Standing waves and traveling waves distinguish two circuits in visual cortex

The visual cortex represents stimuli through the activity of neuronal populations. We measured the evolution of this activity in space and time by imaging voltage-sensitive dyes in cat area V1. Contrast-reversing stimuli elicit responses that oscillate at twice the stimulus frequency, indicating that signals originate mostly in complex cells. These responses stand clear of the noise, whose amplitude decreases as 1/frequency, and yield high-resolution maps of orientation preference and retinotopy. We first show how these maps are combined to yield the responses to focal, oriented stimuli. We then study the evolution of the oscillating activity in space and time. In the orientation domain, it is a standing wave. In the spatial domain, it is a traveling wave propagating at 0.2-0.5 m/s. These different dynamics indicate a fundamental distinction in the circuits underlying selectivity for position and orientation, two key stimulus attributes.     

Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins

Optical imaging of electrical activity using voltage-sensitive dyes has been envisaged for many years as a powerful method to investigate multineuronal representation of information processing in brain tissue. This article describes the advent of novel genetically targeted voltage-sensitive fluorescent proteins. This new class of membrane voltage sensors overcomes previous limitations related to the nonselective staining of membranes associated with conventional voltage-sensitive dyes. Here, we discuss the methodology, applications, and potential advantages of this novel technique.     

Imaging Inhibitory Synaptic Potentials Using Voltage Sensitive Dyes

Studies of the spatio-temporal distribution of inhibitory postsynaptic potentials (IPSPs) in a neuron have been limited by the spatial information that can be obtained by electrode recordings. We describe a method that overcomes these limitations by imaging IPSPs with voltage-sensitive dyes. CA1 hippocampal pyramidal neurons from brain slices were loaded with the voltage-sensitive dye JPW-1114 from a somatic patch electrode in whole-cell configuration. After removal of the patch electrode, we found that neurons recover their physiological intracellular chloride concentration. Using an improved voltage-imaging technique, dendritic GABAergic IPSPs as small as 1 mV could be resolved optically from multiple sites with spatial averaging. We analyzed the sensitivity of the technique, in relation to its spatial resolution. We monitored the origin and the spread of IPSPs originating in different areas of the apical dendrite and reconstructed their spatial distribution. We achieved a clear discrimination of IPSPs from the dendrites and from the axon. This study indicates that voltage imaging is a uniquely suited approach for the investigation of several fundamental aspects of inhibitory synaptic transmission that require spatial information.     

VSDI: a new era in functional imaging of cortical dynamics

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 fundamental level of the cortical column.     

Using voltage-sensitive dye recording to image the functional development of neuronal circuits in vertebrate embryos

Recent developments in the design of voltage-sensitive dyes and of recording apparatuses for detecting voltage-dependent changes in the optical properties of such dyes have established voltage-sensitive dye recording as an important technique for assessing the functional development of neuronal circuits in the brain and spinal cord. Here we discuss general technical issues regarding the recording of voltage-sensitive dye signals and describe studies that have utilized this approach to follow the development of sensory and sensorimotor circuits in the embryonic brain stem. Functional imaging through voltage-sensitive dye recording permits a noninvasive analysis of synaptic development and function at submillisecond temporal resolution in widely distributed circuits. These advantages are particularly valuable in assessing sensorimotor circuit development at early stages when neurons are small and synapses are fragile.     

Real-Time Imaging of Electrical Signals with an Infrared FDA-Approved Dye

Clinical methods used to assess the electrical activity of excitable cells are often limited by their poor spatial resolution or their invasiveness. One promising solution to this problem is to optically measure membrane potential using a voltage-sensitive dye, but thus far, none of these dyes have been available for human use. Here we report that indocyanine green (ICG), an infrared fluorescent dye with FDA approval as an intravenously administered contrast agent, is voltage-sensitive. The fluorescence of ICG can follow action potentials in artificial neurons and cultured rat neurons and cardiomyocytes. ICG also visualized electrical activity induced in living explants of rat brain. In humans, ICG labels excitable cells and is routinely visualized transdermally with high spatial resolution. As an infrared voltage-sensitive dye with a low toxicity profile that can be readily imaged in deep tissues, ICG may have significant utility for clinical and basic research applications previously intractable for potentiometric dyes.     

