Neuroscience is available from oxford university press. Application of active electrode compensation to perform continuous voltageclamp recordings with sharp microelectrodes j f gomezgonzalez1,2, a destexhe1 and t bal1 1unit for neurosciences, information and complexity unic, upr3293 cnrs, f91198 gifsuryvette. Timesharing techniques are used to prevent interactions between the two tasks fig. Negative feedback ensures that the inverting input of the ampli er is always at the command voltage v c. This is a significant advantage in nonslice protocols, such as invivo recording from intact animals, wholecell patch clamping, lfp recording, and spike single unit recording. What is continuous single electrode voltage clamp csevc. Comparison between continuous csevc and discontinuous.
This device is called a voltage clamp because it controls, or clamps, membrane potential at any level desired by the experimenter. This makes use of the current follower opamp con guration see appendix 1. Twoelectrode voltageclamp for large cells with low input resistances r m. Mar 15, 2011 moreover, voltage activated channels in the tip of the patch clamp electrode are subject to nearperfect extracellular voltage control, because of the tight electrical seal between the recording. This category describes a set of techniques in which one electrode is inserted into the cell. To make these recordings, the patch pipette is compared to the ground electrode. In this, an electrode is placed inside a cell, and serves both the voltage recording and currentpassing duties. A smalldiameter, firepolished glass capillary is pressed onto the surface of a cell and suction is used to form a highresistance seal 10 to. It is an electrophysiology patch clamp method that passes a membrane voltage into a cell and measures the change in current as the voltage steps. Continuous single electrode clamp sevcc main article. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc.
In discontinuous single electrode voltage clamp dsevc, the tasks of voltage recording and current passing are allocated to the same micropipette. Apr 25, 2008 this technique, often used with patch clamp electrodes, is called continuous single electrode voltage clamp csevc. The use of a discontinuous single electrode voltage. R e we use the socalled twoelectrode voltage clamp tevc. There are two ways that a singleelectrode voltage clamp can be achieved. Patch clamp technique has been successfully used for measurement of ionic flow caused by the activity of various ion transporting proteins including voltage operated ion channels, ligandoperated ion channels and electrogenic ion exchangers. Dec 17, 2018 patch clamp technique is a laboratory technique first used by neher and sakmann for studying the ion channel activity 12. Extracellular voltage recording single unit and field potential recording in brain or brain slices, electrocardiagrams, encephalograms and oculograms voltage clamp voltage is held constant and the current passing through the cell membrane is measured patchclamp recording two electrode voltage clamp. Lipid bilayer voltage clamp with reconstituted na channels. Wholecell patch clamp 1980, illustration from hille. In csevc, the same electrode is used simultaneously for voltage recording and for current passing. Our approach to rs compensation for a single electrode voltage clamp is based on the two electrode topology in conjunction with a membrane state estimator, as shown in fig.
Discontinuous single electrode voltage clamp sevcd technique is used with penetrating intracellular recording. The transmembrane current is recorded with the large patch electode while the membrane potential is held. The pipette in the photograph has been marked with a slight blue color. In those cases where r e is very small or the membrane current is very small such that the product of the electrode resistance and the membrane current is a few millivolts or even less than a millivolt, the seriesresistance.
This technique, often used with patch clamp electrodes, is called continuous singleelectrode voltage clamp csevc. Introduction the patchclamp is the gold standard in electrophysiology and is a fundamental technique to screen drugs and medical compounds, and ultimately to ensure consumer safety 1, 2. A single ion channel conducts around 10 million ions per second. Discontinuous single electrode voltageclamp sevcd technique is used with penetrating intracellular recording.
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. The important factors that need to be considered are given below. Applied in cell culture, this technique provides accurate control of the. The patch clamp technique allows the investigation of a small set or even single ion channels.
To maximize the performance of the two electrode voltage clamp the high frequency performance of the voltage recording microelectrode must be preserved. A single electrode voltage, current and patchclamp. Patch clamp electrophysiology, voltage clamp, action. This allows a researcher to keep the voltage constant while observing changes in current. 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. An integrated patchclamp potentiostat with electrode. Wholecell patchclamp recordings for electrophysiological. We discuss the two electrode voltage clamp and how hodgkin and huxley used it to elucidate the mechanism of the action potential. Voltage clamp techniques hebrew university of jerusalem. Discontinuous single electrode voltageclamp how is. A single electrode voltage, current and patch clamp amplifier with complete stable series resistance compensation. Patch clamping is voltage clamp but cleverly adapted so that you only need one electrode.
