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{{BioPsy}}
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[[File:Patch Clamp Rig classic.jpg|thumb|right|Classical patch clamp setup, with microscope, antivibration table and micro manipulators]]
[[Image:Patch_Clamp_Rig_classic.jpg|thumb|left|Classical patch clamp setup, with microscope, antivibration table and micro manipulators]]
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[[File:Single channel.png|thumb|270 px|right|A patch clamp recording reveals transitions between two conductance states of a single ion channel: closed (at top) and open (at bottom).]]
[[Image:Planar_Patch_Setup.jpg|thumb|right|Complete miniaturized [[Electrophysiology#Planar_patch_clamp|planar patch clamp]] setup]]
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The '''patch clamp technique''' is a [[laboratory technique]] in [[electrophysiology]] that allows the study of single or multiple [[ion channel]]s in [[cell (biology)|cells]]. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as [[neuron]]s, [[cardiomyocyte]]s, [[muscle fiber]]s and [[pancreas|pancreatic]] [[beta cell]]s. It can also be applied to the study of bacterial ion channels in specially prepared giant [[spheroplasts]].
'''Patch clamp technique''' is a technique in [[electrophysiology]] that allows the study of individual [[ion channel]]s in [[cell (biology)|cells]]. The technique is used to study excitable cells such as [[neuron]]s, [[muscle fiber]]s and the [[beta cell]]s of the [[pancreas]]. In classical patch clamp technique, the [[electrode]] used is a glass [[pipette]], but [[Electrophysiology#Planar_patch_clamp|planar patch clamp]] uses a flat surface punctured with tiny holes.
 
   
Patch clamp technique is a refinement of the [[voltage clamp]]. [[Erwin Neher]] and [[Bert Sakmann]] developed the patch clamp in the late 1970s and early 1980s. They received the [[Nobel Prize in Physiology or Medicine]] in [[1991]] for this work.
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The patch clamp technique is a refinement of the [[voltage clamp]]. [[Erwin Neher]] and [[Bert Sakmann]] developed the patch clamp in the late 1970s and early 1980s. This discovery made it possible to record the currents of single [[ion channel]]s for the first time, proving their involvement in fundamental cell processes such as [[action potential]] conduction. Neher and Sakmann received the [[Nobel Prize in Physiology or Medicine]] in 1991 for this work.
   
 
==Basic technique==
 
==Basic technique==
[[Image:Patchclamp1.png|thumb|right|The cell-attached patch clamp uses a micropipette attached to the cell membrane to allow recording from a single ion channel.]]
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[[File:Pipette Puller-en.svg|thumb|Schematic depiction of a pipette puller device used to prepare micropipettes for patch clamp and other recordings]]
Patch clamp traditionally uses a glass pipette, with an open tip diameter of about one [[micrometre]], and is made such that the tip forms a smooth surfaced circle, rather than a sharp point. This style of electrode is known as a "patch clamp electrode" and is distinct from the "sharp microelectrode" used to impale cells in traditional [[Electrophysiology#Intracellular recording|intracellular recordings]]. The interior of the pipette is filled with different solutions (usually called the pipette solution) depending on the specific technique or variation used (see following). For example, with whole cell recordings, a solution that approximates the [[intracellular fluid]] is used. A metal electrode in contact with this solution conducts the electrical changes to a voltage clamp amplifier. The researcher can change the composition of this solution or add drugs to study the ion channels under different conditions. The patch clamp electrode is pressed against a [[cell membrane]] and suction is applied to the inside of the electrode to pull the cell's membrane inside the tip of the electrode. The suction causes the cell to form a tight seal with the electrode (a so-called "gigaohm seal", since the electrical resistance of that seal is in excess of a [[Ohm (unit)|gigaohm]]).
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[[File:Patchclamp Spheroplast1.jpg|thumb|right|A bacterial [[spheroplast]] patched with a glass pipette]]
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Patch clamp recording uses, as an [[electrode]], a glass [[micropipette]] that has an open tip diameter of about one [[micrometer]], a size enclosing a [[plasma membrane|membrane]] surface area or "patch" that often contains just one or a few ion channel molecules. This type of electrode is distinct from the "sharp microelectrode" used to impale cells in traditional [[Electrophysiology#Intracellular recording|intracellular recordings]], in that it is sealed onto the surface of the [[cell membrane]], rather than inserted through it. In some experiments, the micropipette tip is heated in a microforge to produce a smooth surface that assists in forming a high [[electrical resistance|resistance]] seal with the cell membrane. The interior of the pipette is filled with a solution matching the ionic composition of the bath solution, as in the case of cell-attached recording, or the [[cytoplasm]] for whole-cell recording. A chlorided silver wire is placed in contact with this solution and conducts electric current to the amplifier. The investigator can change the composition of this solution or add drugs to study the ion channels under different conditions. The micropipette is pressed against a cell membrane and suction is applied to assist in the formation of a high resistance seal between the glass and the cell membrane (a "gigaohm seal" or "gigaseal," since the electrical resistance of that seal is in excess of a [[Ohm (unit)|gigaohm]]). The high resistance of this seal makes it possible to electronically isolate the currents measured across the membrane patch with little competing [[electronic noise|noise]], as well as providing some mechanical stability to the recording.
Unlike traditional [[voltage clamp]] recordings, the patch clamp recording uses a single electrode to voltage clamp a cell. This allows a researcher to keep the voltage constant while observing changes in [[Electric current|current]]. Alternately, the cell can be current clamped, keeping current constant while observing changes in membrane [[membrane potential|voltage]].
 