Confocal ratiometric voltage imaging of cultured human keratinocytes reveals layer-specific responses to ATP

Recent evidencesuggests that changes in membrane potential influence the proliferationand differentiation of keratinocytes. To further elucidate the role ofchanges in membrane potential for their biological fate, the electricalbehavior of keratinocytes needs to be studied under complex conditionssuch as multilayered cultures. However, electrophysiological recordingsfrom cells in the various layers of a complex culture would beextremely difficult. Given the high spatial resolution of confocalimaging and the availability of novel voltage-sensitive dyes, wecombined these methods in an attempt to develop a viable alternativefor recording membrane potentials in more complex tissue systems. As afirst step, we used confocal ratiometric imaging of fluorescence resonance energy transfer (FRET)-based voltage-sensitive dyes. We thenvalidated this approach by comparing the optically recorded voltagesignals in HaCaT keratinocytes with the electrophysiological signalsobtained by whole cell recordings of the same preparation. Wedemonstrate 1) that optical recordings allow precisemultisite measurements of voltage changes evoked by the extracellularsignaling molecules ATP and bradykinin and 2) thatresponsiveness to ATP differs in various layers of cultured keratinocytes.

     

Comparison of Responses to Electrical Stimulation and Whisker Deflection Using Two Different Voltage-sensitive Dyes in Mouse Barrel Cortex in Vivo

We examined the spatial structure of noise in optical recordings made with two commonly used voltage-sensitive dyes (RH795 and RH1691) in mouse barrel cortex in vivo, and determined that the signal-to-noise ratio of the two dyes was comparable when averaging over barrel-sized areas, or at single pixels distant from large blood vessels. We examined the spatiotemporal development of whisker- and electrically-evoked optical responses by quantifying the area of activated cortical surface as a function of time. Whisker and electrical stimuli activated cortical areas between 0.2–2.0 mm² depending on intensity. More importantly, both types of activation recruited cortical area at similar rates and showed a linear relationship between the maximal activated area and the peak rate of increase of the activated area. We propose a general rule of supragranular cortical activation in which the initial spreading speed of the response determines the total activated area, independent of the type of activation. Finally, despite comparable single-response kinetics, we observed greater paired-pulse depression of whisker-evoked responses relative to electrically-evoked responses.     

Cortical imaging: capturing the moment

Recent advances in optical imaging techniques using voltage-sensitive dyes have provided new insights into the dynamic of neural processing in distributed cortical networks.     

Genetically encoded fluorescent sensors of membrane potential

Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.     

用电压敏感染料光学记录膜电位

应用传统电生理方法如微电极和膜片钳技术,在记录较小的神经细胞和纤细的神经突起膜电位及同步记录神经细胞群的电活动等方面目前仍是一大难题。随着生理科学和神经生物学的发展,利用电压敏感染料光学记录膜电位技术已成为一种较为理想的新手段。本文对光学记录膜电位技术的发展史、染料特性和作用机制、光学成像及膜电位记录原理、目前的光学方法中某些不足及未来前景等做了较系统的介绍,并且简述了光学记录膜电位在电生理和神经生物学中的应用。     

Fluorescence imaging of cardiac propagation: spectral properties and filtering of optical action potentials

Fluorescence imaging using voltage-sensitive dyes is an important tool for studying electrical propagation in the heart. Yet, the low amplitude of the voltage-sensitive component in the fluorescence signal and high acquisition rates dictated by the rapid propagation of the excitation wave front make it difficult to achieve recordings with high signal-to-noise ratios. Although spatially and temporally filtering the acquired signals has become de facto one of the key elements of optical mapping, there is no consensus regarding their use. Here we characterize the spatiotemporal spectra of optically recorded action potentials and determine the distortion produced by conical filters of different sizes. On the basis of these findings, we formulate the criteria for rational selection of filter characteristics. We studied the evolution of the spatial spectra of the propagating wave front after epicardial point stimulation of the isolated, perfused right ventricular free wall of the pig heart stained with di-4-ANEPPS. We found that short-wavelength (<3 mm) spectral components represent primarily noise and surface features of the preparation (coronary vessels, fat, and connective tissue). The time domain of the optical action potential spectrum also lacks high-frequency components (>100 Hz). Both findings are consistent with the reported effect of intrinsic blurring caused by light scattering inside the myocardial wall. The absence of high-frequency spectral components allows the use of aggressive low-pass spatial and temporal filters without affecting the optical action potential morphology. We show examples where the signal-to-noise ratio increased up to 150 with <3% distortion. A generalization of our approach to the rational filter selection in various applications is discussed.     

Imaging activity of neuronal populations with new long-wavelength voltage-sensitive dyes

We have assessed the utility of five new long-wavelength fluorescent voltage-sensitive dyes (VSD) for imaging the activity of populations of neurons in mouse brain slices. Although all the five were capable of detecting activity resulting from activation of the Schaffer collateral-CA1 pyramidal cell synapse, they differed significantly in their properties, most notably in the signal-to-noise ratio of the changes in dye fluorescence associated with neuronal activity. Two of these dyes, Di-2-ANBDQPQ and Di-1-APEFEQPQ, should prove particularly useful for imaging activity in brain tissue and for combining VSD imaging with the control of neuronal activity via light-activated proteins such as channelrhodopsin-2 and halorhodopsin.     

