Functional Imaging of Nervous Systems Using Voltage‐Sensitive Dyes


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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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. [doi: 10.1002/9780470015902.a0021390]