   
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Unlike traditional two-electrode [[voltage clamp]] recordings, patch clamp recording uses a single electrode to record currents. Many patch clamp amplifiers do not use true voltage clamp circuitry but instead are [[differential amplifier]]s that use the bath electrode to set the zero current level. This allows a researcher to keep the voltage constant while observing changes in [[Electric current|current]]. Alternatively, the cell can be [[electrophysiology#Current clamp|current clamped]] in whole-cell mode, keeping current constant while observing changes in membrane [[membrane potential|voltage]].
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{{clear}}
 
==Variations==
 
==Variations==
Several variations of the basic technique can be applied, depending on what the researcher wants to study. The inside-out and outside-out techniques are called "excised patch" techniques, because the patch is excised (removed) from the main body of the cell. Cell-attached and both excised patch techniques are used to study the behavior of ion channels on the section of membrane attached to the electrode, while whole-cell patch and perforated patch allow the researcher to study the electrical behavior of the entire cell.
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[[File:Patch clamp.svg|thumb|The cell-attached patch clamp uses a micropipette attached to the cell membrane to allow recording from a single ion channel.]]
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Several variations of the basic technique can be applied, depending on what the researcher wants to study. The inside-out and outside-out techniques are called "excised patch" techniques, because the patch is excised (removed) from the main body of the cell. Cell-attached and both excised patch techniques are used to study the behavior of individual ion channels in the section of membrane attached to the electrode.
   
