2012).
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).
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).
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).
Cao, G. et al. Genetically targeted optical electrophysiology in intact neural circuits. Cell 154, 904–913 (2013).
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).
Yang, H. H. H. et al. Subcellular imaging of voltage and calcium signals reveals neural processing in vivo. Cell 166, 245–257 (2016).
Chamberland, S. et al. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. eLife 6, e25690 (2017).
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).
Hochbaum, D. R. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014). This paper highlights the fastest GEVI reporter so far (QuasAr).
Flytzanis, N. C. et al. Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons. Nat. Commun. 5, 4894 (2014).
Piatkevich, K. D. et al. A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat. Chem. Biol. 14, 352–360 (2018).
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).
Zou, P. et al. Bright and fast multicoloured voltage reporters via electrochromic FRET. Nat. Commun. 5, 4625 (2014).
Marshall, J. D. et al. Cell-type-specific optical recording of membrane voltage dynamics in freely moving mice. Cell 167, 1650–1662.e15 (2016).
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).
Mank, M. & Griesbeck, O. Genetically encoded calcium indicators. Chem. Rev. 108, 1550–1564 (2008).
Broussard, G. J., Liang, R. & Tian, L. Monitoring activity in neural circuits with genetically encoded indicators. Front. Mol. Neurosci. 7, 97 (2014).
Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 101, 10554–10559 (2004).
Palmer, A. E. et al. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem. Biol. 13, 521–530 (2006).
Horikawa, K. et al. Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow cameleon-nano. Nat. Methods 7, 729–732 (2010).
Mank, M. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat. Methods 5, 805–811 (2008).
Thestrup, T. et al. Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat. Methods 11, 175–182 (2014).
Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).
Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).
Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013). This is the first report of the most widely applied GECI, GCaMP6.
Muto, A., Ohkura, M., Abe, G., Nakai, J. & Kawakami, K. Real-time visualization of neuronal activity during perception. Curr. Biol. 23, 307–311 (2013).
Ohkura, M. et al. Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals. PLoS One 7, e51286 (2012).
Zhao, Y. et al. An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011).
Lin, M. Z. & Schnitzer, M. J. Genetically encoded indicators of neuronal activity. Nat. Neurosci. 19, 1142–1153 (2016).
Kim, C. K. et al. Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping. Front. Neural Circuits 8, 138 (2014).
Freeman, J. et al. Mapping brain activity at scale with cluster computing. Nat. Methods 11, 941–950 (2014).
Berlin, S. et al. Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging. Nat. Methods 12, 852–858 (2015).
Dreosti, E., Odermatt, B., Dorostkar, M. M. & Lagnado, L. A genetically encoded reporter of synaptic activity in vivo. Nat. Methods 6, 883–889 (2009).
Broussard, G. J. et al. In vivo measurement of afferent activity with axon-specific calcium imaging. Nat. Neurosci. 21, 1272–1280 (2018).
de Juan-Sanz, J. et al. Axonal endoplasmic reticulum Ca2+ content controls release probability in CNS nerve terminals. Neuron 93, 867–881.e6 (2017).
Kim, C. K., Adhikari, A. & Deisseroth, K. Integration of optogenetics with complementary methodologies in systems neuroscience. Nat. Rev. Neurosci. 18, 222–235 (2017).
Packer, A. M., Russell, L. E., Dalgleish, H. W. P. & Häusser, M. Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo. Nat. Methods 12, 140–146 (2015).
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).
Kim, C. K. et al. Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain. Nat. Methods 13, 325–328 (2016).
Wu, J. et al. Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications. ACS Chem. Neurosci. 4, 963–972 (2013).
Inoue, M. et al. Rational design of a high-affinity, fast, red calcium indicator R-CaMP2. Nat. Methods 12, 64–70 (2015).
Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).
Li, Z. et al. Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin. Proc. Natl.Acad. Sci. USA 102, 6131–6136 (2005).
Li, Y. & Tsien, R. W. pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nat. Neurosci. 15, 1047–1053 (2012).
Wang, H., Jing, M. & Li, Y. Lighting up the brain: genetically encoded fluorescent sensors for imaging neurotransmitters and neuromodulators. Curr. Opin. Neurobiol. 50, 171–178 (2018).
Marvin, J. S. et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat. Methods 10, 162–170 (2013).
Xie, Y. et al. Resolution of high-frequency mesoscale intracortical maps using the genetically encoded glutamate sensor iGluSnFR. J. Neurosci. 36, 1261–1272 (2016).
Hefendehl, J. K. et al. Mapping synaptic glutamate transporter dysfunction in vivo to regions surrounding Aβ plaques by iGluSnFR two-photon imaging. Nat. Commun. 7, 13441 (2016).
