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  • Review Article
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VSDI: a new era in functional imaging of cortical dynamics

Nature Reviews Neuroscience volume 5, pages 874–885 (2004)Cite this article

Key Points

  • Imaging based on voltage-sensitive dyes (VSDI) can be used to visualize electrical activity in neurons at a high spatial and temporal resolution. The dye molecules bind to the external surface of cell membranes and transform changes in membrane potential into optical signals. It can be used either in vitro or in vivo, and can be combined with other techniques, such as microstimulation, intracellular or extracellular recording, or tracer injection.

  • VSDI is ideal for studying spatial localization of neocortical activity and dynamics. For example, it has been used to show that subthreshold activity can spread across a large area of cortex following sensory stimulation that directly excites only a small area. It has also provided evidence that orientation tuning in the primary visual cortex depends mainly on thalamic input rather than intracortical processing, although intracortical processing amplifies and modulates the response.

  • In another VSDI study, the neural correlates of the 'line-motion' illusion were investigated. The stationary stimuli that cause illusory motion were shown to give rise to spreading sub-threshold activity in the cat visual cortex, which could produce the perception of motion by dynamically priming areas of cortex.

  • Functional neuronal assemblies were visualized using VSDI. This relies on simultaneous single-unit recordings, followed by spike-triggered averaging to isolate the population of neurons that shows synchronized activity.

  • VSDI has been used to study ongoing cortical activity, which occurs in the absence of sensory input. Although such activity is often thought of as 'noise', VSDI revealed that it can be coherent and of large amplitude. As such fluctuations will affect how far neurons are from their firing threshold, ongoing activity could shape neuronal responses to sensory stimuli. Spontaneous activity measured by VSDI can also be used to predict spiking activity in single neurons, and reveals spontaneously switching cortical states.

  • It is possible to use VSDI to repeatedly image the same area of cortex in awake, behaving monkeys over long periods of time, through implanted 'windows'. Such studies could be used to investigate questions relating to development and plasticity, and higher cognitive functions.

  • The greatest benefit has come when VSDI has been combined with other imaging or electrophysiological techniques.

  • It should also be possible to create improved new dyes and to produce further technical innovations. Genetically engineered in vivo probes should facilitate new types of experiments and improve results. The use of VSDI in the future has the potential to provide new insights into the fundamental principals underlying cortical processing, its development and plasticity.

Abstract

During the last few decades, neuroscientists have benefited from the emergence of many powerful functional imaging techniques that cover broad spatial and temporal scales. We can now image single molecules controlling cell differentiation, growth and death; single cells and their neurites processing electrical inputs and sending outputs; neuronal circuits performing neural computations in vitro; and the intact brain. At present, imaging based on voltage-sensitive dyes (VSDI) offers the highest spatial and temporal resolution for imaging neocortical functions in the living brain, and has paved the way for a new era in the functional imaging of cortical dynamics. It has facilitated the exploration of fundamental mechanisms that underlie neocortical development, function and plasticity at the fundmental level of the cortical column.

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Figure 1: Optical imaging of cortical dynamics in vivo.
Figure 2: Many functional domains are activated during the processing of a small retinal image.
Figure 3: Orientation tuning is constant, but dynamic orientation-selective, intracortical suppression shapes the final response.
Figure 4: Priming by subthreshold spread of synaptic potential can account for the illusion of motion perception.
Figure 5: A spontaneous spike train of the action potentials of a single neuron can be predicted from the similarity of spatio-temporal patterns of spontaneous activity to the functional architecture.
Figure 6: Chronic imaging in behaving monkeys.
Figure 7: Some available tools to meet the challenges of neuroscience.

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Acknowledgements

We thank C. Wijnbergen, Y. Toledo and D. Etner for their assistance, and our previous co-workers, R. Frostig, E. Lieke, D. Shoham, D. Glaser, A. Arieli, E. Seideman, T. Kenet, A. Sterkin, M. Tsodyks,D. Sharon and H. Slovin for their original contributions. This work was supported by grants from the Grodetsky Center, the Goldsmith, Glasberg, Heineman and Korber foundations, BMBF, ISF grants and Ms. Enoch.

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  1. Department of Neurobiology, The Weizmann Institute of Science, Rehovot, 76100, Israel

    Amiram Grinvald & Rina Hildesheim

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  1. Amiram Grinvald
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Correspondence to Amiram Grinvald.

