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. Here, we report the characterization of the flipr membrane potential assay kit fmp in cells expressing voltage and ligandgated ion channels. In order to carry out my protocol, i need the whole cell configuration to remain stable for relatively long periods of time in voltage clamp. Browse electrophysiology patch clamp jobs from 1,000s of job boards and employer web sites in one place. Electrophysiological method for wholecell voltage clamp. The nalcn channel complex is voltage sensitive and. 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. Fluorescence emission spectral shift measurements of membrane potential in single cells. Voltagesensitive dye recording from axons, dendrites and dendritic spines of individual neurons in brain slices. Whole cell voltage clamp recordings from drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of d. Citeseerx modulation of intrinsic circuits by serotonin.
Voltagesensitive rhodol with enhanced twophoton brightness. For dye transfer studies, a given fluorescent dye was introduced into cell 1 of a cell pair through a patch pipette in whole cell voltage clamp mode and the fluorescence intensity of dye was measured in cell 1 and cell 2. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. 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. Use of ratiometric fluorescence measurements of the voltage. In this study, fast voltage sensitive dye imaging and extracellular and whole cell electrophysiology were used on entorhinal corticalhippocampal slice preparations to characterize. Using simultaneous voltage sensitive dye imaging and patch clamp recordings in slices from animals with epilepsy, we assessed the relative degree of synaptic excitation activated by multiple. After establishing the whole cell patch clamp configuration 50ms command voltage u comm steps ranging from. On the day of the experiment, transfected hek293t cells were seeded on polyllysinecoated glass coverslips at least 3 hours before recording. This could provide a suitable tool for the study of signal transduction chains including electrical responses of the guard cell. I then put on wholce cell capacitance to reduce the fast component of my transients the slow components remain. Here, the patch clamp technique in the whole cell and the outside out configuration was employed to monitor current voltage relations of protoplasts derived from the tobacco culture cell line bright yellow2. 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.
Double whole cell recordings and voltage sensitive dye imaging from the dendrites of pyramidal neurons revealed that the membrane of the apical dendrites is electrically leakier than that of other. After removal of the patch elec trode, we found that neurons recover their physiological intracellular chloride concentration. Thus measuring or monitoring the membrane potential is of great importance. Effect of nifedipine and ouabain treatment on electrophysiological and calcium signal from hipscderived cardiomyocytes. Authoritative and practical, advanced patch clamp analysis for neuroscientists aims to ensure successful results in the further study of this vital field. Fluorescence emission spectral shift measurements of. Advanced patchclamp analysis for neuroscientists springerlink. The voltage clamp technique allows an experimenter to clamp the cell potential at a chosen value. We describe a method that overcomes these limitations by imaging ipsps with voltage sensitive dyes. 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. Calcium and voltage sensitive dye signals evoked by olfactory nerve shock in an in vitro turtle olfactory bulb preparation.
During calibrations of the dye, the membrane potential was clamped to different levels ranging from. Pdf in vitro functional imaging in brain slices using. We have applied multiwavelength fluorescence recording with voltage control by whole cell patch clamping. Voltagesensitive dye an overview sciencedirect topics. The whole cell configuration of patch clamp was achieved by gentle suction under voltage clamp. Voltage sensitive dyes, also known as potentiometric dyes, are dyes which change their spectral properties in response to voltage changes. Investigations on alterations of hippocampal circuit. Voltagesensitive dye recording from axons, dendrites and. Decreased hyperpolarizationactivated currents in layer 5. Patch a previously selected neuron and allow free diffusion of the dye from a somatic patch pipette to the soma in the wholecell configuration for 2045 min. In vitro functional imaging in brain slices using fast voltage sensitive dye imaging combined with whole cell patch recording greg c carlson 1 and douglas a coulter 2, 3 1 translational research laboratory, university of pennsylvania school of medicine, room 2226, 125 s 31st street, philadelphia, pennsylvania 191043403, usa. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies.
A critical evaluation of whole cell patch clamp studies on electroporation using the voltage sensitive dye annine6 article in bioelectrochemistry amsterdam, netherlands 92c. The effects of 5ht 3 receptor agonists on cortical circuit response properties were studied in slices of ferret primary visual cortex using highspeed optical imaging of voltage sensitive dye signals and whole cell patch clamp recording. Defined extracellular ionic solutions to study and manipulate. Patch clamp was applied to cells using standard techniques hamill et al. In this study, fast voltage sensitive dye imaging and extracellular and. 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. We then used whole cell patch clamp to investigate the membrane potential of cardiac macrophages attached to cardiomyocytes. Juju makes your electrophysiology patch clamp job search faster and more comprehensive. Inhibitory neuron and hippocampal circuit dysfunction in. In vitro functional imaging in brain slices using fast. This is important because many of the ion channels in the membrane of a neuron are voltage gated ion channels, which open only when the membrane voltage.
