Imaging Nervous System Activity with Voltage‐Sensitive Dyes

Dejan Zecevic1, Maja Djurisic2, Lawrence B. Cohen1, Srdjan Antic1, Matt Wachowiak3, Chun X. Falk3, Michal R. Zochowski1

1 Yale University School of Medicine, New Haven, Connecticut, 2 RedShirtImaging, Fairfield, Connecticut, 3 Warsaw School of Advanced Social Psychology, Warsaw, Poland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.17
DOI:  10.1002/0471142301.ns0617s23
Online Posting Date:  August, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Optical recording with a voltage‐sensitive dye is advantageous where membrane potential must be recorded in many sites at once. This unit describes methods for making voltage‐sensitive dye measurements on different preparations to study (1) how a neuron integrates its synaptic input into its action potential output by measuring membrane potential everywhere synaptic input occurs and where spikes are initiated; (2) how a nervous system generates a behavior in Aplysia abdominal ganglion; and (3) responses to sensory stimuli and generation of motor output in the vertebrate brain by simultaneous measurement of population signals from many areas. The approach is three‐pronged: (1) find the dye with the largest signal‐to‐noise ratio; (2) reduce extraneous sources of noise; and (3) maximize the number of photons measured to reduce the relative shot noise. A discussion of optical recording methods including the choice of dyes, light sources, optics, cameras, and minimizing noise is also provided.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Intracellular Injection of Voltage‐Sensitive Dyes into Neurons in Invertebrate Ganglia
  • Alternate Protocol 1: Intracellular Injection of Voltage‐Sensitive Dyes into Neurons in Mammalian Olfactory Bulb Slice Preparations
  • Basic Protocol 2: Bath Application of Voltage‐Sensitive Dyes for Recording Action Potentials from Individual Cell Bodies in Invertebrate and Vertebrate Ganglia: The Aplysia Abdominal Ganglion
  • Basic Protocol 3: Application of Voltage‐Sensitive Dyes for Recording Population Signals Using in Vivo Vertebrate Preparations: The Turtle Olfactory Bulb
  • Reagents and Solutions
  • Commentary
  • Acknowledgments
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Intracellular Injection of Voltage‐Sensitive Dyes into Neurons in Invertebrate Ganglia

  Materials
  • Helix aspersa terrestrial snails (Sea Life Supply)
  • recipeHelix saline (see recipe)
  • 0.5% (w/v) Type IX trypsin (Sigma) in recipeHelix saline : store up to 2 weeks at −20°C (i.e., frozen) in 0.5‐ml aliquots
  • 0.5% (w/v) Type II‐S trypsin inhibitor (Sigma) in recipeHelix saline : store up to 2 weeks at 4°C in 0.5‐ml aliquots
  • recipeDye solution (see recipe)
  • Dissecting instruments: fine scissors and no. 5 forceps
  • Sylgard‐coated chamber
  • Dissecting microscope (e.g., Olympus SZ 6045)
  • recipeSharp microelectrode (see recipe)
  • Micromanipulator
  • Microelectrode amplifier (e.g., Model ‐ 505A; Warner Instruments)
  • Picospritzer (General Valve)
  • Optical recording device (Fig. ):
  •  Excitation‐interference filter
  •  Dichroic mirror
  •  Barrier filter (RG610; Schott)
  •  464‐element photodiode array data acquisition system (i.e., NeuroPlex; RedShirtImaging) including low‐pass (4‐pole Bessel) filter and RC filter with a cutoff frequency of 1.7 Hz
  •  Computerized data acquisition system (e.g., Model DAP 3200e/214, Microstar Laboratories)
  •  High‐resolution CCD camera with controller (CCD‐300‐RC; Dage‐MTI)
  •  FlashBus frame grabber (Integral Technologies)
  •  NeuroCCD‐SM camera system (RedShirtImaging)
  •  Upright compound microscope (Model E600FN; Nikon)
  •  Vibration‐isolation table (Model 300‐SP‐1; Minus‐k Technology)
  •  250‐W xenon arc‐lamp (Model 770× W/T; Opti Quip, Highland Mills)
  •  Microelectrode amplifier (e.g., Model ‐ 505A; Warner Instruments)
  •  Axoclamp 200B amplifier
  •  Stimulator with stimulus isolation unit (Model A310; WPI)
  •  Oscilloscope, (Model TEK5103N/02/D11; Tektronix)
  •  Micromanipulator for patch‐electrodes (Model MP‐285; Sutter Instrument)
  •  Micromanipulator for stimulating metal electrodes (Model NMN‐21; Narishige)
  • Additional reagents and equipment for backfilling microelectrodes (unit 6.3)