MacDonald, R. B., Kashikar, N. D., Lagnado, L. & Harris, W. A. A novel tool to measure extracellular glutamate in the zebrafish nervous system in vivo. Zebrafish 14, 284–286 (2017).
Helassa, N. et al. Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses. Proc. Natl. Acad. Sci. USA 115, 5594–5599 (2018).
Marvin, J. S. et al. Stability, affinity and chromatic variants of the glutamate sensor iGluSnFR. Nat Methods 15, 936–939 (2017).
Jing, M. et al. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nat. Biotechnol. 36, 726–737 (2018).
Sun, F. et al. A genetically encoded fluorescent sensor enables rapid and specific detection of dopamine in flies, fish, and mice. Cell 174, 481–496.e19 (2018).
Patriarchi, T. et al. Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors. Science 360, eaat4422 (2018).
Masharina, A., Reymond, L., Maurel, D., Umezawa, K. & Johnsson, K. A fluorescent sensor for GABA and synthetic GABAB receptor ligands. J. Am. Chem. Soc. 134, 19026–19034 (2012).
Marvin, J.S. et al. A genetically encoded fluorescent sensor for in vivo imaging of GABA. Preprint at https://doi.org/10.1101/322578 (2018).
Zhang, W. H. et al. Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS. Nat. Chem. Biol. 14, 861–869 (2018).
Alivisatos, A. P. et al. Nanotools for neuroscience and brain activity mapping. ACS Nano 7, 1850–1866 (2013).
Ghosh, K. K. et al. Miniaturized integration of a fluorescence microscope. Nat. Methods 8, 871–878 (2011).
Fosque, B. F. et al. Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators. Science 347, 755–760 (2015). This is the first report of a light- and calcium-gated neuronal activity integrator with a fluorescent protein photoconversion (CaMPARI).
Reijmers, L. G., Perkins, B. L., Matsuo, N. & Mayford, M. Localization of a stable neural correlate of associative memory. Science 317, 1230–1233 (2007). This is the first report of drug-gated immediate early gene reporters for recording neuronal activity.
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).
Gore, F. et al. Neural representations of unconditioned stimuli in basolateral amygdala mediate innate and learned responses. Cell 162, 134–145 (2015).
Fujita, N. et al. Visualization of neural activity in insect brains using a conserved immediate early gene, Hr38. Curr. Biol. 23, 2063–2070 (2013).
Masuyama, K., Zhang, Y., Rao, Y. & Wang, J. W. Mapping neural circuits with activity-dependent nuclear import of a transcription factor. J. Neurogenet. 26, 89–102 (2012).
Gao, X. J. et al. A transcriptional reporter of intracellular Ca2+. Drosophila. Nat. Neurosci. 18, 917–925 (2015).
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).
Sørensen, A. T. et al. A robust activity marking system for exploring active neuronal ensembles. eLife 5, 2 (2016).
Sakurai, K. et al. Capturing and manipulating activated neuronal ensembles with CANE delineates a hypothalamic social-fear circuit. Neuron 92, 739–753 (2016).
Garner, A. R. et al. Generation of a synthetic memory trace. Science 335, 1513–1516 (2012).
Ramirez, S. et al. Creating a false memory in the hippocampus. Science 341, 387–391 (2013).
Wang, W. et al. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons. Nat. Biotechnol. 35, 864–871 (2017). This is one of the two first reports of a light- and calcium-gated neuronal activity integrator with a transcriptional readout (FLARE).
Lee, D., Hyun, J. H., Jung, K., Hannan, P. & Kwon, H.-B. A calcium- and light-gated switch to induce gene expression in activated neurons. Nat. Biotechnol. 35, 858–863 (2017). This is one of the two first reports of a light- and calcium-gated neuronal activity integrator with a transcriptional readout (Cal-Light).
Kim, M. W. et al. Time-gated detection of protein-protein interactions with transcriptional readout. eLife 6, e30233 (2017).
Skocek, O. et al. High-speed volumetric imaging of neuronal activity in freely moving rodents. Nat. Methods 11, 31–46 (2018).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Wang, W., Kim, C.K. & Ting, A.Y. Molecular tools for imaging and recording neuronal activity. Nat Chem Biol 15, 101–110 (2019). https://doi.org/10.1038/s41589-018-0207-0
This article is cited by
Application of optogenetics and in vivo imaging approaches for elucidating the neurobiology of addiction
Molecular Psychiatry (2022)
Nature Photonics (2022)
Communications Biology (2021)
Genetically encoded sensors enable micro- and nano-scopic decoding of transmission in healthy and diseased brains
Molecular Psychiatry (2021)