The patch electrode used for dye loading was attached to the neuron in wholecell configuration for 2530 min. Highthroughput drug profiling with voltage and calcium. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following tbi. Jul 05, 20 i am new to patch clamping and wantd to ask about whole cell capacitance and series resistance. Defined extracellular ionic solutions to study and. Photometric recording of transmembrane potential in outer. Advances in understanding cortical function through combined voltagesensitive dye imaging, whole cell recordings, and analysis of cellular morphology. Oct 21, 2011 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. Figure 4 compares the time courses of a voltagesensitive dye signal using the styryl dye di4anepps and the calcium signal using calcium crimson dextran in an in vitro turtle olfactory bulb preparation. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole cell voltage clamp.
To confirm that membraneassociated rvf5 fluorescence in hek cells is voltage sensitive, hek cells were stained with rvf5 200 nm and subjected to whole cell patch clamp electrophysiology under voltage clamp conditions. Use of ratiometric fluorescence measurements of the. The steadystate and kinetics fluorescence properties of fmp were compared with those of dibac43, using both flipr and whole cell patch clamp. The whole cell patch clamp technique involves a glass micropipette forming a tight gigaohm g. The ohc transmembrane potential induced by the eef is shown to be highly nonuniform along the cell.
Next, we feature three distinct ex vivo recording techniques. Electrophysiology patch clamp jobs juju smarter job search. The amount of staining was determined by measuring the resting fluorescence from the cell body at reduced excitation light intensity. Advanced patchclamp analysis for neuroscientists in. This result clearly exemplified the relevance of using a calcium sensitive dye concomitantly with a voltage sensitive dye on the same plate to differentiate the pharmacological profiles of compounds.
Hek cells is voltage sensitive, hek cells were stained with rvf5 200 nm and subjected to whole cell patch clamp electrophysiology under voltage clamp conditions. Many physiological processes are accompanied by changes in cell. The existing gold or standard methods to measure or monitor the membrane potential are patch clamping and voltage sensitive dyes. Activation of the 5ht 3 receptor decreased the amplitude and lateral extent of excitation throughout postnatal. It is essential to obtain a seal as fast as possible so that the amount of dye free solution in the tip can be kept small. Elucidating the link between structure and function of ion channels and transporters with voltage clamp and patch clamp fluorometry giovanni zifarelli and jana kusch. Citeseerx modulation of intrinsic circuits by serotonin 5. Keywords invivo recordings voltage gated channel recording axonal recordings synaptic current recording advanced fluorescent techniques optogenetics voltage sensitive dye imaging. 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. Heterotypic gap junction channels as voltagesensitive valves. Sep 01, 2009 junctional conductance g j was measured using the dual whole cell voltage clamp method. Patch clamp electrophysiology, voltage clamp, action.
As neurons derived from axolnscs mature over time, the number of cells spiking increased up to 100% of the total number of neurons recorded at one month after plating figure 3a. Many physiological processes are accompanied by changes in cell membrane. Nevertheless, successful vsd recordings from slices require many of the same skills and present many of the same challenges as highquality singlecell electrophysiology in slices. On addition of 60 mm kcl, the kinetics of fluorescence changes of fmp using flipr were identical to those observed in the electrophysiological studies using whole cell current clamp. In vitro functional imaging in brain slices using fast voltagesensitive dye imaging combined with whole cell patch recording greg c carlson 1 and douglas a coulter 2, 3 1 translational research laboratory. Membrane potential measurements of isolated neurons using a. Cells were voltage clamped at room temperature in the whole cell configuration using an axopatch 200b amplifier molecular devices. The effects of 5ht 3 receptor agonists on cortical circuit response properties were studied in slices of ferret primary visual cortex using highspeed optical imaging of voltage sensitive dye signals and whole cell patch clamp. They are able to provide linear measurements of firing activity of single neurons, large neuronal populations or activity of myocytes. However, the most commonly used patch clamp mode is the whole cell mode where the membrane patch is disrupted by briefly applying strong suction to establish electrical and molecular access to the intracellular space. Jan 10, 2017 this work demonstrates that the whole cell patch clamp technique is stabilized by a dynamic passivation mechanism that can be used to control and measure the current and voltage of intact giant unilamellar vesicles guvs, a cell sized model biomimetic system in which the membrane composition, tension, and shape can be readily controlled. These cells enable rapid, electrodefree screening of speed and sensitivity. Spontaneously beating cardiomyocytes displayed typical resting. Advances in understanding cortical function through combined.
A patch of membrane is subsequently ruptured by mild suction so that the glass micropipette provides a lowresistance access to the whole cell, thereby allowing the investigator to control the transmembrane voltage. The first step in our proposed method is to generate a fluorescence voltage calibration curve to obtain the variation of fluorescence for unitary variation of voltage, by combining patch clamp and voltage sensitive dye optical approaches fig. Nonuniform distribution of outer hair cell transmembrane. Wholecell patch clamp can be used to characterize the maturation of neuronal cultures, both at the level of individual cells and at the networks connectivity level. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. When the cell is depolarized, the oxonol dye will associate to the interior of the cell membrane, thus separating the fret pair and disrupting fret figure 1.