Continuous singleelectrode clamp sevcc technique is often used with patchclamp. The patch clamp technique allows the study of individual ion channels. Series resistance compensation for wholecell patchclamp. This category describes a set of techniques in which one electrode is used for voltage clamp. Continuous single electrode voltage clamp csevc discontinuous single electrode voltage clamp dsevc pipette.
Moreover, voltageactivated channels in the tip of the patchclamp electrode are subject to nearperfect extracellular voltage control, because of the tight electrical seal between the recording. Dsevc discontinuous single electrode voltage clamp. However, you dont see much if you hold the voltage constant so the voltage. To do this you need one electrode to measure the voltage and another electrode to pass current, positive or negative to stop the voltage from changing. Population patch clamp ppc is a novel high throughput planar array electrophysiology technique that allows ionic currents to be recorded from populations of cells under voltage clamp. Note that the value of zg based on vmeas is closer to the patch. Voltage clamp is one, if not the most powerful, approach to the study of the. Continuous single electrode voltageclamp csevc discontinuous single electrode. Voltage at the tip of the electrode and the ruptured patch benefits from series. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Oct 28, 20 the single electrode voltage clamp is a special purpose circuit clamping arrangement where a single microelectrode is used not only to measure the membrane voltage but also to pass the current necessary to control the voltage level. The patch clamp technique was originally developed in the late 1970s 25 and further improved by hamill et al.
The same pipette is used simultaneously for voltage recording and current passing. Keeping the membrane voltage constant by voltage clamp is key to. The basic principle of voltage clamp and system setup are illustrated as below reference dale purves,1997,neuroscience,sinauer associates, inc. Change the resistance of the electrode the patch clamp. It allows highresolution current recordings not only of whole cells, but also of excised cellular patches. This single electrode carries out the functions of both current injection and recording. Measurement of cellular excitability by whole cell patch. Continuous single electrode voltageclamp csevc molecular. Change the resistance of the electrode the patch clamp technique has greatly from biology 376 at university of waterloo. Application of active electrode compensation to perform.
The stem with the constant diameter is the longest part of. Send the appropriate oscilloscope setting commands whenever amplifier commands are issued from the interface. Continuous single electrode clamp sevc edit main article. The patch clamp technique has extended the application of voltage clamp methods to the recording of ionic currents. Circuit drawing of the discontinuous single electrode voltage clamp.
The csevc circuit ensures that the pipette potential vp is equal to vcmd. Continuous single electrode voltageclamp csevc is an electrophysiological patch clamping method to pass a membrane voltage into a cell and measure the change in current as the voltage steps. Since the membrane resistance r m changes in response to various stimuli during an experiment, the membrane potential v m is continuously monitored via the potential electrode pe, and then can be. Continuous single electrode voltageclamp csevc is an electrophysiological patchclamping method to pass a membrane voltage into a cell and measure the change in current as the voltage steps. How exactly does current and voltage clamping work. R m can be accurately measured in dual patch single cell recording.
Continuous single electrode clamp sevcc technique is often used with patch clamp recording. A computerassisted multielectrode patchclamp system. Errors in the measurement of voltageactivated ion channels. The holder houses both an optical fiber and an electrode enabling simultaneous patch clamp recording and optogenetic activation. The integrated patchclamp system will be used to fabricate highthroughput planar patchclamp systems. The low r m of passive astrocytes is the cause of poor voltage clamp quality. Dual patch voltage clamp study of low membrane resistance. It is discontinuous single electrode voltage clamp. The patch clamp technique, designed by neher and sakmann 1976. Continuous single electrode voltageclamp csevc is an electrophysiological patchclamping technique to pass a membrane voltage into a cell and measure the. Determine the oscilloscope scale, coupling and temporal resolution for the different steps of the patch clamp procedure in voltage clamp e. A single electrode voltage, current and patchclamp amplifier with. In dual patch recording, the v cinduced i and the actual voltage drop across the membrane v m, are measured separately by the two electrodes, so that r m can be calculated accurately from r m v m i. The same micropipette is used and shares time to inject current and record voltage.
A fast electronic switch alternates the connection to the microelectrode between these two functions. Likewise in the continuous single electrode voltage clamp mode the performance of the clamp is governed by the patch pipette. Action potentials recorded with patchclamp amplifiers. Twoelectrode voltage clamp with sharp intracellular electrodes. Intracellular recording cnrs delegation iledefrance sud. Patch clamp of a nerve cell within a slice of brain tissue. Oct, 2014 we discuss the two electrode voltage clamp and how hodgkin and huxley used it to elucidate the mechanism of the action potential. Pdf action potentials recorded with patchclamp amplifiers. The most common application of the wholecell patch clamp is the analysis. The voltage vm at all times is exactly clamped to the battery voltage vcmd.
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