The patch clamp technique, an electrophysiological technique that has been developed in the late 1970s 1,2, is a primary tool for studying single or multiple ion channel functions in live tissue. Patchclamp is the gold standard technique for highfidelity analysis of the electrical properties and functional connectivity of neurons. While suitable for a broad range of ionic currents, the series resistance rs of the recording pipette limits the bandwidth of the whole cell configuration, making it difficult to. The whole cell patchclamp technique involves a glass micropipette forming a tight gigaohm g. However, compared with in vitro whole cell recording, in vivo whole cell recording often suffers from low success rates and high access resistance, preventing its wide. The whole cell patch configuration is achieved when additional negative pressure is applied to the cell membrane through the pipette as it is in the cell attached configuration. This article briefly summarizes the use of this variant of the patch clamp technique in isolated cardiac myocytes with an emphasis on practical suggestions. This breaks the membrane and forms the whole cell configuration. Change in holding current but not membrane resistance.
Such a method is the socalled loose patch clamp almers, stan. 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. Why does seal resistance fall to less than 50 mohms when i do whole cell patch clamp. Wholecell patchclamp recordings in brain slices protocol. Whole cell patch clamp wholecell recording requires the experimenter to patch on to the membrane of a single neuron with a patch pipette, remove the small patch with suction, and maintain a seal with the neuron for the duration of recording. 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. Why does seal resistance fall to less than 50 mohms when i do. This screencast describes iv and vi relations derived from patch or current clamp data, and how to calculate conductance or resistance from the data. The cell attached patch configuration is a noninvasive approach which is used to measure the currents current clamp of single ion channels of the intact cell. Clip detecting with series resistance compensation using. Wholecell patchclamp recordings of isolated primary. Perforated patch clamp analysis of the passive membrane properties of.
Whole cell patch clamp techniques are widely used to measure membrane currents from isolated cells. While suitable for a broad range of ionic currents, the. Data analysis was accomplished with the qpatch assay software. The technician would position the glass pipette near a cell and apply the appropriate suction to create an electrical seal between the pipette and the cell membrane. While suitable for a broad range of ionic currents, the series resistance r s of the recording pipette limits the bandwidth of the whole cell configuration, making it difficult to measure rapid ionic currents. 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. In particular, permeabilised patch clamp wholecell recordings using nystatin or amphotericin b usually have 2 to 3fold higher series resistance compared to normal wholecell recordings.
Wholecell recording of neuronal membrane potential during. In vivo wholecell recording with high success rate in. Series resistance compensation for wholecell patchclamp. Im studying a population of cortical neurons in rodent brain slices using whole cell patchclamp technique. Calculating conductanceresistance from patchcurrent. 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. First, the open patch pipette isinserted into the extracellular solution of the recording chamber with positive pressure inside the pipette.
One i break into my cell, i clamp and 60mv and input a pulse of 4mv. Whole cell voltage clamp of isolated heart cells has led to an explosion of information about ionic currents in the heart. Series resistance compensation for wholecell patchclamp studies. These things are bad, but what is worse is when it changes over time. Understand whole cell capacitance and series resistance. C, membrane capacitance in picofarads onlinefit, wholecell model. The whole cell patch clamp technique involves a glass micropipette forming a tight gigaohm g.
The patchclamp amplifier is set to voltageclamp mode, and the pipette resistance is monitored by applying voltage steps v while measuring. To prepare a membrane in the excised patch mode, the pipette is pulled away from the cell. As a result, input resistance is also much more uniform less dependent on location, and, if you knew the total surface area of the cell. Limitations of quantitative wholecell voltage clamp posted on may 9, 2017 by p. Automated wholecell patch clamp electrophysiology of. Since its introduction by professors bert sakmann and erwin neher in the mid 1970s, patch clamp has been the classic method for studying ion channel function. Series resistance compensation for wholecell patch clamp studies using a membrane state estimator adam j. The activity of the channels in the entire plasma membrane can be monitored in this whole cell mode. Data analysis recorded ion channel whole cell currents were stored in an integrated database oracle.
D wholecell recordings are typically established in threesteps. As a critical technique for dissection of synaptic and cellular mechanisms, whole cell patch clamp recording has become feasible for in vivo preparations including both anaesthetized and awake mammalian brains. Series resistance compensation for whole cell patchclamp studies using a membrane state estimator. This method provides a current resolution several orders of magnitude larger than previous current measurement methods. Structural biochemistrymembrane proteinsion channels. Wholecell patch clamp recording has been successfully used in identifying the voltagedependent gating and conductance properties of ion.
Several patch clamp configurations can be used depending on the research interests, but in all cases, electrophysiological recordings are produced using a glass micropipette in contact with a patch of the neurons membrane. The resulting electrical current and changes in membrane potential can be measured using the patchclamp technique, in which a membrane patch is aspirated into a clean glass micropipette so that it adheres to the pipette surface and creates a highresistance 1 g. Series resistance compensation for wholecell patchclamp studies using a membrane state estimator. I am performing whole cell current clamp in ca1 hippocampal neurons from slices. Only a negligible amount of current will flow across the seal, as the seal resistance is at least an order of magnitude larger than the membrane resistance now, the membrane resistance is determined by the entire cells membrane area, not just the membrane patch within the. I then put on wholce cell capacitance to reduce the fast component of my transients the slow components remain. Measurement of cellular excitability by whole cell patch. I am new to electrophysiology in general and i am doing whole cell patch clamp at the moment to investigate. The wholecell technique involves rupturing a patch of membrane with mild suction to provide lowresistance electrical access, allowing control of transmembrane. Secondly, a high seal resistance reduces the current noise of the recording. I wanted to know what would be the best way to calculate the input resistance of the neurons from the iv curve.
