Use of Fast-Responding Voltage-Sensitive Dyes for Large-Scale Recording of Neuronal Spiking Activity with Single-Cell Resolution

  • William N. Frost
  • Jean Wang
  • Christopher J. Brandon
  • Caroline Moore-Kochlacs
  • Terrence J. Sejnowski
  • Evan S. Hill


Optical recording with fast voltage sensitive dyes makes it possible, in suitable preparations, to simultaneously monitor the action potentials of large numbers of individual neurons. Here we describe methods for doing this, including considerations of different dyes and imaging systems, methods for correlating the optical signals with their source neurons, procedures for getting good signals, and the use of Independent Component Analysis for spike-sorting raw optical data into single neuron traces. These combined tools represent a powerful approach for large-scale recording of neural networks with high temporal and spatial resolution.


Independent Component Analysis Independent Component Analysis Optical Recording Spike Sorting Multiple Neuron 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Supported by NS060921, Dart Foundation and Grass Foundation Marine Biological Laboratory summer fellowships, the Fred B. Snite Foundation, Rosalind Franklin University of Medicine and Science (WF), and the Howard Hughes Medical Institute (TS). We thank JY Wu, LB Cohen and L Eliot for comments on the manuscript.


  1. Bell AJ, Sejnowski TJ (1995) An information-maximization approach to blind separation and blind deconvolution. Neural Comput 7:1129–1159.PubMedCrossRefGoogle Scholar
  2. Boyle MB, Cohen LB, Macagno ER, Orbach H (1983) The number and size of neurons in the CNS of gastropod molluscs and their suitability for optical recording of activity. Brain Res 266:305–317.PubMedCrossRefGoogle Scholar
  3. Brown GD, Yamada S, Sejnowski TJ (2001) Independent component analysis at the neural cocktail party. Trends Neurosci 24:54–63.PubMedCrossRefGoogle Scholar
  4. Brown GD, Yamada S, Nakashima M, Moore-Kochlacs C, Sejnowski TJ (2008) Independent component analysis of optical recordings from Tritonia swimming neurons. In: Technical Report INC-08-001, Institute for Neural Computation, University of California at San Diego.Google Scholar
  5. Carlson GC, Coulter DA (2008) In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording. Nat Protoc 3:249–255.PubMedCrossRefGoogle Scholar
  6. Chang PY, Jackson MB (2003) Interpretation and optimization of absorbance and fluorescence signals from voltage-sensitive dyes. J Membr Biol 196:105–116.PubMedCrossRefGoogle Scholar
  7. Cohen L, Hopp HP, Wu JY, Xiao C, London J (1989) Optical measurement of action potential activity in invertebrate ganglia. Annu Rev Physiol 51:527–541.PubMedCrossRefGoogle Scholar
  8. Delorme A, Makeig S (2004) EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods 134:9–21.PubMedCrossRefGoogle Scholar
  9. Ebner TJ, Chen G (1995) Use of voltage-sensitive dyes and optical recordings in the central nervous system. Prog Neurobiol 46:463–506.PubMedCrossRefGoogle Scholar
  10. Falk CX, Wu J, Cohen LB, Tang AC (1993) Nonuniform expression of habituation in the activity of distinct classes of neurons in the Aplysia abdominal ganglion. J Neurosci 13:4072–4081.PubMedGoogle Scholar
  11. Frost WN, Wang J, Brandon CJ (2007) A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity. J Neurosci Methods 162:148–154.PubMedCrossRefGoogle Scholar
  12. Greenberg DS, Houweling AR, Kerr JN (2008) Population imaging of ongoing neuronal activity in the visual cortex of awake rats. Nat Neurosci 11:749–751.PubMedCrossRefGoogle Scholar
