Laser excitation at nearoptimal wavelength regarding signal size improved the sensitivity of recording by a factor of 50 over previous approaches. Voltage clamp, current clamp, patch clamp and dynamic clamp systems. In this article, we focus on a refinement of the traditional voltage clamp methods that are used to measure current from whole cells, or relatively large areas of membrane, called the patch clamp. Voltagesensitive dye vsd imaging directly assays the spatiotemporal dynamics of. Calcium and voltage sensitive dye signals evoked by olfactory nerve shock in an in vitro turtle olfactory bulb preparation. Patch clamp solutions intracellular and extracellular. Cortical dendritic spine heads are not electrically.
One current alternative is to use calcium imaging, as the agents are robust and sensitive, come in a variety of colors, and can be used in a wide. Jul 28, 2004 experiments were performed on 4 mitral cells. Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch clamp characterization. The fivephoton ion channel membrane potential assay kit provides an easytouse, highly sensitive, accurate quantitative method to measure changes in ion flux and the cellular membrane potential using fluorescent voltage sensitive dyes in eukaryotic and prokaryotic cells. Gandini,1 alejandro sandoval,2 and ricardo felix1,3 1department of cell biology, center for research and advanced studies of the national polytechnic institute. Imaging membrane potential with two types of genetically. Spontaneously spiking hek cells screening fluorescent. Long wavelength voltage sensitive dyes available technology. J m nooney department of pharmacology and clinical pharmacology, ninewells hospital, university of dundee.
The staining level was determined empirically as a compromise that attains a high level of fluorescence without causing damage by prolonged dialysis from the patch pipette. The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level. The signaltonoise ratio in the voltage sensitive dye recording from neuronal processes varied as a function of 1 the amount of the dye injected, 2 the morphology of the cell, and 3 the proximity of the cell to the surface of the slice. Dendritic signals from rat hippocampal ca1 pyramidal neurons. Chapters focus on invivo recordings, voltage gated channel recording and analysis, dendritic and axonal recordings, synaptic current recording and analysis, advanced fluorescent techniques, optogenetics and voltage sensitive dye imaging, and finally channel and neuronal modeling. School of biological sciences illinois state university. Imaging membrane potential with two types of genetically encoded fluorescent voltage sensors. Comprehensive protocol for effective patch clamp analysis. The classical means to monitor membrane potentials is patch clamp electrophysiology, a lowthroughput and highly invasive technique. Membrane potential measurements of isolated neurons using a.
This fretbased measurement is restricted to the plasma membrane and is therefore more relevant than nonfret single dye based approaches that measure signals. We first applied patch clamp electrophysiology and voltage sensitive dye vsd imaging to characterize the waveform and reproducibility of the spiking behavior, and recorded movies of voltage waves in syncytial monolayers. Use of ratiometric fluorescence measurements of the voltage. Slices, neuron selection, patchclamp recording and intracellular application of dyes experiments were carried out on somatosensory cortex. Voltage sensor probes vsps are a fluorescence resonance energy transfer fretbased voltagesensing assay technology, used to measure changes in cells membrane potential.
Many physiological processes are accompanied by changes in cell membrane potential which can be detected with voltage. Elucidating the link between structure and function of ion channels and transporters with voltage clamp and patch clamp fluorometry giovanni zifarelli and jana kusch. In vitro functional imaging in brain slices using fast. Recording from fine neuronal structures using patch clamp and imaging techniques sonia gasparini and lucy m palmer. Voltage sensitive dye how is voltage sensitive dye abbreviated.
Jan 19, 2016 what does voltage sensitive dye imaging mean in english. In this study, fast voltage sensitive dye imaging and extracellular and wholecell electrophysiology were used on entorhinal corticalhippocampal slice preparations to characterize hippocampal network activity in 1216 month old female appswepsen1deltae9 apde9 mice mice. We then developed assays to use the spiking hek cells to test vsds and. Patch clamp electrophysiology, voltage clamp, action. A patch clamp study of the nicotinic acetylcholine. Somatic wholecell recordings were done using a multiclamp 700a amplifier axon instruments. The voltage is normally stepped to a family of levels, matching preset command patterns, and the current supplied or absorbed by the circuit to hold the voltage at each level is measured. Vsd imaging also records voltage changes from processes too small to access with patchclamp electrodes in intact cells. Rapid changes in the membrane potential of neurons and cardiomyocytes define the unique physiology of these cells. In vitro functional imaging in brain slices using fast voltagesensitive. In pharmacological research, automated patch clamping is used to screen potent substances for ion channel modifications. Screening fluorescent voltage indicators with spontaneously. Voltagesensitive dyes, also known as potentiometric dyes, are dyes which change their spectral properties in response to voltage changes. Genetically encoded voltage indicators gevis are emerging tools to elucidate the inner workings of the brain.
