Functional Imaging of Nervous Systems Using Voltage‐Sensitive Dyes

Abstract

Voltage‐sensitive dyes are small molecules that bind to cell membranes and change their optical properties, absorption or fluorescence, in response to changes in membrane potential, the voltage across the membrane. These optical changes are extremely rapid and tend to be linear reporters of local electrical events. Thus, these ‘potentiometric probes’ behave as molecular voltmeters and can be used, with a high‐speed camera or other image dissector, for functional imaging of electrical activity including action potentials, synaptic potentials, and passive (electrotonic) signals. No physical contact with the object is required and spatial and temporal resolution is limited primarily by signal‐to‐noise considerations. Such millisecond time‐resolved functional imaging can be of single cells, of regions of cells or of nervous systems, of cardiac muscle or of any other electrically active tissue. In the nervous system, functional imaging of electrical activity can lead to an understanding of the spatio‐temporal relationships between neurons and of their functional connectivity.

Key Concepts:

  • In electrically excitable cells, the membrane potential can change extremely rapidly.

  • Some coloured molecules (dyes) change their optical properties in response to changes in the local electric field.

  • Changes in the optical properties of the molecules that stain‐cell membranes can be used to follow rapid changes in membrane potential.

  • A high‐speed camera, usually with a microscope, can be used together with these dyes to monitor the electrical activity at hundreds or even thousands of sites, virtually simultaneously.

  • Functional imaging of electrical activity can lead to an understanding of the spatio‐temporal relationships in nervous systems, and to the functional connectivity among neurons.

Keywords: imaging; fluorescence; voltage‐sensitive dye; membrane potential; action potential

Figure 1.

First optical recording of an action potential from an individual neuron. (a) ΔF/F recorded in a single sweep from a leech (Hirudo medicinalis) nociceptive neuron that had been stained with the first practical VSD, Merocyanine 540 (Salzberg et al., ; Davila et al., ). (b) Simultaneous microelectrode recording of the membrane potential change in this neuron. The response time constant of the light measuring system was 2.2 ms. The optical recording system sampled at 2 kHz and this accounts for the jagged character of the records. Modified from Salzberg et al.. Reproduced with permission from BM Salzberg.

Figure 2.

Linearity and temporal fidelity of a fast VSD. (a) Linearity of the extrinsic optical change exhibited by a (nonfluorescent) absorption VSD with voltage‐clamp steps in the absence of all nonlinear membrane currents. The fractional change in transmitted light intensity through a squid (Loligo pealeii) giant axon is plotted as a function of the size of the applied voltage step from a holding potential of −70 mV. The axon had been stained for 10 min in artificial seawater containing 10 μg ml−1 of the VSD WW 375 (Ross et al., ). Tetrodotoxin (300 nM) and 3,4‐diaminopyridine (4 mM) were used to block most of the sodium and potassium currents, respectively. The potential steps were 5 ms in duration. The response time constant of the light measuring system was 17.5 μs. λ=722.4±20 nm. (b) Microsecond optical response to a fast hyperpolarizing voltage clamp step applied to a squid (L. pealeii) giant axon stained with the merocyanine–oxazolone VSD natural killer (NK) 2367 (Salzberg et al., ). The top trace shows the change in transmitted light intensity at 722.4±21.5 nm in response to the 250 μs duration 100‐mV step, from a holding potential of −40 mV, shown in the middle trace. The bottom trace shows the leak and capacitance currents during the voltage clamp step. The VSD was applied at a concentration of 500 μg mL−1 for 20 min. The artificial seawater here contained 500 nM tetrodotoxin and 1 mM 3,4‐diaminopyridine. The optical signal is the sum of 2000 sweeps, and the response time constant of the light measuring system was 250 ns. As is evident, the optical response at room temperature (21°C) was essentially instantaneous (<2 μs delay). For (a) Copyright Salzberg and Bezanilla, 1983. Originally published in The Journal of General Physiology. 82(6): 807–817. Reproduced with permission from BM Salzberg. (b) Modified from Salzberg et al.. Reproduced with permission from The Japanese Journal of Physiology.

Figure 3.

Raster diagram illustrating optical recording of action potentials from many (83) individual neurons in the buccal ganglion of Aplysia californica. The time of occurrence of each spike is indicated by a tick. The ganglion was stained for 10 min with 1–4 mg ml−1 of the merocyanine rhodanine dye JPW 1124 (equivalent to Dye XXIII of (Gupta et al., )), dissolved in Aplysia L‐15 medium. Modified from Morton et al.. Reproduced with permission from Elsevier.

Figure 4.

