Voltage-sensitive dye

Post-publication activity

Curator: Lawrence B. Cohen

Voltage-sensitive dyes are organic molecules or proteins which reside in a cell membrane and change their optical properties in response to a change in membrane potential. They have been widely used in combination with fast (1 kfps frames rate) cameras to monitor membrane potential in processes of individual neurons and from multiple cell bodies in localized brain regions. In more global measurements they have been used to follow population changes in membrane potential over large regions of the brain and the heart. The organic voltage sensitive dyes are fast, with response times less than 10 µsec, and their signals are often linearly related to the change in membrane potential.

Introduction

Optical recording techniques use molecular probes to transduce electrical events into changes in light intensity. There are several potentially powerful advantages to optical recording. First, the measurements are in principle noninvasive. Therefore, they can be used to record from small cells or cell processes without impalement damage. Second, it is easy to record from multiple sites simultaneously because many detectors can be placed over the image of a preparation. Third, different kinds of physiological responses related to neuronal activity can be detected using appropriate indicators. Electrical activity can be monitored directly with voltage-sensitive dyes, or indirectly using ion indicators or intrinsic optical properties.

Figure 1: An optical recording from a squid axon

Organic voltage-sensitive dyes change their absorbance, fluorescence, or birefringence when there is a change in the membrane potential of the cell to which they are bound. The dyes that have this property have been divided into two practical groups, slow dyes and fast dyes. The slow dyes respond to changes in potential with a time constant of milliseconds to seconds and have been used to measure the resting potential of large populations of identical cells. Their slow response time makes them unsuitable for recording the more rapid kinds of neuronal activity. The fast dyes respond in microseconds with optical signals that are linearly related to changes in membrane potential. The changes are small, but several dyes are sensitive enough that changes in light intensity can be detected without averaging when a neuron fires an action potential. However, it is often not easy to calibrate the magnitude of the intensity change in terms of membrane potential without reference to electrode measurements. Signals have been obtained when the cell is stained from either the inside or the outside, and both methods have been exploited in physiological measurements. An optical recording from a squid axon stained with a merocyanine dye is shown in Figure 1. It is clear that the absorbance change is a very good representation of the action potential. Several different areas of neurobiology are now taking advantage of these optical methods.

Regional properties of neurons

Figure 2: Action potential signals from axons and dendrites

With a photodiode array or a very fast CCD camera it is possible to record simultaneously voltage changes from many parts of a neuron, especially if the records can be signal averaged. Profiles of propagating action potentials and subthreshold potentials have been examined using both bath applied and intracellularly injected dyes. Figure 2 shows optical recordings of an action potential detected from different sites on axonal and dendritic processes of a neuron stained with a styryl dye. The response time of the dye and apparatus are fast enough to detect fast neuronal impulses without distortion. When these kinds of optical recordings are combined with measurements of the neuron geometry it is possible, in principle, to determine the linear and nonlinear membrane properties of different parts of the cell. It is also possible to follow the generation and spread of synaptic potentials. Recent improvements in indicator dyes and measuring techniques have enabled investigators to detect signals from individual spines.

Large optical signals are obtained if a large group of almost identical neurons are simultaneously active. If these cells are stimulated simultaneously, the resulting optical signal is the sum of the signals from each of the cells and representative of the potential change in any one of them. This approach has been used in experiments on hippocampal slices, the vertebrate neurohypophysis, and cardiac tissue.

Circuit analysis of small nervous systems

In favorable invertebrate preparations action potentials from a large fraction of the cells in a ganglion can be detected optically without signal averaging (e.g., [Zecevic et al., 1989]). Because the signal size is proportional to the area of the stained membrane, this action potential signal is usually dominated by the response from the soma. Consequently, signals from different cells can be spatially resolved. Further separation of action potential signals can be achieved by timing differences. These techniques are being used to work out the circuits underlying simple behaviors and to determine how these circuits are modified by experience.

Mapping of receptive fields in cortical areas

Figure 3: Measurement from somatosensory cortex

Many events in the vertebrate brain occur in layers of the cortex close to the surface. Optical signals can be detected by measuring fluorescence changes from brains stained with voltage-sensitive dyes. Figure 3 illustrates results from a measurement from somatosensory cortex of an anesthetized rat stained with a styryl dye. Movement of a whisker resulted in a signal seen only in the center of the field, a region presumed to be associated with a whisker barrel. Similar experiments have been performed in the auditory and visual cortices and in the olfactory pathway.

Detection of electrical activity using ion-sensitive indicators

Figure 4: Fluorescence changes in a dendrite of a Purkinje cell

As a consequence of electrical activity the intracellular concentration of $$Ca^{2+}$$ and $$Na^+$$ ions change. Using suitable optical indicators these concentration changes can be detected and used to reveal information about the electrical events that caused them and the channels that were active during this activity. For example, Figure 4 shows fluorescence changes in a dendrite of a Purkinje cell that was injected with fura-2. The jumps in fluorescence correspond to the electrically recorded calcium action potentials, which are active in the dendrites, and not to the fast sodium spikes, which are active in the somatic region. This technique has been used with $$Na^+$$ and $$Ca^{2+}$$ sensitive indicators to illuminate properties of dendrites and presynaptic terminals. Another approach is to load large numbers of cells with a $$Ca^{2+}$$ indicator and to use the temporal and spatial pattern of transient fluorescence changes to illuminate circuit properties of the stained neurons (e.g., [O'Donovan et al., 1993; Stosiek et al, 2003]). The development of high-speed CCD cameras and two-photon scanning microscopy has enhanced the spatial and temporal resolution of these kinds of measurements.

