Compared to singlecell electrophysiology, our ability to directly study. After introducing one or more such compounds into tissue via perfusion. Voltagesensitive dye recording from axons, dendrites and. Membrane potential measurements of isolated neurons using a. Because fast vsds respond to membrane potential changes with microsecond temporal resolution, these are better suited than calcium indicators for recording rapid neural signals. Series resistance compensation for wholecell patchclamp studies using a membrane state estimator adam j. In a series of studies with a model membrane system, a spherical lipid bilayer, we showed that the potential develops on the membrane in good agreement with a time. Advanced patchclamp analysis for neuroscientists in. Throughout the history of neuroscience, electrophysiological and imaging techniques have been utilized to observe neural signals at various spatial and temporal scales. Chapters focus on invivo recordings, voltagegated channel recording and analysis. Rapid changes in the membrane potential of neurons and cardiomyocytes define the unique physiology of these cells. Single cell voltage sensitive dye vsd imaging zecevic. Voltage sensor probes vsps are a fluorescence resonance energy transfer fretbased voltage sensing assay technology, used to measure changes in cells membrane potential. Thus, in a series of experiments, we investigated the effect of the.
Fast voltagesensitive dye imaging of excitatory and inhibitory. 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. Experimental ulcers alter voltagesensitive sodium currents in. I am new to patch clamping and wantd to ask about whole cell capacitance and series resistance. Optogenetics and electrophysiology oxford medicine. Patch clamp electrophysiology instruments used to evaluate ion channel behavior. Elucidating the link between structure and function of ion channels and transporters with voltage clamp and patch clamp fluorometry giovanni zifarelli and jana kusch. Additional evidence for voltage sensing by pcnrs was provided by simultaneous patchclamp and fluorescence measurements. Defined extracellular ionic solutions to study and. It involves measurements of voltage changes or electric current or. Electrophysiology is the branch of physiology that studies the electrical properties of biological cells and tissues. This volume presents current adaptations of the patchclamp technique to neuroscience. It brings useful information for deciding whether voltagesensitive dye imaging is an appropriate method for your experimental question, and how to determine if a student is ready to. A critical evaluation of whole cell patch clamp studies on.
In contrast to action potentials where the membrane potential is free to spontaneously change, voltage clamp enables the membrane potential to be controlled i. Imaging inhibitory synaptic potentials using voltage sensitive dyes. Cortical dendritic spine heads are not electrically. I do most of my recordings in current clamp whereas most people in my lab use voltage clamp, i have often heard it said that changes in series resistance are more of a problem in voltage clamp. Dendritic spines located on individual neurons process information, but our understanding of the electrical behaviour of spines is still limited.
After the dye diffusion was completed, the preparation was typically incubated for an additional 1. However, it has been difficult to manipulate the activity of specific cells or neural circuits with the spatial and temporal resolutions relevant to neural coding. 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. Heterotypic gap junction channels as voltagesensitive. We show here that the experimental approach is open to testing by whole cell patch clamping of cells stained with the voltage sensitive dye annine6. Series resistance compensation for wholecell patchclamp. 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. In vitro functional imaging in brain slices using fast. All of these experiments could not be accomplished with conventional electrical measurements using microelectrode or patch clamp techniques. A new electrooptical approach for conductance measurement. In this respect, it would be desirable for a consortium to standardize the haphazard nomenclature of new indicators to enable easier access to this increasingly complex body of work and to prevent confusion arch vs arclight, or vsd as acronym used for both voltagesensitive domain and voltagesensitive dye, for example or colorful, but not.
With the calciumsensitive dye fluo4 labelled isolated cardiomyocytes from trout were stimulated by a trigger pulsing regime using patch clamping heka epc 10 double. Imaging inhibitory synaptic potentials using voltage. 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. Cell membranes can also be stained with voltage sensitive dyes. In vitro functional imaging in brain slices using fast voltagesensitive. Written for the popular neuromethods series, chapters include the kind of detail and key implementation advice that ensures successful results. Voltage sensitive dye imaging vsd can be used to record neural activity in hundreds of locations in preparations ranging from mammalian cortex to invertebrate ganglia. The portfolio of amplifiers includes axopatch 200b for ultra lownoise singlechannel recordings, multiclamp 700b for wholecell voltageclamp and highspeed currentclamp recordings. A novel technique called optogenetics, has recently been. After 1525 min, fine dendritic structures appeared. Fivephoton membrane potential ion channel assay kit fluorescent voltage sensitive dye kit kw. Using simultaneous voltage and calcium imaging to study fast.
Single cell voltagesensitive dye vsd imaging zecevic, 1996 has the potential to. What is the difference between vclamp and iclamp normal. 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. Voltagesensitive dye vsd imaging directly assays the spatiotemporal dynamics of. Voltagesensitive dye an overview sciencedirect topics. Vsd imaging also records voltage changes from processes too small to access with patchclamp electrodes in intact cells. Slices that show little surface damage and a large number of healthy cells when. Patchclamp amplifiers from single channels to large macroscopic recordings. 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.
