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. It has been applied to measure the action potentials of a single ttubule of cardiomyocytes by guixue bu et al. For each chromophore, approximately 100 analogues have been synthesized in an attempt to optimize the signaltonoise ratio that can be. When using ionsensitive indicator dyes such as fura2, no additional substances that buffer the ion under study i. Patchclamp fluorometry recording of conformational. To address the need for a simple and replicable technique for functional rmp. This has been partly achieved using organic voltagesensitive dyes in vitro, or in invertebrate preparations yet unspecific staining has prevented singlecell resolution measurements from mammalian preparations in vivo. 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 dyes di2anepeq jpw1114, di4anepps jpw211, and di8anepps jpw1153 are commercially available from invitrogen carlsbad, ca, usa. 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. In vitro functional imaging in brain slices using fast. Final concentration of the dye solution used in slice staining should be between 0.
We tested a range of 4 different anepbased voltagesensitive dyes with increasing lipophylic properties dye screening in rohr and salzberg, 1994. Finally, applications of patch clamp recording are provided to demonstrate how the biophysical properties of ion channels, cell excitability, and neuroactive compounds are evaluated in neurophysiology labs today. Patch clamp of a nerve cell within a slice of brain tissue. Optical recording of membrane potential on isolated spiral ganglion cells of newborn mice using a voltagesensitive dye. Matsumoto a, doi t, asako m, yang sm, and yamashita t. Since photometry combined with voltage clamp provides the most accurate measurement of the dye response to membrane potential changes, a plot of. This approach, named voltageclamp fluorometry vcf by its inventors, allows direct observations of the realtime motions of protein structures that are exposed on the extracellular side of the cell 4, 5, 7 19. In vitro functional imaging in brain slices using fast voltagesensitive. Wholecell recording of neuronal membrane potential. The voltagesensitive dye captures any changes to the resting membrane potential, and trpv1 channels are activated prior to compound addition, representing a more physiological scenario when channels would be activated prior to compound being available. Fast voltagesensitive dye imaging of excitatory and. As a holy grail of neuroscience, optical imaging of membrane potential could enable high resolution measurements of spiking and synaptic activity in neuronal populations. 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. Simultaneous recordings of voltage and current waveforms from.
Software tools for applications in electrophysiology. Offset voltages of various origins have to be considered in patchclamp experiments. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in. In vivo voltagesensitive dye imaging of subcortical brain. Among these techniques, voltagesensitive dye imaging vsd imaging is of particular interest due to its good spatial resolution. Laser excitation at nearoptimal wavelength regarding signal size improved the sensitivity of recording by a factor of 50 over previous approaches. After a significant amount of dye diffuses away from the soma into dendritic and axonal processes, the waveform of the ap is completely restored. Measurement of cellular excitability by whole cell patch. 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. Comparing data generated using the flipr membrane potential assay kits with results from the manual patch clamp method shows good correlation. This article describes a voltage sensitive dye recording method for monitoring electrical activity of individual neurons with submicrometer and submillisecond spatiotemporal resolution. Voltage clamp methods for the study of membrane currents.
To understand how a neuron can be excited, or to study the behavior of membrane potential, a current clamp is used. The patchclamp technique is a versatile electrophysiological tool for. The membrane current and changes in fluorescence are recorded simultaneously. B moving the c2 whiskers evokes transient increases in fluorescence, first in s1 black, then in m1 blue. When using ion sensitive indicator dyes such as fura2, no additional substances that buffer the ion under study i. To understand the classical current clamp experiment and to study the nature of spiking using current injection. Next, the key steps of a typical wholecell patch clamp experiment are outlined, in which a current voltage iv curve is generated. Similar to fura2 and related compounds, voltagesensitive dyes. A a craniotomy was performed over somatosensory cortex s1 and mortor cortex m1 in anesthetized mice to deliver the voltage sensitive dye rh1691.
Similar experiments can be performed with ph or clsensitive dyes. To maintain this command voltage level, an amplifier must inject current. This method was the best basic biophysical technique used for studying the ion channels. After establishing the whole cell patch clamp configuration 50ms command voltage u comm steps ranging from.