Voltagesensitive dye an overview sciencedirect topics. Most patchclamp recordings from neurons have been made from the soma, the. They are able to provide linear measurements of firing activity of single neurons, large neuronal populations or activity of myocytes. Oct 21, 2011 another problem is dye bleaching which will result in declining signals as a function of illumination duration. This volume presents current adaptations of the patchclamp technique to neuroscience. Voltage sensitive dye how is voltage sensitive dye. To examine the spatial distribution and time course of inhibitory responses observed in coronal slices in voltagesensitive dye vsd experiments, we performed patchclamp recordings from interneurons in layer 1 or ls neurons in layers 23 of the grs. The study of ion channelmediated changes in membrane potential using the conventional bisoxonol fluorescent dye dibac43 has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. The field of view is 3x6 mm and the playback is slowed fivefold from real time. Finally we do histological and immunohistochemical analysis of. Apr 12, 20 we use a variety of electrophysiological techniques e.
The biological sciences microscopy facility provides a range of instruments for light and electron microscopy, including a new, stateoftheart scanning electron microscope. Voltagesensitive dye recording from axons, dendrites and dendritic spines of individual neurons in brain slices. Ccd or vidicon camera and controller for gain and offset. Visualize neuronal processes under low light level at a frame rate of 1040 hz in the continuous recording mode of the ccd for voltage imaging. Gandini,1 alejandro sandoval,2 and ricardo felix1,3 1department of cell biology, center for research and advanced studies of the national polytechnic institute cinvestavipn, mexico city, mexico. Fast voltagesensitive dye imaging of excitatory and. Use of ratiometric fluorescence measurements of the. Posters publications presentations how to videos and guides blog. Imaging inhibitory synaptic potentials using voltage sensitive dyes. Inhibitory neuron and hippocampal circuit dysfunction in.
Membrane insertion ofand membrane potential sensing by. It is based on the principle that the current flowing through a cell membrane can be estimated by clamping the membrane potential to a constant value using the injection of a mirror current a current of the same amplitude but reversed polarity. Preformulated patch clamp solutions with compositions that promote seal formation and recording. Oct 10, 2012 the staining level was determined empirically as a compromise that attains a high level of fluorescence without causing damage by prolonged dialysis from the patch pipette. Cortical dendritic spine heads are not electrically isolated. Chapters focus on invivo recordings, voltagegated channel recording and analysis, dendritic and axonal recordings, synaptic current recording and analysis, advanced fluorescent techniques, optogenetics and voltagesensitive dye imaging, and finally channel and neuronal modeling. Precisely formulated and matched intracellular and extracellular solutions for patch clamp technique. Vsd imaging also records voltage changes from processes too small to access with patch clamp electrodes in intact cells.
Videos s1s4 show more propagation patterns in spiking hek cells. Compared to singlecell electrophysiology, our ability to directly study neuronal. It is based on the principle that the current flowing through a cell membrane can be. However, the unresolved multicomponent origin of the optical signal encourages us to perform a detailed analysis of the method limitation and the existing models. The membrane current and changes in fluorescence are recorded simultaneously.
In vitro functional imaging in brain slices using fast voltagesensitive dye imaging combined with wholecell patch recording. We introduce a line of nonfluorescent hek cells that stably express nav 1. A video of voltage recorded using the voltage sensitive dye vf2. Intracellular solutions for wholecell patch clamping see reagent setup voltage sensitive dye. The signaltonoise ratio in the voltagesensitive dye recording from neuronal processes varied as a function of 1 the amount of the dye injected, 2 the morphology of the cell, and 3 the proximity of the cell to the surface of the slice.
As a tradeoff of having a high na objective lens, the working distance decreases. Spontaneously spiking hek cells screening fluorescent voltage. What does voltagesensitive dye imaging mean in english. We attribute this poor performance to imperfect membrane insertion for a large fraction of. Ccd camera for infrared dic videomicroscopy ir, dage mti, usa. A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture. Vsd allows to measure membrane potential variations at a network level. D voltagesensitive dye images showing electrical wave propagation in a culture of spiking.
Wholecell recording of neuronal membrane potential. One current alternative is to use calcium imaging, as the agents are robust and sensitive, come in a variety of colors, and can be used in a wide range of biological contexts. The voltage clamp is a technique used to control the voltage across the membrane of a small or isopotential area of a nerve cell by an electronic feedback circuit. Voltage clamp is an electrophysiological technique to record electrical currents in biological membranes.
Our school has extensive microscopy facilities and training for graduate students. The possibility offered for in vivo and in vitro brain imaging is unprecedented in terms of spatial and temporal resolution. C patch clamp recording of membrane voltage in a single spiking hek cell. However, the unresolved multicomponent origin of the optical signal encourages us to perform a detailed analysis of the method limitation and. Additional evidence for voltage sensing by pcnrs was provided by simultaneous patchclamp and fluorescence measurements. Using vsd imaging to probe neuronal circuitry function has continued over the last decade with increasing. Imaging activity in slices, using fast voltagesensitive dyes. Voltage sensitive dyes, also known as potentiometric dyes, are dyes which change their spectral properties in response to voltage changes. How exactly does current and voltage clamping work. Since the cell membrane is a likely primary site for the cascade of events resulting in a biological response to electromagnetic fields, methodologies for monitoring the membrane voltage will be critical. Membrane potential measurements of isolated neurons using. We first applied patch clamp electrophysiology and voltagesensitive dye vsd imaging to characterize the waveform and reproducibility of the spiking behavior, and recorded movies of voltage waves in syncytial monolayers. A basic voltage clamp will iteratively measure the membrane potential, and then change the membrane potential voltage to a desired value by adding the necessary current. The ability to monitor changes in membrane potential is a useful tool for studying neuronal function, but there are only limited options available at present.