  13. Grinvald A, Salzberg BM, Cohen LB (1977) Simultaneous recording from ­several neurones in an invertebrate central nervous system. Nature (Lond) 268:140–142.CrossRefGoogle Scholar
  14. Hickie C, Cohen LB, Balaban PM (1997) The synapse between LE sensory neurons and gill motoneurons makes only a small contribution to the Aplysia gill-withdrawal reflex. Eur J Neurosci 9:627–636.PubMedCrossRefGoogle Scholar
  15. Jin W, Zhang RJ, Wu JY (2002) Voltage-sensitive dye imaging of population neuronal activity in cortical tissue. J Neurosci Methods 115:13–27.PubMedCrossRefGoogle Scholar
  16. Kerr JN, Greenberg D, Helmchen F (2005) Imaging input and output of neocortical networks in vivo. Proc Natl Acad Sci U S A 102:14063–14068.PubMedCrossRefGoogle Scholar
  17. Kojima S, Hosono T, Fujito Y, Ito E (2001) Optical detection of neuromodulatory effects of conditioned taste aversion in the pond snail Lymnaea stagnalis. J Neurobiol 49:118–128.PubMedCrossRefGoogle Scholar
  18. Kosmidis EK, Cohen LB, Falk CX, Wu JY, Baker BJ (2005) Imaging with voltage-sensitive dyes: spike signals, population signals, and retrograde transport. In: Yuste R, Konnerth A (eds) Imaging in neuroscience and development. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  19. London JA, Zecevic D, Cohen LB (1987) Simultaneous optical recording of activity from many neurons during feeding in Navanax. J Neurosci 7:649–661.PubMedGoogle Scholar
  20. Momose-Sato Y, Sato K et al. (1999) Evaluation of voltage-sensitive dyes for long-term recording of neural activity in the hippocampus. J Membr Biol 172:145–157.PubMedCrossRefGoogle Scholar
  21. Nakashima M, Yamada S, Shiono S, Maeda M, Satoh F (1992) 448-Detector optical recording system: development and application to Aplysia gill-withdrawal reflex. IEEE Trans Biomed Eng 39:26–36.PubMedCrossRefGoogle Scholar
  22. Neunlist M, Peters S, Schemann M (1999) Multisite optical recording of excitabi­lity in the enteric nervous system. Neurogastroenterol Motil 11:393–402.PubMedCrossRefGoogle Scholar
  23. Nikitin ES, Balaban PM (2000) Optical recording of odor-evoked responses in the olfactory brain of the naive and aversively trained terrestrial snails. Learn Membr 7:422–432.CrossRefGoogle Scholar
  24. Obaid AL, Koyano T, Lindstrom J, Sakai T, Salzberg BM (1999) Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus. J Neurosci 19:3073–3093.PubMedGoogle Scholar
  25. Obaid AL, Loew LM, Wuskell JP, Salzberg BM (2004) Novel naphthylstyryl-pyridium potentiometric dyes offer advantages for neural network analysis. J Neurosci Methods 134:179–190.PubMedCrossRefGoogle Scholar
  26. Parsons TD, Salzberg BM, Obaid AL, Raccuia-Behling F, Kleinfeld D (1991) Long-term optical recording of patterns of electrical activity in ensembles of cultured Aplysia neurons. J Neurophysiol 66:316–333.PubMedGoogle Scholar
  27. Salzberg BM, Grinvald A, Cohen LB, Davila HV, Ross WN (1977) Optical recording of neuronal activity in an invertebrate central nervous system: simultaneous monitoring of several neurons. J Neurophysiol 40:1281–1291.PubMedGoogle Scholar
  28. Sato TR, Gray NW, Mainen ZF, Svoboda K (2007) The functional microarchitecture of the mouse barrel cortex. PLoS Biol 5:e189.PubMedCrossRefGoogle Scholar
  29. Schemann M, Michel K, Peters S, Bischoff SC, Neunlist M (2002) Cutting-edge technology. III. Imaging and the gastrointestinal tract: mapping the human enteric nervous system. Am J Physiol Gastrointest Liver Physiol 282:G919–G925.PubMedGoogle Scholar