In an experiment using the voltageclamp method, the investigator controls the membrane voltage in a cell and measures the transmembrane current required to maintain that voltage. The patchclamp recording technique was developed by erwin. Pipette and bath solutions were identical in patch clamp experiments and. Voltagesensitive dye recording from axons, dendrites and dendritic spines of individual neurons in brain slices. Biolistic delivery of voltagesensitive dyes for fast. Unlike extracellular electrophysiology techniques, it is possible to detect subthreshold synaptic potentials in addition to spiking activity. 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.
Use of ratiometric fluorescence measurements of the voltage. Annine6plus is the latest voltage sensitive dye with fast response ns response time and high voltage sensitivity. Comparison of patch clamp and flipr membrane potential assay kit data the manual patch clamp method is able to detect fast responses allowing detection of very rapid changes in membrane 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. However the unresolved multicomponent origin of the optical signal encourages us to perform a detailed analysis of the method limitation. The dyes that have this property have been divided into two practical groups, slow dyes and fast dyes. In the voltage clamp mode the recorded signal is the transmembrane current and the controlled input is the. Voltage clamping activates voltagesensitive ion channels, as well as allows monitoring of the functional states of the channels. For patch clamp and voltage sensitive dye measurements, rptectert1 cells were seeded at 50% density into 35 mm dish and 12well plate respectively and were grown for 3 days before starting the experiments. Voltagesensitive dye recording from axons, dendrites and.
Rather than penetrating the cell with sharp electrodes as is traditionally performed in voltageclamp experiments, in the patchclamp technique, blunttipped glass pipettes are used in such a way that, when pressed gently against the membrane of a cell, they isolate a small area of membrane. The patch clamp technique, however, is routinely used to measure. Defined extracellular ionic solutions to study and manipulate. Neurophysiology in cockroach distinguish extracellular, intracellular, patchclamp and optical imaging of electrical activity. I compensate my series resistance 7580%, i have sodium currents that range from 0. Patch clamp technique has been successfully used for measurement of ionic flow caused by the activity of various ion transporting proteins including voltageoperated ion channels, ligandoperated ion channels and electrogenic ion exchangers. Voltagesensitive dye an overview sciencedirect topics. In vitro functional imaging in brain slices using fast voltage sensitive dye imaging combined with wholecell 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.
Figure 2 illustrates four different chromophores the merocyanine dye, xvii, was used for the measurement illustrated in fig. Intensity graph top, electrical data middle and images bottom recorded during a patch clamp experiment in an isolated cardiomyocyte. Plasticity of representational maps in somatosensory cortex observed by in vivo voltagesensitive dye imaging. Understanding the spatiotemporal organization of longterm synaptic plasticity in neuronal networks demands techniques capable of monitoring changes in synaptic responsiveness over extended multineuronal structures. Fill glass patch pipettes from the tip with dye free intracellular solution by applying negative pressure for about 15 s up to 23 of the electrode taper. Highthroughput drug profiling with voltage and calcium. Imaging inhibitory synaptic potentials using voltage sensitive dyes. Oct 28, 20 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. While patch clamp remains the gold standard for its precision and control, it requires specific. The development of the patchclamp technique in the late 1970s has. Voltagesensitive dye vsd imaging directly assays the spatiotemporal.
Longterm spatiotemporal reconfiguration of neuronal. Rhodol voltagefluor5 rvf5 is a voltagesensitive dye with improved 2p crosssection for use in thick tissue or brain samples. Mar 14, 2017 we have designed, synthesized, and applied a rhodolbased chromophore to a molecular wirebased platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using twophoton 2p illumination. A voltage clamp experiment can give information about the current. Defined extracellular ionic solutions to study and. Although increasing the dye concentration to enhance vsd signal is tempting, higher dye concentrations are associated with higher. A voltagesensitive dyebased assay was developed using ph 5.
The renal proximal tube epithelial cell rptectert1 line was obtained from atcc. New standard in electrophysiology and deep tissue imaging. Voltage clamp protocol can be used to study a minute patch of neuronal membrane sealed to the end of glass pipette by measuring the current. Neuroscience is available from oxford university press. Patch pipettes were pulled on a horizontal puller p97 sutter instruments, novato, ca, usa from borosilicate glass gb 150f8p, science products gmbh, germany with a tip resistance of 67 m. Patch clamp electrophysiology instruments used to evaluate ion channel behavior. How can i calculate the voltage error in a wholecell patch. The wholecell patch clamp and the two electrode voltage clamp techniques are appropriate methods to study ec coupling in short fdb muscle fibres from the mouse. Patchclamp is the gold standard technique for highfidelity analysis of the. Next, the key steps of a typical wholecell patch clamp experiment are outlined, in which a currentvoltage iv curve is generated. The voltage sensitivity of an ensemble of membraneinserted pcnrs in selfspiking hek293 cells was quite poor, with a.