We use a variety of electrophysiological techniques e. Fivephoton membrane potential ion channel assay kit fluorescent voltage sensitive dye kit kw. Brightness is proportional to voltageinduced change in fluorescence. Dye indicators of membrane potential have been available for the past 15 years and have been employed in numerous studies of cell physiology. Voltagesensitive dye recording from axons, dendrites and. The voltage sensitive dye vsd is a molecule that, once bound to the external surface of cell membranes, acts as a molecular transducer transforming changes in membrane potential into optical signals changes in absorption or emitted fluorescence. A novel membrane potentialsensitive fluorescent dye. Here, we have investigated the potential of a commercially available flipr membrane potential fmp dye, developed originally for high throughput screening using a plate reader, for imaging the membrane potential of cultured cells using. The voltage sensitivity was most marked for hexamethonium. Advanced patchclamp analysis for neuroscientists in. This is a disadvantage of the inverted microscope setup. Here, we report the characterization of the flipr membrane potential assay kit fmp in cells. Dendritic signals from rat hippocampal ca1 pyramidal.
The aim of the current study was to demonstrate the utility of the voltagesensitive dye approach to characterize the baseline electrophysiology of spontaneously active hipsccm and in particular determine the sensitivity of the technique to detect changes in depolarization and repolarization phases of the action potential. Cl showing a plane wave, the most common electrical propagation pattern. In this article, we focus on a refinement of the traditional voltageclamp methods that are used to measure current from whole cells, or relatively large areas of membrane, called the patchclamp. There is evidence that some voltage sensitive dyes respond differently on different preparations. Rh795 invitrogen or jpw 3031 or jpw 5029 leslie loew and joe p. Voltage sensitive dye recording from axons, dendrites and dendritic spines of individual neurons in brain slices. At this time, voltageclamp could only be applied to rather big cells as. The voltage sensitive dye vsd is a molecule that, once bound to the external surface of cell membranes, acts as a molecular transducer transforming changes in membrane potential into optical signals changes in. These cells enable rapid, electrodefree screening of speed and sensitivity. Patch clamp electrophysiology instruments used to evaluate ion channel behavior. The development of the patchclamp technique in the late 1970s has. Patch clamp electrophysiology, voltage clamp, action potential. The aim of the current study was to demonstrate the utility of the voltage sensitive dye approach to characterize the baseline electrophysiology of spontaneously active hipsccm and in particular determine the sensitivity of the technique to detect changes in depolarization and repolarization phases of the action potential.
Advanced patchclamp analysis for neuroscientists walmart. Patch clamping is voltage clamp but cleverly adapted so that you only need one electrode. Saltatory conduction along myelinated axons involves a. The calcium crimson dextran signal was recorded first, then the olfactory bulb was stained with di4anepps and the voltage sensitive dye signal recorded using the same wavelength filters, stimulus intensity, and detectors. The technique, based on laser excitation of voltagesensitive dyes, allows. Locate the soma of the stained neuron under low light level fluorescence and re patch the neuron with a standard no dye patch electrode under dic. A patch clamp study of the nicotinic acetylcholine receptor. All of these experiments could not be accomplished with conventional electrical measurements using microelectrode or. The patchclamp amplifier is set to voltageclamp mode, and the pipette resistance is. Imaging activity in slices, using fast voltagesensitive dyes, was pioneered using high temporal but low spatial resolution photodiode arrays 1 6, although for slower signals, higher resolution video cameras were also developed 7.
We also perform invivo and in vitro patchclamp recordings in brain slices, voltagesensitive dye imaging and optogenetic approaches in vivo and in vitro. The traditional goldstandard method of patch clamping provides singlecell data. Your browser does not currently recognize any of the video formats available. Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patchclamp characterization.
This volume presents current adaptations of the patch clamp technique to neuroscience. The voltage sensitivity of an ensemble of membraneinserted pcnrs in selfspiking hek293 cells was quite poor, with a. We combine behavioural experiments with different disease animal models, andor with systemic and stereotaxic injections. Video articles in jove about voltage sensitive dye include optical imaging of neurons in the crab stomatogastric ganglion with voltagesensitive dyes, preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices, investigations on alterations of hippocampal circuit function following mild traumatic brain injury. Patchclamp recording and computational modeling combined with highspeed voltagecalibrated optical recordings and em analysis reveal a second longitudinal conducting pathway formed by the periaxonal and paranodal submyelin spaces that are integral to reproducing the spatiotemporal profile of action potential saltation. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The axopatch 200b patch clamp offers the lowestnoise patchclamp amplifier technology. After the dye diffusion was completed, the preparation was typically incubated for an additional 1. Most of the work of the icp groups involves direct recordings from neurons in living brain slices.
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