*'''Cell-attached patch''': The electrode remains sealed to the patch of membrane. This allows for the recording of currents through single ion channels in that patch of membrane. For ligand-gated channels or channels that are activated through the action of drug molecules, the drug of choice is usually included in the pipette solution. While the resulting channel activity can be attributed to the drug used, it is not possible to then change the drug concentration. The technique is thus limited to one point in a dose response curve per patch. Usually, the dose response is accomplished through several cells and patches. However, voltage-gated channels or channels that are activated through changes in the potential across the membrane, can be clamped at different membrane potentials using the same patch. This results in graded channel activation, and a proper I-V(current-voltage) curve can be established with only one patch.
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Whole-cell patch and perforated patch allow the researcher to study the electrical behavior of the entire cell, instead of single channel currents. The whole-cell patch, which allows for low resistance electrical access to the inside of a cell, has now largely replaced [[Electrophysiology#Sharp electrode technique|high resistance microelectrode]] recording techniques to record currents across the entire cell membrane.
*'''"Inside-out" patch''': After the gigaseal is formed, the electrode is quickly withdrawn from the cell, thus ripping the patch of membrane off the cell, leaving the patch of membrane attached to the electrode exposing the [[intracellular]] surface of the membrane to the external media. This is useful when an experimenter wishes to manipulate the environment affecting the inside of ion channels. For example, channels that are activated by intracellular ligands like cGMP can then be studied through a range of ligand concentrations.
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*'''Whole-cell recording''' or '''whole-cell patch''': The electrode is left in place, but more suction is applied to rupture the portion of the cell's membrane that is inside the electrode, thus providing access to the intracellular space of the cell. 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. A disadvantage of this technique is that the volume of the electrode is larger than the cell, so the soluble contents of the cell's interior will slowly be replaced by the contents of the electrode. This is referred to as the electrode [[dialysis|"dialyzing"]] the cell's contents. Thus, any properties of the cell that depend soluble intracellular contents will be altered. The pipette solution used usually approximates the high-potassium environment of interior of the cell. Generally speaking, there is a "grace period" at the beginning of a whole-cell recording, lasting approximately 10 minutes, when one can take measurements ''before'' the cell has been dialyzed. Whole-cell recordings involve recording currents through multiple channels at once.
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[[File:Patchmodes.svg|260px|left|thumb|Diagram showing variations of the patch clamp technique]]
*'''"Outside-out" patch''': After the aforementioned whole cell patch is formed, the electrode can be slowly withdrawn from the cell, allowing a bulb of membrane to [[bleb]] out from the cell. When the electrode is pulled far enough away, this bleb will detach from the cell and reform as a ball of membrane on the end of the electrode, with the outside of the membrane being the surface of the ball. Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is protected from the outside environment, but not in contact with its usual environment. In this conformation, the experimenter can perfuse the same patch with different solutions, and if the channel is activated from the extracellular face, a dose-response curve can then be studied. Single channel recordings are possible in this conformation if the bleb of membrane is small enough. This is the distinct advantage the outside-out patch variation possesses relative to the cell-attached method. However, it is more difficult to accomplish as more steps are involved in the patching process.
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=== Cell-attached or on-cell patch ===
*'''Perforated patch''': In this variation of whole-cell recording, the experimenter forms the gigaohm seal, then adds a new solution to the electrode containing small amounts of an antibiotic, such as [[Amphothericin-B]] or [[Gramicidin]] into the electrode solution to punch small perforations on the bit of membrane attached to the electrode. This has the advantage of reducing the dialysis of the cell that occurs in whole cell recordings, but also has several disadvantages. First, the access [[Electrical resistance|resistance]] is higher (access resistance being the sum of the electrode resistance and the resistance at the electrode-cell junction). This will decrease current resolution, increase recording noise, and magnify any series resistance error. Second, it can take a significant amount of time (10-30 minutes) for the antibiotic to perforate the membrane. Third, the membrane under the electrode tip is weakened by the perforations formed by the antibiotic and tends to rupture. When the patch ruptures, the recording is essentially in whole-cell mode, except with antibiotic inside the cell. All of these problems tend to limit the time-length of experiments, and so this technique is most appropriate for short-duration experiments of about an hour.
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The electrode is sealed to the patch of membrane, and the cell remains intact. This allows for the recording of currents through single ion channels in that patch of membrane, without disrupting the interior of the cell. For [[ligand-gated ion channels]] or channels that are modulated by [[receptor (biochemistry)#Metabotropic receptors|metabotropic receptors]], the [[neurotransmitter]] or drug being studied is usually included in the pipette solution, where it can contact what had been the external surface of the membrane. While the resulting channel activity can be attributed to the drug being used, it is usually not possible to then change the drug concentration. The technique is thus limited to one point in a [[dose-response relationship|dose response curve]] per patch. Usually, the dose response is accomplished using several cells and patches. However, [[voltage-gated ion channel]]s can be clamped at different membrane potentials using the same patch. This results in graded channel activation, and a complete [[Ohm's law|I-V (current-voltage) curve]] can be established with only one patch.
  +
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=== Inside-out patch ===
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After the gigaseal is formed, the micropipette is quickly withdrawn from the cell, thus ripping the patch of membrane off the cell, leaving the patch of membrane attached to the micropipette, and exposing the [[intracellular]] surface of the membrane to the external media. This is useful when an experimenter wishes to manipulate the environment at the intracellular surface of ion channels. For example, channels that are activated by [[second messenger system|intracellular ligands]] can then be studied through a range of ligand concentrations.
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=== Whole-cell recording or whole-cell patch ===
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[[File:WholeCellPatchClamp-03.jpg|thumb|right|Whole cell recording of a nerve cell from the [[hippocampus]]. The pipette in the photograph has been marked with a slight blue colour.]]
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Whole-cell recordings, in contrast, involve recording currents through multiple channels at once, over the membrane of the entire 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. 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. A disadvantage of this technique is that the volume of the electrode is larger than the cell, so the soluble contents of the cell's interior will slowly be replaced by the contents of the electrode. This is referred to as the electrode [[dialysis (biochemistry)|"dialyzing"]] the cell's contents. Thus, any properties of the cell that depend on soluble intracellular contents will be altered. The pipette solution used usually approximates the high-potassium environment of the interior of the cell. Generally speaking, there is a period at the beginning of a whole-cell recording, lasting approximately 10 minutes, when one can take measurements before the cell has been dialyzed.
  +
=== Outside-out patch ===
  +
After the whole-cell patch is formed, the electrode can be slowly withdrawn from the cell, allowing a bulb of membrane to [[bleb (cell biology)|bleb]] out from the cell. When the electrode is pulled far enough away, this bleb will detach from the cell and reform as a convex membrane on the end of the electrode (like a ball open at the electrode tip), with the original outside of the membrane facing outward from the electrode. Single channel recordings are possible in this conformation if the bleb of membrane is small enough. Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is isolated from the cell, and exposed to different solutions on the [[extracellular]] surface of the membrane. The experimenter can perfuse the same patch with different solutions, and if the channel is activated from the extracellular face, a dose-response curve can then be obtained. This is the distinct advantage of the outside-out patch relative to the cell-attached method. However, it is more difficult to accomplish, as more steps are involved in the patching process.
  +
  +
=== Perforated patch ===
  +
In this variation of whole-cell recording, the experimenter forms the gigaohm seal, but does not use suction to rupture the patch membrane. Instead, the electrode solution contains small amounts of an antifungal or antibiotic agent, such as [[amphothericin-B]], [[nystatin]], or [[gramicidin]]. As the antibiotic molecules diffuse into the membrane patch, they form small perforations in the membrane, providing electrical access to the cell interior. This has the advantage of reducing the dialysis of the cell that occurs in whole-cell recordings, but also has several disadvantages. First, the access resistance is higher, relative to whole-cell, due to the partial membrane occupying the tip of the electrode (access resistance being the sum of the electrode resistance and the resistance at the electrode-cell junction). This will decrease electrical access and thus decrease current resolution, increase recording noise, and magnify any [[voltage clamp#Continuous single-electrode clamp (SEV-c)|series resistance error]]. Second, it can take a significant amount of time for the antibiotic to perforate the membrane (10–30 minutes, though this can be reduced with properly shaped electrodes). Third, the membrane under the electrode tip is weakened by the perforations formed by the antibiotic and can rupture. If the patch ruptures, the recording is then in whole-cell mode, with antibiotic contaminating the inside of the cell.
  +
  +
=== Loose patch ===
  +
Loose patch clamp is different in that it employs a loose seal rather than the tight gigaseal used in the conventional technique. A significant advantage of the loose seal is that the pipette that is used can be repeatedly removed from the membrane after recording, and the membrane will remain intact. This allows for repeated measurements in a variety of locations on the same cell without destroying the integrity of the membrane. A major disadvantage is that the resistance between the pipette and the membrane is greatly reduced, allowing current to leak through the seal. This leakage can be corrected for, however, which offers the opportunity to compare and contrast recordings made from different areas on the cell of interest.
  +
  +
=== Automatic patch clamping ===
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[[Automated patch clamp]] systems have recently been developed, in order to inexpensively collect large amounts of data in a shorter period of time. Such systems typically include a single-use [[microfluidics|microfluidic]] device, either an [[injection molding|injection molded]] or a [[Polydimethylsiloxane|PDMS]] cast chip, to capture a cell or cells, and an integrated electrode.
   