Simultaneous recordings of voltage and current waveforms. Rather than penetrating the cell with sharp electrodes as is traditionally performed in voltage clamp experiments, in the patch clamp technique, blunttipped glass pipettes are used in such a way that, when pressed gently against the membrane of a cell, they isolate a small area of membrane. With this optical system, we were able to run the camera at a 400hz frame rate 2. A membrane potentialsensitive dye for vascular smooth muscle cells assays. The pipette in the photograph has been marked with a slight blue color. Patch clamp electrophysiology, voltage clamp, action potential. Fill glass patch pipettes from the tip with dyefree intracellular solution by applying negative pressure for about 15 s up to 23 of the electrode taper. Rhodol voltagefluor5 rvf5 is a voltage sensitive dye with improved 2p crosssection for use in thick tissue or brain samples.
For electrodes invented for experiment in vitro, sheet electrode and electrode cuvette, electrodes with highspeed fluorescence imaging system, electrodes with patchclamp, and electrodes with confocal laser scanning microscopy are introduced. We have designed, synthesized, and applied a rhodolbased chromophore to a molecular wirebased platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using twophoton 2p illumination. 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 most common application of the wholecell patch clamp is the analysis. Now you will find a voltage pulse in the upper diagram which you can change in the same way as the current pulse in the currentclamp lab.
The voltagesensitive dye experiment provides an independent test of the interpretation of wholecell patch clamp data presented so far. Many patch clamp amplifiers do not use true voltage clamp circuitry, but instead are differential amplifiers that use the bath electrode to set the zero current ground level. Current recorded purple, trigger pulses for stimulation of cells red and line trigger from the scanner green. K1016 and k1046 provide sufficient reagent volumes for performing approximately 20 sets each set is a 96well assay plate done in 100l volume of assays, each using 5 m cc2dmpe and 10 m of either disbac23 or disbac43. The voltage clamp has had been used as the best biophysical technique for the last few decades.
The voltage sensitive dye experiment provides an independent test of the interpretation of wholecell patch clamp data presented so far. 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. For simplicity we illustrate the voltage clamp experiment in an axon with poisoned na channels and we ignore the small leakage currents. Patch clamp electrophysiology, voltage clamp, action. Optical recording of membrane potential on isolated spiral ganglion cells of newborn mice using a voltage sensitive dye. At this time, voltageclamp could only be applied to rather big cells as. Rather than penetrating the cell with sharp electrodes as is traditionally performed in voltageclamp experiments, in the patchclamp technique, blunttipped glass pipettes are used in such a way that, when pressed gently against the membrane of a. Similar to the performance of dibac 4 3 with the plasma membrane, the relationship between the clamped potential and the fluorescence intensity of the tonoplast was sigmoidal figure 6b.
The whole cell patch clamp can operate in two recording modes depending on the configuration of a patch clamp amplifier. 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. Currentand voltageclamp experiments in virtual laboratories. B, timing of the current injection pulse used to trigger ap green trace and current. Rapid changes in the membrane potential of neurons and cardiomyocytes define the unique physiology of these cells. The electrode modality development in pulsed electric. Voltage offsets in patchclamp experiments springerlink. Voltagesensitive dyes shift their absorption or emission fluorescence based on the membrane potential, allowing a scientist to gauge the global electrical state of a neuron. Experiments, approved by basel cantonal authorities, were done in 250. Which methods used to observe neuron activity can be used on single cells. Example of a voltage sensitive dye imaging experiment. How can i calculate the voltage error in a wholecell. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. Use of ratiometric fluorescence measurements of the.
The patch clamp technique in the whole cell configuration is. Membrane potential measurements of isolated neurons using. Flipr membrane potential assay kits molecular devices. A critical evaluation of whole cell patch clamp studies on. We attribute this poor performance to imperfect membrane insertion for a large fraction of.
285 1046 1294 971 334 192 1125 1180 717 350 348 111 645 653 438 829 770 1112 1456 1461 1218 620 542 27 1138 565 207 108 890 838 140 668 843 819 1091 958 1147