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A.G. holds equity in a company that manufactures imagers to map cortical activity based on intrinsic signals or voltage sensitive dyes.

Supplementary information

Supplementary information S1 (JPG 96 kb)

Supplementary information S2 (JPG 67 kb)

Supplementary information S3 (JPG 90 kb)

Supplementary information S4

Movie S4 | Lateral spread of activity in the barrel cortex. Visualization of the Spatio–temporal dynamics of the response to whisker C3 stimulation using a 3–ms air–puff. Initially, activation is seen only in a restricted area similar to the whisker representation in layer IV. (Temporal resolution 0.6 ms. Average of 128 trials.) Data by Derdikman et al., JNS 2003. (AVI 616 kb)

Supplementary information S5

Movie S5 | Surround inhibition in the barrel cortex. The VSD response to strong whisker or cutaneous stimulus triphasic. Visualization of a frame sequence of averaged functional maps depicting a depolarization phase followed surround hyperpolarization phase and a depolarizing rebound. N = 192 trials; Scale bar, 1 mm. Data from REF. 67. (AVI 2607 kb)

Supplementary information S6

Movie S6 | Dynamics of orientation tuning (differential map). Visualization of the appearance of orientation patches cat area 18 in response to visual presentation of drifting gratings. The difference between the cortical responses vertical and horizontal gratings is shown (differential maps). Time resolution, 10 ms. Data from REF. 71. (MOV 385 kb)

Supplementary information S7

Movie S7 | Dynamics of orientation tuning: polar movie plus time course. Time–course of ‘polar orientation maps’: colour represents the preferred orientation of each pixel (0–180º from bottom to top of colour bar on right), and brightness represents the modulation depth of its tuning curve (0–0.5‰ from left to right of colour bar). After peaking at 74 ms, map strength declines gradually to about 65% of maximal at 120 ms (not shown). Data from REF. 71. (MOV 663 kb)

Supplementary information S8

Movie S8 | The line motion illusion. Small squares (cues) are presented at different locations followed by a bar. All stimuli shown are flashed AND stationary. However, illusory motion is perceived, drawn away from the cue locations to form the full bar. Data from REF. 81. (AVI 262 kb)

Supplementary information S9

Movie S9 | Cortical correlates of the line motion illusion. Optically detected activity within 5.5 mm x 2.9 mm cortical surface. The first frames show the rapid spread of low amplitude activity (light blue, green, yellow; presumably subthreshold) followed by high–level activity (red, brown; presumably spiking) that gradually propagates towards the end of the cortical bar representation, therefore reporting motion in the stationary flashed bar. A drawn bar or a moving square at appropriate speed produce similar spatio–temporal patterns to that observed here with the line–motion stationary stimuli (not shown). (Sampling rate, 9.6 ms.) Data from REF. 81. (AVI 4135 kb)

Supplementary information S10

Movie S10 | A single neuron is spontaneously active when the pattern of on–going activity resembles its related orientation map. Comparison between the patterns of cortical activity during spontaneous and evoked regimes. The preferred cortical states (PCS) of the population activity are obtained by averaging over the time samples when the recorded neuron emitted an action potential. The left movie shows the spatial patterns for evoked activity by oriented gratings that optimally drive that neuron. This pattern corresponds to the orientation map (not shown). The right movie shows the results of spike–triggered averaging of spontaneous activity when the eyes were closed. The two patterns are nearly identical at time zero, when the action potentials occurred. Data from REF. 85. (MOV 507 kb)

Supplementary information S11

Movie S11 | Dynamics of cortical states. The left panel displays a sequence of a single frame (10–ms snapshots, without averaging) from a movie showing instantaneous cortical activity in the absence of stimulation (raw data; not processed). The time relative to the first frame is displayed in the centre. Whenever a state that is significantly correlated with one of the orientation maps emerges, that orientation map appears on the right panel, its corresponding orientation is displayed by a bar in the centre, and the correlation between the two frames is displayed over the left panel. When the maximal correlation for a state is reached, the movie is briefly paused. To facilitate the perception of the similarity between the structures, outlines of the main patches have been added to both panels. Data from REF. 89. (MOV 2366 kb)