Alternate Protocol 1: Intracellular Injection of Voltage‐Sensitive Dyes into Neurons in Mammalian Olfactory Bulb Slice Preparations

  • 18‐ to 25‐day‐old Wistar rats
  • Halothane
  • recipeVertebrate extracellular solution (see recipe)
  • recipeIntracellular solution (see recipe)
  • Rodent guillotine
  • 5‐ to 7‐MΩ patch pipets (unit 6.3)
  • Infrared differential interference contrast (IR‐DIC) video microscope
  • Additional reagents and equipment for anesthetizing rodents ( appendix 4B), vibratome sectioning (unit 1.1), back‐filling microelectrodes (unit 6.3), and patch‐clamping (unit 6.6)
NOTE: All protocols using live vertebrates must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Basic Protocol 2: Bath Application of Voltage‐Sensitive Dyes for Recording Action Potentials from Individual Cell Bodies in Invertebrate and Vertebrate Ganglia: The Aplysia Abdominal Ganglion

  Materials
  • 5‐ to 20‐g Aplysiacalifornica (Marinus or University of Miami Aplysia Resource Facility)
  • Isotonic (345 mM) MgCl 2
  • 50:50 recipeartificial sea water (see recipe)/isotonic MgCl 2
  • 0.15 mg/ml RH 155 dye (Nippon Kankoh‐Shikiso Kenkyusho) in recipeartificial sea water
  • Artificial sea water with and without low calcium and high magnesium (see reciperecipes)
  • Dissection equipment: fine scissors, Dumont no. 5 forceps, pins
  • Lucite chamber with separate compartments
  • Petroleum jelly (e.g., Vaseline)
  • Computer‐controlled galvanometer motor
  • 0.6‐mm‐diameter glass rod with rounded tip
  • 25× 0.4‐NA microscope objective
  • 464‐element photodiode array camera system (i.e., NeuroPlex; RedShirtImaging)
  • Videotape recorder
  • Threshold discriminator
  • Additional reagents and equipment for dissection of Aplysia siphon, abdominal ganglion, and gill (Kupfermann, )
NOTE: Contact Dr. L. Cohen at Yale University ( ) for more information concerning construction of the apparatus.

Basic Protocol 3: Application of Voltage‐Sensitive Dyes for Recording Population Signals Using in Vivo Vertebrate Preparations: The Turtle Olfactory Bulb