 
==See also==
 
==See also==
*[[Electrophysiology#Planar_patch_clamp|Planar Patch Clamp]]
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{{Columns-list|2|
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*[[Bioelectronics]]
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*[[Cable theory]]
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*[[Channelome]]
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*[[Channelomics]]
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*[[GHK flux equation]]
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*[[Goldman equation]]
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*[[Multielectrode array]]
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*[[Electrophysiology#Planar patch clamp|Planar patch clamp]]
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*[[Slice preparation]]
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}}
   
 
==References==
 
==References==
* Kandel E.R., Schwartz, J.H., Jessell, T.M. (2000). ''Principles of Neural Science'', 4th ed., pp.152-153. McGraw-Hill, New York.
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* {{cite book |author=Kandel E.R., Schwartz, J.H., Jessell, T.M. |year=2000 |title=Principles of Neural Science, 4th ed. |publisher=McGraw-Hill, New York. |url=http://books.google.com/?id=yzEFK7Xc87YC&pg=PR35&dq=Principles+of+Neural+Science |isbn=978-0-8385-7701-1}} pp. 152–153.
* Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85-100.
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* {{cite journal |author=Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. |title=Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches |journal=Pflügers Archiv European Journal of Physiology |year=1981 |volume=391 |issue=2 |pages=85–100 |url=http://www.springerlink.com/content/h348734770442854/ |doi=10.1007/BF00656997 |pmid=6270629}}
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* {{cite journal |author=Tanzi S, Østergaard PF, Matteucci M, Christiansen TL, Cech J, Marie R, Taboryski RJ|title=Fabrication of combined-scale nano- and microfluidic polymer systems using a multilevel dry etching, electroplating and molding process |journal=Journal of Micromechanics and Microengineering|year=2012|volume=22|issue=11|pages=115008|url=http://iopscience.iop.org/0960-1317/22/11/115008 |doi=10.1088/0960-1317/22/11/115008
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}}
   