Nasal chemosensory-stimulation evoked activity patterns in the rat trigeminal ganglion visualized by in vivo voltage-sensitive dye imaging

Mammalian nasal chemosensation is predominantly mediated by two independent neuronal pathways, the olfactory and the trigeminal system. Within the early olfactory system, spatiotemporal responses of the olfactory bulb to various odorants have been mapped in great detail. In contrast, far less is known about the representation of volatile chemical stimuli at an early stage in the trigeminal system, the trigeminal ganglion (TG), which contains neurons directly projecting to the nasal cavity. We have established an in vivo preparation that allows high-resolution imaging of neuronal population activity from a large region of the rat TG using voltage-sensitive dyes (VSDs). Application of different chemical stimuli to the nasal cavity elicited distinct, stimulus-category specific, spatiotemporal activation patterns that comprised activated as well as suppressed areas. Thus, our results provide the first direct insights into the spatial representation of nasal chemosensory information within the trigeminal ganglion imaged at high temporal resolution.     

Imaging Brain Activity With Voltage- and Calcium-Sensitive Dyes

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio.Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations.Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors.Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb.Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise.Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras.The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.     

pH-Insensitive FRET voltage dyes

Many high-throughput ion channel assays require the use of voltage-sensitive dyes to detect channel activity in the presence of test compounds. Dye systems employing F?rster resonance energy transfer (FRET) between 2 membrane-bound dyes are advantageous in combining high sensitivity, relatively fast response, and ratiometric output. The most widely used FRET voltage dye system employs a coumarin fluorescence donor whose excitation spectrum is pH dependent. The authors have validated a new class of voltage-sensitive FRET donors based on a pyrene moiety. These dyes are significantly brighter than CC2-DMPE and are not pH sensitive in the physiological range. With the new dye system, the authors demonstrate a new high-throughput assay for the acid-sensing ion channel (ASIC) family. They also introduce a novel method for absolute calibration of voltage-sensitive dyes, simultaneously determining the resting membrane potential of a cell.     

Quantitative imaging of the heterogeneity of membrane activation of mammalian neurons and glial cells

By using quantitative imaging with an ultra-high sensitivity, it was possible to observe the simultaneous action of multiple patches unevenly distributed over the membranes of neurons and glial cells in culture. We used a voltage-sensitive probe to stain vitally the cells. The instrumentation consisted of a liquid-nitrogen cooled matrix of 222,530 photodetectors with a spatial resolution of 0.25 microns 2, a photodynamic range of 10(5), a detection level of a few tens of photons and a maximum time resolution of 500 microseconds. Electrical and pharmacological stimulations were applied to produce the activation of the cells which was accompanied by large variations of the level of fluorescence, giving a precise spatial localization of active domains over the soma-neuritic membranes. These images of fluorescent signals are interpreted as corresponding to the plasmalemmal localization of voltage-dependent channels. This finding, which had not been previously observed with voltage-sensitive probes in fluorescent dye imaging indicates the possibility of measuring the activity of independently functioning domains in single neurons.     

High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes

An improved method for making fast quantitative determinations of membrane potential with voltage-sensitive dyes is presented. This method incorporates a high-speed, random-access, laser-scanning scheme (Bullen et al., 1997. Biophys. J. 73:477-491) with simultaneous detection at two emission wavelengths. The basis of this ratiometric approach is the voltage-dependent shift in the emission spectrum of the voltage-sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS). Optical measurements are made at two emission wavelengths, using secondary dichroic beamsplitting and dual photodetectors (<570 nm and >570 nm). Calibration of the ratiometric measurements between signals at these wavelengths was achieved using simultaneous optical and patch-clamp measurements from adjacent points. Data demonstrating the linearity, precision, and accuracy of this technique are presented. Records obtained with this method exhibited a voltage resolution of approximately 5 mV, without any need for temporal or spatial averaging. Ratiometric recordings of action potentials from isolated hippocampal neurons are used to illustrate the usefulness of this approach. This method is unique in that it is the first to allow quantitative determination of dynamic membrane potential changes in a manner optimized for both high spatiotemporal resolution (2 micrometers and <0.5 ms) and voltage discrimination.     

Cutting-edge technology. III. Imaging and the gastrointestinal tract: mapping the human enteric nervous system

Monitoring membrane potentials by multisite optical recording techniques using voltage-sensitive dyes is ideal for direct analysis of network signaling. We applied this technology to monitor fast and slow excitability changes in the enteric nervous system and in hundreds of neurons simultaneously at cellular and subcellular resolution. This imaging technique presents a powerful tool to study activity patterns in enteric pathways and to assess differential activation of nerves in the gut to a number of stimuli that modulate neuronal activity directly or through synaptic mechanisms. The optical mapping made it possible to record from tissues such as human enteric nerves, which were, until now, inaccessible by other techniques.