High‐speed camera VSDFI from a guinea pig submucosal ganglion stained with the naphthylstyryl pyridinium dye di‐3‐ANEPPDHQ (JPW 3031). (a) A NeuroCCD‐SM (RedShirtImaging, LLC) image (inverted grey‐scale). Here, the relatively high spatial resolution of the camera reduced the ambiguity in the assignment of groups of pixels to individual neurons. The colours indicate the pixels binned for each neuron soma. (b) Pixel map of the camera. The coloured pixels identify all of the eleven somata in the selected ganglion. (c) Spatially averaged optical outputs from all the neurons identified in B. Trains of eight stimuli at 10 Hz, acquired in single sweeps 12 min apart. The illumination was reduced 32‐fold by insertion of a neutral density filter in the light path, and was limited to 1.8 s per trial. (d) Higher‐gain display of the first response from neuron 2. Modified from Obaid et al.. Reproduced with permission from Elsevier.

Figure 5.

Chronic VSDFI in behaving monkeys. (a) A large area of exposed monkey cortex in perfect condition after nearly a years worth of repeated VSDFI. (b) VSDFI maps of ocular dominance (left and right) and orientation (centre), recorded from the same patch of cortex in optical recording sessions separated by up to one year were virtually identical (compare top and bottom rows of images) in this individual monkey. (c) Dynamics of retinotopic activation. Top: retinotopic activation of areas V1 and V2 (width of cortical area about 14 mm). Bottom: The spread of cortical dynamics along horizontal (upper) and vertical (lower) axes as a function of time (ms) – marked by different colours. (d) Single‐trial evoked responses in the awake monkey. As in the anaesthetized animal, the response was somewhat variable. Modified from Grinvald and Hildesheim , modified, with permission, from Slovin et al.. Reprinted by permission from Macmillan Publishers Ltd.

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References

Antic S and Zecevic D (1995) Optical signals from neurons with internally applied voltage‐sensitive dyes. Journal of Neuroscience 15(2): 1392–1405.

Ataka K and Pieribone VA (2002) A genetically targetable fluorescent probe of channel gating with rapid kinetics. Biophysical Journal 82(1 Pt 1): 509–516.

Baker BJ, Lee H, Pieribone VA et al. (2007) Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells. Journal of Neuroscience Methods 161(1): 32–38.

Braddick HJJ (1960) Photoelectric Phytomety. Reports on Progress in Physics 23: 154.

Bullen A and Saggau P (1998) Indicators and optical configuration for simultaneous high‐resolution recording of membrane potential and intracellular calcium using laser scanning microscopy. Pflugers Archiv: European Journal of Physiology 436(5): 788–796.

Cohen LB and Salzberg BM (1978) Optical measurement of membrane potential. Reviews of Physiology, Biochemistry and Pharmacology 83: 35–88.

Cohen LB, Salzberg BM, Davila HV et al. (1974) Changes in axon fluorescence during activity: molecular probes of membrane potential. Journal of Membrane Biology 19(1): 1–36.

Cohen LB, Salzberg BM and Grinrald A (1978) Optical methods for monitoring neuron activity. Annual Review of Neuroscience 1: 171–182.

Cohen LB, Keynes RD and Hille B (1968) Light scattering and birefringence changes during nerve activity. Nature 218(5140): 438–441.

Dainty JC (1984) Laser Speckle and Related Phenomena. New York: Springer‐Verlag.

Davila HV, Cohen LB, Salzberg BM et al. (1974) Changes in ANS and TNS fluorescence in giant axons from Loligo. Journal of Membrane Biology 15(1): 29–46.

Davila HV, Salzberg BM, Cohen LB et al. (1973) A large change in axon fluorescence that provides a promising method for measuring membrane potential. Nature: New Biology 241(109): 159–160.

Dillon S and Morad M (1981) A new laser scanning system for measuring action potential propagation in the heart. Science 214(4519): 453–456.

Dimitrov D, He Y, Mutoh H et al. (2007) Engineering and characterization of an enhanced fluorescent protein voltage sensor. PLoS One 2(5): e440.

Efimov IR, Nikolski VP and Salama G (2004) Optical imaging of the heart. Circulation Research 95(1): 21–33.

Fisher JA, Barchi JR, Welle CG et al. (2008) Two‐photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ. Journal of Neurophysiology 99(3): 1545–1553.

Fluhler E, Burnham VG and Loew LM (1985) Spectra, membrane binding, and potentiometric responses of new charge shift probes. Biochemistry 24(21): 5749–5755.

Furness JB and Costa M (1987) The Enteric Nervous System. New York: Churchill Livingstone.

Griesbeck O, Baird GS, Campbell RE et al. (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. Journal of Biological Chemistry 276(31): 29188–29194.

Grinvald A (1985) Real‐time optical mapping of neuronal activity: from single growth cones to the intact mammalian brain. Annual Review of Neuroscience 8: 263–305.

Grinvald A, Anglister L, Freeman JA et al. (1984) Real‐time optical imaging of naturally evoked electrical activity in intact frog brain. Nature 308(5962): 848–850.