In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. In vivo whole cell recordings bojana kokinovic, stylianos papaioannou, and paolo medini. This makes it possible to measure how much ionic current crosses a cells membrane at any given voltage. A novel membrane potentialsensitive fluorescent dye. Calcium and voltagesensitive dye signals evoked by olfactory nerve shock in an in vitro turtle olfactory bulb preparation. This protocol describes an in vivo technique for combining whole cell wc recordings of membrane potentials of individual neurons with voltage sensitive dye vsd imaging of the. This volume presents current adaptations of the patch clamp technique to neuroscience. Start by practicing fast patching without the dye in the electrode. Membrane electric properties by combined patch clamp and. Serotonergic projections are widespread in the developing neocortex, but their functions are obscure. Protoplasts derived from tobacco culture cell lines bright yellow2, by2. 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.
Ca1 hippocampal pyramidal neurons from brain slices were loaded with the voltage sensitive dye jpw1114 from a somatic patch electrode in whole cell. One i break into my cell, i clamp and 60mv and input a pulse of 4mv. Fast voltagesensitive dye imaging of excitatory and. While patch clamp remains the gold standard for its precision and control, it requires specific. Advances in understanding cortical function through combined voltage sensitive dye imaging, whole cell recordings, and analysis of cellular morphology carl c. The use of ratiometric fluorescence measurements of the voltage sensitive dye di4anepps to examine action potential characteristics and drug effects on human induced pluripotent stem cell derived cardiomyocytes. A patch clamp study on the electropermeabilization of higher. Virginia bright italian0, vbi0 were stained with the voltage sensitive dye annine6. After establishing the whole cell patch clamp configuration 50ms command voltage.
In vitro functional imaging in brain slices using fast voltagesensitive dye imaging combined with wholecell patch recording. The calcium signal in response to an olfactory nerve shock was measured first using dye in olfactory receptor neuron nerve terminals that had been transported from the cell bodies in the nose. In addition, kcl concentrationdependent increases in fmp fluorescence correlated with the changes of membrane potential recorded in whole cell patch clamp. Understand whole cell capacitance and series resistance. A critical evaluation of whole cell patch clamp studies on. We introduce a line of nonfluorescent hek cells that stably express nav 1. We developed a method to optically measure the phase of the ohc transmembrane potential using the voltage sensitive dye vsd di8anepps. The wholecell configuration of patch clamp was achieved by gentle suction under voltage clamp. It is essential to obtain a seal as fast as possible so that the amount of dyefree solution in the tip can be kept small. Sep 12, 2019 this greatly improves the targeting and accumulation of the modified electrochromic dye in the membrane of the phosphataseexpressing cell. The calcium crimson dextran signal was recorded first, then the olfactory bulb was stained with di4anepps and the voltagesensitive dye. In vitro functional imaging in brain slices using fast voltage sensitive dye imaging combined with whole cell patch recording. Ratiometric measurement of endothelial depolarization in arterioles with a potential sensitive dye. Dynamic depolarization fields in the cerebral cortex.
Cells were exposed to a sequence of voltage pulses including supraphysiological voltages. Tranexamic acid impairs hippocampal synaptic transmission. The aminostyrylpyridinium asp chromophore is an example of a voltage sensitive dye precursor with a phosphate group attached to its head group 30, 65. The membrane current and changes in fluorescence are recorded simultaneously. The dyes that have this property have been divided into two practical groups, slow dyes and fast dyes. I recorded mitral cells using whole cell patch clamp. Vsd imaging also records voltage changes from processes too small to access with patchclamp electrodes in intact cells. Wholecell recording and voltagesensitive dye imaging in vivo. Here, we have presented a method to investigate whole populations of guard cell protoplasts in a non. Screening fluorescent voltage indicators with spontaneously. Patch clamp electrophysiology instruments used to evaluate ion channel behavior. Advances in understanding cortical function through. Macrophages facilitate electrical conduction in the heart.
We used intracellular solutions containing either 40 or 5 mm cl. Briefly, patch electrodes of kimax capillary tubing 1. American scientific products, golden, ca were pulled on a twostage list lm3pa patch. 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. Cells are exposed to the voltage pulses of various sizes and the actual transmembrane voltage. In acute slices of the murine hippocampus, fast depolarizationmediated imaging signals fdss and postsynaptic currents pscs were recorded using voltage sensitive dye imaging and whole cell patch clamp. Imaging inhibitory synaptic potentials using voltage. The vsp fret pair consists of a mobile, voltage sensitive. A critical evaluation of whole cell patch clamp studies on electroporation using the voltage sensitive dye annine6. Patch a previously selected neuron and allow free diffusion of the dye from a somatic patch pipette to the soma in the whole cell configuration for 2045 min. The whole cell patchclamp technique involves a glass micropipette forming a tight gigaohm g. At the end of staining period, carefully pull the patch pipette away from the soma in voltage clamp configuration ensuring that the transition from whole cell to outsideout patch configuration is attained in the process. A novel membrane potentialsensitive fluorescent dye improves.
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