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==External links==
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* [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=neurosci.box.229 Alternative images for patch clamp variations]
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{{DEFAULTSORT:Patch Clamp}}
 
[[Category:Neurophysiology]]
 
[[Category:Neurophysiology]]
 
[[Category:Physiology]]
 
[[Category:Physiology]]
 
[[Category:Electrophysiology]]
 
[[Category:Electrophysiology]]
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[[Category:Laboratory techniques]]
   
 
{{Link FA|uk}}
 
{{Link FA|uk}}
 
:de:Patch-Clamp-Technik
 
[[fr:Patch-clamp]]
 
[[nl:Patch-clamp techniek]]
 
[[ja:パッチクランプ法]]
 
[[ru:Метод локальной фиксации потенциала]]
 
[[uk:Patch-clamp]]
 
{{enWP|Patch clamp }}
 

Latest revision as of 12:30, October 29, 2013

Patch Clamp Rig classic
Classical patch clamp setup, with microscope, antivibration table and micro manipulators
Dr Joe KiffAdded by Dr Joe Kiff
File:Single channel.png
A patch clamp recording reveals transitions between two conductance states of a single ion channel: closed (at top) and open (at bottom).

The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers and pancreatic beta cells. It can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts.

The patch clamp technique is a refinement of the voltage clamp. Erwin Neher and Bert Sakmann developed the patch clamp in the late 1970s and early 1980s. This discovery made it possible to record the currents of single ion channels for the first time, proving their involvement in fundamental cell processes such as action potential conduction. Neher and Sakmann received the Nobel Prize in Physiology or Medicine in 1991 for this work.

Basic techniqueEdit

File:Pipette Puller-en.svg
Schematic depiction of a pipette puller device used to prepare micropipettes for patch clamp and other recordings
File:Patchclamp Spheroplast1.jpg
A bacterial spheroplast patched with a glass pipette

Patch clamp recording uses, as an electrode, a glass micropipette that has an open tip diameter of about one micrometer, a size enclosing a membrane surface area or "patch" that often contains just one or a few ion channel molecules. This type of electrode is distinct from the "sharp microelectrode" used to impale cells in traditional intracellular recordings, in that it is sealed onto the surface of the cell membrane, rather than inserted through it. In some experiments, the micropipette tip is heated in a microforge to produce a smooth surface that assists in forming a high resistance seal with the cell membrane. The interior of the pipette is filled with a solution matching the ionic composition of the bath solution, as in the case of cell-attached recording, or the cytoplasm for whole-cell recording. A chlorided silver wire is placed in contact with this solution and conducts electric current to the amplifier. The investigator can change the composition of this solution or add drugs to study the ion channels under different conditions. The micropipette is pressed against a cell membrane and suction is applied to assist in the formation of a high resistance seal between the glass and the cell membrane (a "gigaohm seal" or "gigaseal," since the electrical resistance of that seal is in excess of a gigaohm). The high resistance of this seal makes it possible to electronically isolate the currents measured across the membrane patch with little competing noise, as well as providing some mechanical stability to the recording.

Unlike traditional two-electrode voltage clamp recordings, patch clamp recording uses a single electrode to record currents. 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 level. This allows a researcher to keep the voltage constant while observing changes in current. Alternatively, the cell can be current clamped in whole-cell mode, keeping current constant while observing changes in membrane voltage.

VariationsEdit

File:Patch clamp.svg
The cell-attached patch clamp uses a micropipette attached to the cell membrane to allow recording from a single ion channel.

Several variations of the basic technique can be applied, depending on what the researcher wants to study. The inside-out and outside-out techniques are called "excised patch" techniques, because the patch is excised (removed) from the main body of the cell. Cell-attached and both excised patch techniques are used to study the behavior of individual ion channels in the section of membrane attached to the electrode.

Whole-cell patch and perforated patch allow the researcher to study the electrical behavior of the entire cell, instead of single channel currents. The whole-cell patch, which allows for low resistance electrical access to the inside of a cell, has now largely replaced high resistance microelectrode recording techniques to record currents across the entire cell membrane.