  30. Senseman DM (1996) High-speed optical imaging of afferent flow through rat olfactory bulb slices: voltage-sensitive dye signals reveal periglomerular cell activity. J Neurosci 16:313–324.PubMedGoogle Scholar
  31. Sinha SR, Saggau P (1999) Optical recording from populations of neurons in brain slices. In: Johanson H, Windhorst U (eds) Modern techniques in neuroscience research. Springer Verlag, Berlin.Google Scholar
  32. Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100:7319–7324.PubMedCrossRefGoogle Scholar
  33. Takahashi N, Sasaki T, Usami A, Matsuki N, Ikegaya Y (2007) Watching neuronal circuit dynamics through functional multineuron calcium imaging (fMCI). Neurosci Res 58:219–225.PubMedCrossRefGoogle Scholar
  34. Tsau Y, Wu J, Hopp H, Cohen LB, Schiminovich D, Falk CX. (1994) Distributed aspects of the response to siphon touch in Aplysia: spread of stimulus information and cross-correlation analysis. J Neurosci 14:4167–4184.PubMedGoogle Scholar
  35. Vanden Berghe P, Bisschops R, Tack J (2001) Imaging of neuronal activity in the gut. Curr Opin Pharmacol 1:563–567.PubMedCrossRefGoogle Scholar
  36. Wang Y, Jing G, Perry S, Bartoli F, Tatic-Lucic S (2009) Spectral characterization of the voltage-sensitive dye di-4-ANEPPDHQ applied to probing live primary and immortalized neurons. Opt Express 17:984–990.PubMedCrossRefGoogle Scholar
  37. Wu J, Cohen LB, Falk CX (1994a) Neuronal activity during different behaviors in Aplysia: a distributed organization? Science 263:820–823.PubMedCrossRefGoogle Scholar
  38. Wu J, Tsau Y, Hopp H, Cohen LB, Tang AC, Falk CX (1994b) Consistency in nervous systems: trial-to-trial and animal-to-animal variations in the responses to repeated applications of a sensory stimulus in Aplysia. J Neurosci 14:1366–1384.PubMedGoogle Scholar
  39. Yagodin S, Collin C, Alkon DL, Sheppard NF Jr, Sattelle DB (1999) Mapping membrane potential transients in crayfish (Procambarus clarkii) optic lobe neuropils with voltage-sensitive dyes. J Neurophysiol 81:334–344.PubMedGoogle Scholar
  40. Yang S, Doi T, Asako M, Matsumoto-Ono A, Kaneko T, Yamashita T (2000) Multiple-site optical recording of mouse brainstem evoked by vestibulocochlear nerve stimulation. Brain Res 877:95–100.PubMedCrossRefGoogle Scholar
  41. Yuste R (2008) Circuit neuroscience: the road ahead. Front Neurosci 2:6–9.PubMedCrossRefGoogle Scholar
  42. Zecevic D, Wu J, Cohen LB, London JA, Hopp H, Falk CX (1989) Hundreds of neurons in the Aplysia abdominal gang­lion are active during the gill-withdrawal reflex. J Neurosci 9:3681–3689.PubMedGoogle Scholar
  43. Zochowski M, Cohen LB, Fuhrmann G, Kleinfeld D (2000a) Distributed and partially separate pools of neurons are correlated with two different components of the gill-withdrawal reflex in Aplysia. J Neurosci 20:8485–8492.PubMedGoogle Scholar
  44. Zochowski M, Wachowiak M, Falk CX, Cohen LB, Lam YW, Antic S, Zecevic D (2000b) Imaging membrane potential with voltage-sensitive dyes. Biol Bull 198:1–21.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • William N. Frost
    • 1
  • Jean Wang
    • 1
  • Christopher J. Brandon
    • 1
  • Caroline Moore-Kochlacs
    • 2
  • Terrence J. Sejnowski
    • 2
    • 3
  • Evan S. Hill
    • 1
  1. 1.Department of Cell Biology and Anatomy, The Chicago Medical SchoolRosalind Franklin University of Medicine and ScienceNorth ChicagoUSA
  2. 2.Howard Hughes Medical Institute, The Salk Institute for Biological StudiesLa JollaUSA
  3. 3.Division of Biological SciencesUniversity of California San DiegoLa JollaUSA

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