Supplementary information S12

Movie S12 | Cortical representations of a small object during a saccade in the primary visual cortex of an awake monkey. Activity in primary visual cortex and V2 of the behaving monkey during a saccade. Time series of the average optical responses are triggered at the onset of a saccade to the visual stimulus. The first few frames show the fully developed evoked response to the small (0.5º) single drifting grating, which was turned on 500–800 ms earlier. After a saccadic eye movement to the stimulus (t = 0), the activity on the cortex is shifted to a more foveal location (lateral) (n = 17 trials). The right panel shows the average eye movement during the imaging of cortical responses. The border between V1 and V2 is labelled by a thin line. Data from REF. 90. (MOV 743 kb)

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FURTHER INFORMATION

Encyclopedia of Life Sciences

Brain imaging: observing ongoing neuronal activity

Grinvald's laboratory

Glossary

MACROSCOPE

An improvised microscope made of photographic lenses that provides much larger numerical aperture (NA) for low magnification (for example, one or less) than a standard microscope objective. This is crucial for fluorescence imaging of large areas (for example, 10 mm) because illumination intensity and fluorescence collections depend on the square of the NA.

VOLTAGE-SENSITIVE DYES

The ones discussed here are organic molecules with a molecular weight of about 500 Da and a length shorter than 20 Å These molecules usually have a hydrophobic portion that sticks to the membrane and a charged chromophore that prevents a 'flip' to the cell interior. The dyes have a high absorbtion coefficient and, usually, a high quantum efficiency for fluorescence when they bind to the neuronal membranes.

FUCTIONAL OPTICAL IMAGING

A means of recording neural activity by measuring the optical properties of brain tissue, using either voltage-sensitive dyes or intrinsic signals relating to the oxygen saturation of haemoglobin or light scattering.

BARREL

A cylindrical column of neurons found in the rodent somatosensory neocortex. Each barrel receives sensory input from a single whisker follicle, and the topographical organization of the barrels corresponds precisely to the arrangement of whisker follicles on the face.

PHARMACOLOGICAL SIDE EFFECTS

Organic molecules may bind to ion channels or other important components of the neuronal machinery, and as a result they have the potential to modify ion conductances, neurotransmitter receptors and other membrane properties. These modifications could change the electrical behaviour of single neurons and/or neuronal networks.

PHOTOTOXICITY

(Photodynamic damage.) Once excited by illumination, certain organic molecules collide with oxygen molecules, which can create singlet oxygen radicals. Singlet oxygen is highly reactive, and can oxidise proteins and other membrane components, causing damage to cell membranes.

FRONTAL EYE FIELD

An area in the frontal lobe that receives visual inputs and controls movements of the eye.

ORIENTATION SELECTIVITY

A property of visual cortex neurons that allows the detection of bars and edges within visual images and the encoding of their orientations. As the cortex is organized in columns, neurons that belong to the same column share the same orientation tuning.

SIGNAL AVERAGING

A standard procedure used to improve the signal-to-noise ratio. Adding the results of repeated trials means that if a signal is reproducible, it adds up, whereas random noise is averaged out. However, if the signal is variable the true dynamics cannot be explored. It is, therefore, highly significant when VSDI provides large signals in a single trial — as is shown here.

HYPERCOLUMN

In the visual cortex, an orientation hypercolumn refers to a patch of cortex containing several cortical columns, in which neurons which have similar spatial receptive fields but that cover all possible preferred orientations are found. This concept can be generalized to other visual attributes and to other sensory and motor areas.

BEHAVING MONKEYS

The behaving monkey is often the ideal model for studying higher cognitive functions in relation to human behaviour. Unlike rodents, monkeys can be readily trained to perform complicated tasks that are difficule even for talented students. This model can be used to combine various techniques that are not applicable to human studies, and to give insight into the workings of the primate brain.

MULTI-PHOTON MICROSCOPY

A form of microscopy in which a fluorochrome that would normally be excited by a single photon is stimulated quasi-simultaneously by several photons of lower energy. Under these conditions, fluorescence increases as a function of the square of the light intensity, and decreases approximately as the square of the distance from the focus. Because of this behaviour, only fluorochrome molecules near the plane of focus are excited, greatly reducing light scattering and photodamage to the sample.

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Grinvald, A., Hildesheim, R. VSDI: a new era in functional imaging of cortical dynamics. Nat Rev Neurosci 5, 874–885 (2004). https://doi.org/10.1038/nrn1536

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