  Materials
  • Terepene carolina and ornata turtles (Charles D. Sullivan Co.)
  • 1% (w/v) lidocaine (Sigma) in turtle saline
  • Tubocurarine (Sigma)
  • Cyanoacrylate glue (e.g., Krazy Glue)
  • Epoxy
  • 0.01 to 0.2 mg/ml RH 414 (Molecular Probes; Fig. D) in turtle saline
  • recipeTurtle saline (see recipe)
  • Odorant (e.g., cineole)
  • Dremel tool with small round bit
  • Dumont no. 5 forceps
  • Polyethylene tubing with outer diameter of 2 mm and an inner diameter of 1 mm
  • Flexible plastic
  • Tape
  • Olfactometer (Kauer and Moulton, ; Fig. A)
  • CO 2 detector (e.g., Medical Gas Analyzer LB‐2; Beckman)
  • Optical measuring and recording device (Fig. C):
  •  4× macroscope (RedShirtImaging) with 25‐mm focal length, 0.95‐f‐stop, C‐mount camera lens
  •  100‐W tungsten‐halogen lamp (Osram)
  •  520 ± 45‐nm excitation filter
  •  580‐nm long‐pass dichroic mirror (Omega Optical)
  •  464‐element photodiode array (NeuroPlex; RedShirtImaging)
  •  RG610 long‐pass filter (Schott Glass Technologies)
  •  CCD camera (e.g., RC300; DAGE‐MTI)
NOTE: All protocols using vertebrates must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Adrian, E.D. 1942. Olfactory reactions in the brain of the hedgehog. J. Physiol. 100:459‐473.
   Antic, S. and Zecevic, D. 1995. Optical signals from neurons with internally applied voltage‐sensitive dyes. J. Neuroscience 15:1392‐1405.
   Antic, S., Major, G., and Zecevic, D. 1999. Fast optical recording of membrane potential changes from dendrites of pyramidal neurons. J. Neurophysiol. 82:1615‐1621.
   Antic, S., Wuskell, J.P., Loew, L., and Zecevic, D. 2000. Functional profile of the giant metacerebral neuron of Helix aspersa: Temporal and spatial dynamics of electrical activity in situ. J. Physio. 527:55‐69.
   Blasdel, G.G. and Salama, G. 1986. Voltage‐sensitive dyes reveal a modular organization in monkey striate cortex. Nature 321:579‐585.
   Braddick, H.J.J. 1960. Photoelectric photometry. Rep. Prog. Physics 23:154‐175.
   Chen, W.R., Midtgaard, J., and Shepherd, G.M. 1997. Forward and backward propagation of dendritic impulses and their synaptic control in mitral cells. Science 278:463‐467.
   Cohen, L.B. and Lesher, S. 1986. Optical monitoring of membrane potential: Methods of multisite optical measurement. Soc. Gen. Physiol. Ser. 40:71‐99.
   Dainty, J.C. 1984. Laser speckle and related phenomena. Springer‐Verlag, New York.
   Denk, W., Piston, D.W., and Webb, W. 1995. Two‐photon molecular excitation in laser‐scanning microscopy. In Handbook of Biological Confocal Microscopy. (J.W. Pawley, ed.) pp.445‐458. Plenum Press, New York.
   Fromherz, P., Dambacher, K.H., Ephardt, H., Lambacher, A., Muller, C.O., Neigl, R., Schaden, H., Schenk, O., and Vetter, T. 1991. Fluorescent dyes as probes of voltage transients in neuron membranes: Progress report. Ber. Bunsenges. Phys. Chem. 95:1333‐1345.
   Gonzalez, J.E. and Tsien, R.Y. 1995. Voltage sensing by fluorescence energy transfer in single cells. Biophys. J. 69:1272‐1280.
   Grinvald, A., Hildesheim, R., Farber, I.C., and Anglister, L. 1982. Improved fluorescent probes for the measurement of rapid changes in membrane potential. Biophys. J. 39:301‐308.
   Grinvald, A., Salzberg, B.M., Lev‐Ram, V., and Hildesheim, R. 1987. Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes. Biophys. J. 51:643‐651.
   Grinvald, A., Lieke, E.E., Frostig, R.D., and Hildesheim, R. 1994. Cortical point‐spread function and long‐range lateral interactions revealed by real‐time optical imaging of Macaque monkey primary visual cortex. J. Neurosci. 18:9977‐9988.
   Gross, E., Bedlack, R.S. Jr, and Loew, L.M. 1994. Dual‐wavelength ratiometric fluorescence measurement of the membrane dipole potential. Biophys J. 67:208‐216.
   Gupta, R.K., Salzberg, B.M., Grinvald, A., Cohen, L.B., Kamino, K., Lesher, S., Boyle, M.B., Waggoner, A.S., and Wang, C.H. 1981. Improvements in optical methods for measuring rapid changes in membrane potential. J. Memb. Biol. 58:123‐137.
   Hamer, F.M. 1964. The Cyanine Dyes and Related Compounds. John Wiley & Sons, New York.
   Hirota, A., Sato, K., Momose‐Sato, Y., Sakai, T., and Kamino, K. 1995. A new simultaneous 1020‐site optical recording system for monitoring neural activity using voltage‐sensitive dyes. J. Neurosci. Methods 56:187‐194.
   Iijima, T., Ichikawa, M., and Matsumoto, G. 1989. Optical monitoring of LTP and related phenomena. Abstracts Soc. for Neuroscience 15:398.
   Inoue, S. 1986. Video Microscopy, p.128. Plenum Press, New York.
   Kauer, J.S. and Moulton, D.G. 1974. Responses of olfactory bulb neurones to odor stimulation of small nasal areas in the salamander. J. Physiol. 243:717‐737.