How to calculate input resistance in current clamp. I am new to electrophysiology in general and i am doing whole cell patch. Patch clamp electrophysiology, voltage clamp, action. Methodology open access dual patch voltage clamp study. The whole cell technique involves rupturing a patch of membrane with mild suction to provide low resistance electrical access, allowing control of transmembrane voltage. Patch clamp system all measurements have been performed on a qpatch automated patch clamp system. Why does seal resistance fall to less than 50 mohms when i.
Standard whole cell voltage clamp and capacitance recordings were performed using an epc9 patch amplifier heka electronics, lambrechtpfalz, germany and patchmaster software. Abstract whole cell patch clamp techniques are widely used to measure membrane currents from isolated cells. Applications of the patch clamp method from the four patch clamp techniques, the cell. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. While suitable for a broad range of ionic currents, the series resistance rs of the recording pipette limits the bandwidth of the wholecell configuration, making it difficult to measure rapid ionic currents. Using an equivalent circuit mimicking simultaneous whole cell voltage clamp and cell attached current clamp recordings we have defined both mathematically and experimentally the relationship between the membrane patch resistance, the seal resistance, and the fraction of the whole cell potential recorded across an intact membrane patch. Whole cell voltageclamp experiments were performed with an intracellular. Dual patch voltage clamp study of low membrane resistance. It allows direct measurement of single channel currents as well as of the total current across the entire cell membrane. Whole cell patch clamp recording is a wellestablished technique for examining the intrinsic properties of both excitable and nonexcitable cells, and is particularly helpful for studying the functions of primarily dissociated cells in heterogeneous cell samples. Clip detecting with series resistance compensation. The interpretation of currentclamp recordings in the cell. Electrodes in the pipette and chamber can then be used to control the voltage across the membrane patch. Loose patch clamp in certain situations, however, a low seal resistance can be an advantage.
While suitable for a broad range of ionic currents, the series resistance rs of the recording pipette limits the bandwidth of the whole cell configuration, making it difficult to measure rapid ionic currents. Dual patch voltage clamp study of low membrane resistance astrocytes in situ baofeng ma1, guangjin xu2, wei wang1, john j enyeart1 and min zhou1 abstract whole cell patch clamp recording has been successfully used in identifying the voltagedependent gating and conductance properties of ion channels in a variety of cells. Among the different patch configurations that can be achieved, wholecell patchclamp recordings allow the study of the electrical behavior of a substantial part of the neuron. The advantage of whole cell patch clamp recording over sharp microelectrode recording is that the larger opening at the tip of the patch clamp electrode provides lower resistance and thus better electrical access to the inside of the cell. The electrode is left in place on the cell, but more suction is applied to rupture the membrane patch, thus providing access to the intracellular space of the cell. Abstract wholecell patchclamp techniques are widely used to measure membrane currents from isolated cells. Alternatively, investigators can pull a patch of membrane away from the cell and evaluate currents through single channels via the insideout or outsideout patch clamp technique. This video describes the details of patch clamp technique starting from very basics and the utility of this technique in neuroscience. A patch clamp recording of current reveals transitions between two conductance states of a single ion channel. Patch clamp techniques for single channel and wholecell. The traditional manual method to patch clamp using glass pipettes was developed by erwin neher and bert sakmann and required a highly skilled technician.
A patch of membrane is subsequently ruptured by mild suction so that the glass micropipette provides a low resistance access to the whole cell, thereby allowing the investigator to control the transmembrane voltage. While suitable for a broad range of ionic currents, the series resistance r s of the recording pipette limits the bandwidth of the wholecell configuration, making it difficult to measure rapid ionic currents. I am new to patch clamping and wantd to ask about whole cell capacitance and series resistance. Under these conditions the background leak current caused by flow of random ions between the cell surface and the surface of the pipette tip. Because the volume of the electrode is larger than the volume of the cell, the soluble contents of the cell s interior will slowly be replaced by the contents of the electrode. However, the most frequently employed patch clamp mode is the whole cell mode where the membrane patch is interrupted by applying strong suction for a. Series resistance compensation for whole cell patch clamp studies using a membrane state estimator adam j. Sherman, alvin shrier, and ellis cooper department of physiology, mcgill university, montre. Im studying a population of cortical neurons in rodent brain slices using wholecell patchclamp technique. Rupprecht before i first dived into experimental neuroscience, i imagined whole cell voltage clamp recordings to be the holy grail of precision. Imagine a spherical cell to which a patch clamp electrode is attached.
Whole cell patch clamp an overview sciencedirect topics. Patch clamp techniques for single channel and wholecell recording. Measurement of cellular excitability by whole cell patch clamp. Patch clamp electrophysiology, voltage clamp, action potential. Wholecell patchclamp techniques are widely used to measure membrane currents from isolated cells. This automatically comes with correspondingly greater problems to achieve the command voltage. This micropipette contains a wire bathed in an electrolytic solution to. Isolation and wholecell patch clamping of arabidopsis. Whole cell patch larger opening at the tip of the patch clamp electrode provides lower resistance and thus better electrical access to the inside of the cell. To increase bandwidth, it is necessary to compensate for r s. The advantage of whole cell patch clamp recording over sharp microelectrode recording is that the larger opening at the tip of the patch clamp electrode provides lower resistance.
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