Grinvald A, Cohen LB, Lesher S et al. (1981) Simultaneous optical monitoring of activity of many neurons in invertebrate ganglia using a 124‐element photodiode array. Journal of Neurophysiology 45(5): 829–840.

Grinvald A and Hildesheim R (2004) VSDI: a new era in functional imaging of cortical dynamics. Nature Reviews Neuroscience 5(11): 874–885.

Grinvald A, Salzberg BM and Lev‐Ram V (1987) Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes. Biophysical Journal 51(4): 643–651.

Gupta RK, Salzberg BM, Grinvald A et al. (1981) Improvements in optical methods for measuring rapid changes in membrane potential. Journal of Membrane Biology 58(2): 123–137.

Hamer FM (1964) The Cyanine Dyes and Related Compounds. New York: John Wiley and Sons, Inc.

Horowitz P and Hill W (1980) The Art of Electronics. Cambridge: Cambridge University Press.

Ichikawa I, Iijima T and Matsumoto G (1993) Real‐time optical recording of neural activities in the brain. In: Ono T, Squire LR, Raichle ME, Perrett DI, Fukuda M (eds) Brain mechanisms of perception and memory, pp. 638–648. New York: Oxford University Press.

Junge W and Witt HT (1968) On the ion transport system of photosynthesis‐‐investigations on a molecular level. Zeitschrift für Naturforschung. Teil B 23(2): 244–254.

Loew LM, Cohen LB, Salzberg BM et al. (1985) Charge‐shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon. Biophysical Journal 47(1): 71–77.

Matiukas A, Mitrea BG and Qin M (2007) Near‐infrared voltage‐sensitive fluorescent dyes optimized for optical mapping in blood‐perfused myocardium. Heart Rhythm 4(11): 1441–1451.

Milojkovic BA, Wuskell JP, Loew LM et al. (2005) Initiation of sodium spikelets in basal dendrites of neocortical pyramidal neurons. Journal of Membrane Biology 208(2): 155–169.

Morton DW, Chiel HJ, Cohen LB et al. (1991) Optical methods can be utilized to map the location and activity of putative motor neurons and interneurons during rhythmic patterns of activity in the buccal ganglion of Aplysia. Brain Research 564(1): 45–55.

Murata Y, Iwasaki H, Sasaki M et al. (2005) Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature 435(7046): 1239–1243.

Obaid AL, Koyano T, Lindstrom J et al. (1999) Spatiotemporal patterns of activity in an intact mammalian network with single‐cell resolution: optical studies of nicotinic activity in an enteric plexus. Journal of Neuroscience 19(8): 3073–3093.

Obaid AL, Loew LM, Wuskell JP et al. (2004) Novel naphthylstyryl‐pyridinium potentiometric dyes offer advantages for neural network analysis. Journal of Neuroscience Methods 134(2): 179–190.

Obaid AL and Salzberg BM (1996) Micromolar 4‐aminopyridine enhances invasion of a vertebrate neurosecretory terminal arborization: optical recording of action potential propagation using an ultrafast photodiode‐MOSFET camera and a photodiode array. Journal of General Physiology 107(3): 353–368.

Obaid AL, Zou D‐J, Rohr S et al. (1992) Optical recording with single cell resolution from a simple mammalian nervous system: electrical activity in ganglia from the submucous plexus of the guinea pig ileum. Biological Bulletin 183: 344–346.

Orbach HS and Cohen LB (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. Journal of Neuroscience 3(11): 2251–2262.

Orbach HS, Cohen LB and Grinvald A (1985) Optical mapping of electrical activity in rat somatosensory and visual cortex. Journal of Neuroscience 5(7): 1886–1895.

Petersen CC, Grinvald A and Sakmann B (2003) Spatiotemporal dynamics of sensory responses in layer 2/3 of rat barrel cortex measured in vivo by voltage‐sensitive dye imaging combined with whole‐cell voltage recordings and neuron reconstructions. Journal of Neuroscience 23(4): 1298–1309.

Pooler J (1972) Photodynamic alteration of sodium currents in lobster axons. Journal of General Physiology 60(4): 367–387.

Rizzo MA, Springer GH, Granada B et al. (2004) An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnology 22(4): 445–449.

Rohr S and Salzberg BM (1994) Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale. Biophysical Journal 67(3): 1301–1315.

Ross WN, Salzberg BM, Cohen LB et al. (1977) Changes in absorption, fluorescence, dichroism, and birefringence in stained giant axons: optical measurement of membrane potential. Journal of Membrane Biology 33(1‐2): 141–183.

Rumyantsev SL, Shur MS, Kosterin PV, Bilenko Yu and Salzberg BM (2004) Low frequency noise and long‐term stability of non‐coherent light sources. Journal of Applied Physics 96: 966–969.