   Kerkut, G.A. and Ridge, R.M.A. 1962. The effect of temperature changes on the activity of the neurons of the snail Helix aspersa. Com. Biochem. Physiol. 5:283‐296.
   Kleinfeld, D. and Delaney, K. 1996. Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage‐sensitive dyes. J. Comp. Neurol. 375:89‐109.
   Kupfermann, I., Pinsker, H., Castellucci, V., and Kandel, E.R. 1971. Central and peripheral control of gill movements in Aplysia. Science 174:1252‐1256.
   Lam, Y.‐W., Cohen, L.B., Wachowiak, M., and Zochowski, M.R. 2000. Odors elicit three different oscillations in the turtle olfactory bulb. J. Neurosci. 20:749‐762.
   Loew, L.M., Cohen, L.B., Dix, J., Fluhler, E.N., Montana, V., Salama, G., and Wu, J.Y. 1992. A napthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations. J. Memb. Biol. 130:1‐10.
   London, J.A., Zecevic, D., and Cohen, L.B. 1987. Simultaneous optical recording of activity from many neurons during feeding in Navanax. J. Neurosci. 7:649‐661.
   Mainen, Z.F., Carnevalle, N.T., Zador, A.M., Claiborne, B.J., and Brown, T.H. 1996. Electronic architecture of hippocampal CA1 pyramidal neurons based on three‐dimensional reconstructions. J. Neurophysiol. 76:1904‐1923.
   Momose‐Sato, Y., Sato, K., Sakai, T., Hirota, A., Matsutani, K., and Kamino, K. 1995. Evaluation of optimal voltage‐sensitive dyes for optical measurement of embryonic neural activity. J. Memb. Biology 144:167‐176.
   Nakashima, M., Yamada, S., Shiono, S., Maeda, M., and Sato, F. 1992. 448‐detector optical recording system: development and application to Aplysia gill‐withdrawal reflex. IEEE Trans. Biomed. Eng. 39:26‐36.
   Nirenberg, S. and Cepko, C. 1993. Targeted ablation of diverse cell classes in the nervous system in vivo. J. Neurosci. 13:3238‐3251.
   Orbach, H.S. and Cohen, L.B. 1983. Optical monitoring of activity from many areas of the in vitro and in vivo salamander olfactory bulb: A new method for studying functional organization in the vertebrate central nervous system. J. Neurosci. 3:2251‐2262.
   Orbach, H.S., Cohen, L.B., and Grinvald, A. 1985. Optical mapping of electrical activity in rat somatosensory and visual cortex. J. Neurosci. 5:1886‐1895.
   Petran, M. and Hadravsky, M. 1996. Czechoslovakian patent 7720.
   Ratzlaff, E.H. and Grinvald, A. 1991. A tandem‐lens epifluorescence microscope: Hundred‐fold brightness advantage for wide‐field imaging. J. Neurosci. Methods 36:127‐137.
   Rohr, S. and Salzberg, B.M. 1994. Multiple site optical recording of transmembrane voltage in patterned growth heart cell cultures: Assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale. Biophys. J. 67:1301‐1315.
   Salama, G. 1988. Voltage‐sensitive dyes and imaging techniques reveal new patterns of electrical activity in heart and cortex. SPIE Proceedings 94:75‐86.
   Salzberg, B.M., Grinvald, A., Cohen, L.B. Davila, H.V., and Ross, W.N. 1977. Optical recording of neuronal activity in an invertebrate central nervous system: Simultaneous monitoring of several neurons. J. Neurophysiol. 40:1281‐1291.
   Shoham, D., Glaser, D.E., Arieli, A., Kenet, T., Wijnbergen, C., Toledo, Y., Hildesheim, R., and Grinvald, A. 1999. Imaging cortical dynamics at high spatial and temporal resolution with novel blue voltage‐sensitive dyes. Neuron 24:791‐802.
   Siegel, M.S. and Isacoff, E.Y. 1997. A genetically encoded optical probe of membrane voltage. Neuron 19:735‐741.
   Tsau, Y., Wenner, P., O'Donovan, M.J., Cohen, L.B., Loew, L.M., and Wuskell, J.P. 1996. Dye screening and signal‐to‐noise ratio for retrogradely transported voltage‐sensitive dyes. J. Neurosci. Methods 70:121‐129.
   Waggoner, A.S. and Grinvald, A. 1977. Mechanisms of rapid optical changes of potential sensitive dyes. Annu. N.Y. Acad. Sci. 303:217‐241.
   Wu, J.Y. and Cohen, L.B. 1993. Fast multisite optical measurements of membrane potential. In Fluorescent and Luminescent Probes for Biological Activity. (W.T. Mason, ed.) Academic Press, London.
   Wu, J‐Y., Lam, Y‐W., Falk, C.X, Cohen, L.B., Fang, J., Loew, L., Prechtl, J.C., Kleinfeld, D., and Tsau, Y. 1998. Voltage‐sensitive dyes for monitoring multineuronal activity in the intact central nervous system. Histochem. J. 30:169‐187.
   Zecevic, D. 1996. Multiple spike‐initiation zones in single neurons revealed by voltage‐sensitive dyes. Nature 381:322‐325.
   Zecevic, D., Wu, J.Y., Cohen, L.B., London, J.A., Hopp, H.P., and Falk, C.X. 1989. Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill‐withdrawal reflex. J. Neurosci. 9:3681‐3689.
   Zochowski, M., Wachowiak, D.M., Falk, C. X., Cohen, L. B., Lam, Y.‐W., Antic, S., and Zecevic, D. 2000. Imaging membrane potential with voltage‐sensitive dyes. Biol. Bull. 198:1‐21.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library