Some cautions

While voltage-sensitive dyes give an accurate representation of the potential change in the cells to which they are bound, they may have pharmacological and photodynamic effects. Although dyes have been found with minimal pharmacological and photodynamic consequences in several preparations, subtle effects may still exist. Another problem is dye bleaching which will result in declining signals as a function of illumination duration. There is evidence that some voltage-sensitive dyes respond differently on different preparations. Although these effects have limited some applications, it is the small size of the optical signals that most often restricts the kinds of experiments that can be done. For all the experiments, careful attention to details of the apparatus is required.

Use of ion indicators also requires caution. Because they bind ions the indicators are buffers, and therefore can alter cell physiology and affect the magnitude and time course of the concentration changes they are reporting. The rate constants and equilibrium constants of these indicators in the intracellular environment are often unknown since they are sensitive to small changes in viscosity, solvent polarity, and binding to intracellular constituents.

Other approaches

In addition to these extrinsic indicators of electrical activity, there are additional neuronal parameters that are closely linked to neuronal events and that can be detected optically. Light scattering and birefringence changes measured in stimulated squid axons have been related to combinations of membrane potential, membrane current, and gating currents. In experiments on the vertebrate neurohypophysis light scattering changes have been correlated with stimulated secretion. In the visual and somatosensory cortices reflectance changes, partially related to underlying changes in blood flow or oxygenation, have been used to detect dynamic receptive field patterns. These are called intrinsic imaging signals. The optical technique has also been extended to report events unrelated to changes in the external membrane potential. Voltage sensitive dyes have been used to measure potential changes in mitochondria and calcium indicators have been used to monitor the flow of calcium into and out of the endoplasmic reticulum and nucleus. Recently, a number of genetically encoded indicators for voltage, calcium, and pH have been developed and are starting to be applied (Siegel and Isacoff, 1997; Tsutsui et al, 2008).

• Cinelli AR, Kauer JS (1992): Voltage-sensitive dyes and functional activity in the olfactory pathway. Annu Rev Neurosci 15:321–351
• Cohen LB, Salzberg BM (1978): Optical measurement of membrane potential. Rev Physiol Biochem Pharmacol 83:35–88
• Grinvald A (1985): Real time optical mapping of neuronal activity: From growth cones to the intact mammalian brain. Annu Rev Neurosci 8:263–305
• Murphy TH, Baraban JM, Wier WG, Blatter LA (1994): Visualization of quantal synaptic transmission by dendritic calcium imaging. Science 263: 529–532
• Ross WN (1989): Changes in intracellular calcium during neuron activity. Annu Rev Physiol 51:491–506

Internal references

• Jan A. Sanders (2006) Averaging. Scholarpedia, 1(11):1760.
• Valentino Braitenberg (2007) Brain. Scholarpedia, 2(11):2918.
• Eugene M. Izhikevich (2007) Equilibrium. Scholarpedia, 2(10):2014.
• Rodolfo Llinas (2008) Neuron. Scholarpedia, 3(8):1490.
• Jose-Manuel Alonso and Yao Chen (2009) Receptive field. Scholarpedia, 4(1):5393.

References

• [Antic S, Zecevic, 1995] Antic S, Zecevic (1995): Optical signals from neurons with internally applied voltage-sensitive dyes. J Neurosci 15:1392–1405
• [Lev-Ram et al., 1992] Lev-Ram V, Miyakawa H, Lasser-Ross N, Ross WN (1992): Calcium transients in cerebellar Purkinje neurons evoked by intracellular stimulation. J Neurophysiol 68:1167–1177
• [O'Donovan et al., 1993] O'Donovan MJ, Ho S, Sholomenko G, Yee W (1993): Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes. J Neurosci Methods 46:91–106
• [Orbach et al., 1985] Orbach H, Cohen LB, Grinvald A (1985): Optical mapping of electrical activity in rat somatosensory and visual cortex. J Neurosci 5:1886–1895
• [Ross et al., 1977] Ross WN, Salzberg BM, Cohen LB, Grinvald A, Davila HV, Waggoner A, Wang CH (1977): Changes in absorption, fluorescence, dichroism and birefringence in stained giant axons: Optical measurement of membrane potential. J Membr Biol 33:141–183.
• [Siegel, MS, Isacoff, EY, 1997] A genetically encoded optical probe of membrane voltage. Neuron 19:735-41.
• [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.
• [Tsutsui H, Karasawa S, Okamura Y, Miyawaki A., 2008] Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat Methods. 8:683-5.
• [Zecevic et al., 1989] Zecevic D, Wu JY, Cohen LB, London JA, Hopp HP, Falk XC (1989): Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex. J Neurosci 9:3681–3689