Sacconi L, Dombeck DA and Webb WW (2006) Overcoming photodamage in second‐harmonic generation microscopy: real‐time optical recording of neuronal action potentials. Proceedings of the National Academy of Sciences of the USA 103(9): 3124–3129.

Sakai R, Repunte‐Canonigo V and Raj CD (2001) Design and characterization of a DNA‐encoded, voltage‐sensitive fluorescent protein. European Journal of Neuroscience 13(12): 2314–2318.

Salama G, Choi BR, Azour G et al. (2005) Properties of new, long‐wavelength, voltage‐sensitive dyes in the heart. Journal of Membrane Biology 208(2): 125–140.

Salzberg BM (1983) Optical recording of electrical activity in neurons using molecular probes. In: Barker J and McKelvy J (eds) Current Methods in Cellular Neurobiology, pp. 139–187. New York: Wiley.

Salzberg BM and Bezanilla F (1983) An optical determination of the series resistance in Loligo. Journal of General Physiology 82(6): 807–817.

Salzberg BM, Cohen LB, Grinvald A et al. (1978) Potentiometric probes for simultaneous optical recordings from multiple sites in neural networks. In: Dutton PL, Leigh JS and Scarpa A (eds) Frontiers of Biological Energetics, Vol. 2: Electrons to Tissues, p. 1313. New York: Academic Press.

Salzberg BM, Davila HV, Cohen LB et al. (1972) A large change in axon fluorescence, potentially useful in the study of simple nervous systems. Biological Bulletin of the Marine Biological Laboratory 143: 475.

Salzberg BM, Davila HV and Cohen LB (1973) Optical recording of impulses in individual neurones of an invertebrate central nervous system. Nature 246(5434): 508–509.

Salzberg BM, Grinvald A, Cohen LB et al. (1977) Optical recording of neuronal activity in an invertebrate central nervous system: simultaneous monitoring of several neurons. Journal of Neurophysiology 40(6): 1281–1291.

Salzberg BM, Kosterin PV, Muschol M et al. (2005) An ultra‐stable non‐coherent light source for optical measurements in neuroscience and cell physiology. Journal of Neuroscience Methods 141(1): 165–169.

Salzberg BM, Obaid AL and Bezanilla F (1993) Microsecond response of a voltage‐sensitive merocyanine dye: fast voltage‐clamp measurements on squid giant axon. Japanese Journal of Physiology 43(Suppl 1): S37–41.

Senseman DM and Salzberg BM (1980) Electrical activity in an exocrine gland: optical recording with a potentiometric dye. Science 208(4449): 1269–1271.

Sherrington C (1963) Man on His Nature: The Gifford Lectures Edinburgh 1937–1938. Cambridge: Cambridge University Press.

Siegel MS and Isacoff EY (1997) A genetically encoded optical probe of membrane voltage. Neuron 19(4): 735–741.

Slovin H, Arieli A, Hildesheim R et al. (2002) Long‐term voltage‐sensitive dye imaging reveals cortical dynamics in behaving monkeys. Journal of Neurophysiology 88(6): 3421–3438.

Tasaki I, Watanabe A, Sandlin R et al. (1968) Changes in fluorescence, turbidity, and birefringence associated with nerve excitation. Proceedings of the National Academy of Sciences of the USA 61(3): 883–888.

Tasaki I, Watanabe A and Hallet M (1972) Fluorescence of squid axon membrane labelled with hydrophobic probes. Journal of Membrane Biology 8(2): 109–132.

Vranesic I, Iijima T, Ichikawa M et al. (1994) Signal transmission in the parallel fiber‐Purkinje cell system visualized by high‐resolution imaging. Proceedings of the National Academy of Sciences of the USA 91(26): 13014–13017.

Wuskell JP, Boudreau D et al. (2006) Synthesis, spectra, delivery and potentiometric responses of new styryl dyes with extended spectral ranges. Journal of Neuroscience Methods 151(2): 200–215.

Zecevic D, Wu JY, Cohen LB et al. (1989) Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill‐withdrawal reflex. Journal of Neuroscience 9(10): 3681–3689.

Zhou WL, Yan P, Wuskell JP et al. (2007) Intracellular long‐wavelength voltage‐sensitive dyes for studying the dynamics of action potentials in axons and thin dendrites. Journal of Neuroscience Methods 164(2): 225–239.

Zochowski M, Wachowiak M and Falk CX (2000) Imaging membrane potential with voltage‐sensitive dyes. Biological Bulletin 198(1): 1–21.

Further Reading

Canepari M and Zecevic D (eds) (2009) The Voltage Imaging Book (in press). Springer.

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Salzberg, Brian M(Dec 2009) Functional Imaging of Nervous Systems Using Voltage‐Sensitive Dyes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021390]