The first step in our proposed method is to generate a fluorescencevoltage calibration curve to obtain the variation of fluorescence for unitary variation of voltage, by combining patch clamp and voltagesensitive dye optical approaches fig. At the same time, the membrane potential was changed with a series of voltageclamp pulses from. While patch clamp remains the gold standard for its precision and control, it requires specific. Patch clamp electrophysiology, voltage clamp, action.
This fretbased measurement is restricted to the plasma membrane and is therefore more relevant than nonfret single dye based approaches that measure signals. 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. Membrane insertion ofand membrane potential sensing by. Axon instruments patchclamp amplifiers molecular devices. In an initial series of experiments, we randomly chose 5 coverslips to determine the percentage of dii. Junctional conductance g j was measured using the dual wholecell voltage clamp method. For dye transfer studies, a given fluorescent dye was introduced into cell1 of a cell pair through a patch pipette in wholecell voltage clamp mode and the fluorescence intensity of. In vitro functional imaging in brain slices using fast voltagesensitive dye imaging combined with wholecell patch recording. I then put on wholce cell capacitance to reduce the fast component of my transients the slow components remain.
They are able to provide linear measurements of firing activity of single neurons, large neuronal populations or activity of myocytes. We used borosilicate electrodes for wholecell patch clamp recordings 1. Here, we show how this approach can be used to investigate the function of ca. The classical means to monitor membrane potentials is patch clamp electrophysiology, a lowthroughput and highly invasive technique. Similar to fura2 and related compounds, voltagesensitive dyes. In voltage clamp, series resistance prevents your amplifier from charging the membrane capacitor, and in current clamp, series resistance stops your cell from being able to charge the capacitance of your pipette. This chapter discloses disadvantages associated with voltagesensitive dyes. Here we show the results of successfully combining high resolution vsd.
We tested the hypothesis that nearinfrared fluorescent voltagesensitive dye di4anbdqbs exhibits minimal phototoxicity and high precision to allow for moderatethroughput analysis of human. Voltage clamp techniques represent the gold standard in the field and provide. Membrane potential measurements of isolated neurons using. A fine control of membrane ion fluxes through the ion channels is a basic requirement for the maintenance of cellular homeostasis and it is also at the basis of specialized cellular functions as. One i break into my cell, i clamp and 60mv and input a pulse of 4mv. A critical evaluation of whole cell patch clamp studies on electroporation using the voltage sensitive dye annine6. Many physiological processes are accompanied by changes in cell membrane potential which can be detected with voltage.
Voltage clamp is one, if not the most powerful, approach to the study of the voltagegated na channel. Can someone advise on series resistance in current clamp. Examples are voltage sensitive dyes and fluorescing proteins. To measure the fmp fluorescence under voltage clamp conditions, the fmp dye was excited at a rate of 4 hz with a 530 nm wavelength exposure time 6 ms. This volume presents current adaptations of the patch clamp technique to neuroscience. Pipette and bath solutions were identical in patch clamp experiments and. Somatic wholecell recordings were done using a multiclamp 700a amplifier. Understand whole cell capacitance and series resistance. Highsensitivity fluorometry to resolve ion channel conformational. The stimulation protocol on the patch clamp setup was synchronised with the confocal time lapse series using. Combined optogenetics and voltage sensitive dye imaging at. Finally, we define a series of extracellular solutions using nonpermeable. The mitochondriaspecific voltagesensitive dye rh123 was applied via the patch pipette. Elucidating the link between structure and function of ion channels and transporters with voltageclamp and patchclamp fluorometry giovanni zifarelli and jana kusch.
Advanced patchclamp analysis for neuroscientists alon. We used the dibac voltagesensitive dye which signal is stable while recording. We used borosilicate electrodes for wholecell patchclamp. Fast voltagesensitive dye imaging of excitatory and. Voltagesensitive dye recording from axons, dendrites and dendritic spines of individual neurons in brain slices. Historically the patch clamp technique was based on the work of alan hodgkin and andrew huxley who, in the 1950s, conducted a series of elegant voltage clamp experiments that allowed the recording of macroscopic currents in the squid giant axon by controlling the voltage of the membrane hodgkin and huxley, 1952. New standard in electrophysiology and deep tissue imaging. There were no lesions in the control and 3 6 1 ulcers in. 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. 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. Electrical behaviour of dendritic spines as revealed by.
The patchclamp amplifier is set to voltageclamp mode, and the pipette resistance is monitored by applying voltage. Voltagesensitive dyes, also known as potentiometric dyes, are dyes which change their spectral properties in response to voltage changes. If you want to know more there is a rg thread discussing the difference between vclamp and. Within 510 min after establishing the wholecell recording mode, the somata and the primary dendrites, but not the nuclear areas, became fluorescent, showing a bright, dotted staining pattern. The use of environmentally sensitive dyes in voltageclamp fluorometry vcf. Xx and quantified fluorescent signal for a field of view and patch clamp electrophysiology to measure the rmp of individual cells. Advanced patch clamp analysis for neuroscientists pdf. The patch clamp technique is too laborious and the inherent phototoxicity of most voltagesensitive dyes limits their use in single cells. Keeping the membrane voltage constant by voltage clamp is key to understanding the behavior of ion channels. Imaging activity in slices, using fast voltagesensitive dyes.
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