File:Patchmodes.svg
Diagram showing variations of the patch clamp technique

Cell-attached or on-cell patch Edit

The electrode is sealed to the patch of membrane, and the cell remains intact. This allows for the recording of currents through single ion channels in that patch of membrane, without disrupting the interior of the cell. For ligand-gated ion channels or channels that are modulated by metabotropic receptors, the neurotransmitter or drug being studied is usually included in the pipette solution, where it can contact what had been the external surface of the membrane. While the resulting channel activity can be attributed to the drug being used, it is usually not possible to then change the drug concentration. The technique is thus limited to one point in a dose response curve per patch. Usually, the dose response is accomplished using several cells and patches. However, voltage-gated ion channels can be clamped at different membrane potentials using the same patch. This results in graded channel activation, and a complete I-V (current-voltage) curve can be established with only one patch.

Inside-out patch Edit

After the gigaseal is formed, the micropipette is quickly withdrawn from the cell, thus ripping the patch of membrane off the cell, leaving the patch of membrane attached to the micropipette, and exposing the intracellular surface of the membrane to the external media. This is useful when an experimenter wishes to manipulate the environment at the intracellular surface of ion channels. For example, channels that are activated by intracellular ligands can then be studied through a range of ligand concentrations.

Whole-cell recording or whole-cell patch Edit

File:WholeCellPatchClamp-03.jpg
Whole cell recording of a nerve cell from the hippocampus. The pipette in the photograph has been marked with a slight blue colour.

Whole-cell recordings, in contrast, involve recording currents through multiple channels at once, over the membrane of the entire 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. 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. A disadvantage of this technique is that the volume of the electrode is larger than the cell, so the soluble contents of the cell's interior will slowly be replaced by the contents of the electrode. This is referred to as the electrode "dialyzing" the cell's contents. Thus, any properties of the cell that depend on soluble intracellular contents will be altered. The pipette solution used usually approximates the high-potassium environment of the interior of the cell. Generally speaking, there is a period at the beginning of a whole-cell recording, lasting approximately 10 minutes, when one can take measurements before the cell has been dialyzed.

Outside-out patch Edit

After the whole-cell patch is formed, the electrode can be slowly withdrawn from the cell, allowing a bulb of membrane to bleb out from the cell. When the electrode is pulled far enough away, this bleb will detach from the cell and reform as a convex membrane on the end of the electrode (like a ball open at the electrode tip), with the original outside of the membrane facing outward from the electrode. Single channel recordings are possible in this conformation if the bleb of membrane is small enough. Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is isolated from the cell, and exposed to different solutions on the extracellular surface of the membrane. The experimenter can perfuse the same patch with different solutions, and if the channel is activated from the extracellular face, a dose-response curve can then be obtained. This is the distinct advantage of the outside-out patch relative to the cell-attached method. However, it is more difficult to accomplish, as more steps are involved in the patching process.

Perforated patch Edit

In this variation of whole-cell recording, the experimenter forms the gigaohm seal, but does not use suction to rupture the patch membrane. Instead, the electrode solution contains small amounts of an antifungal or antibiotic agent, such as amphothericin-B, nystatin, or gramicidin. As the antibiotic molecules diffuse into the membrane patch, they form small perforations in the membrane, providing electrical access to the cell interior. This has the advantage of reducing the dialysis of the cell that occurs in whole-cell recordings, but also has several disadvantages. First, the access resistance is higher, relative to whole-cell, due to the partial membrane occupying the tip of the electrode (access resistance being the sum of the electrode resistance and the resistance at the electrode-cell junction). This will decrease electrical access and thus decrease current resolution, increase recording noise, and magnify any series resistance error. Second, it can take a significant amount of time for the antibiotic to perforate the membrane (10–30 minutes, though this can be reduced with properly shaped electrodes). Third, the membrane under the electrode tip is weakened by the perforations formed by the antibiotic and can rupture. If the patch ruptures, the recording is then in whole-cell mode, with antibiotic contaminating the inside of the cell.

Loose patch Edit

Loose patch clamp is different in that it employs a loose seal rather than the tight gigaseal used in the conventional technique. A significant advantage of the loose seal is that the pipette that is used can be repeatedly removed from the membrane after recording, and the membrane will remain intact. This allows for repeated measurements in a variety of locations on the same cell without destroying the integrity of the membrane. A major disadvantage is that the resistance between the pipette and the membrane is greatly reduced, allowing current to leak through the seal. This leakage can be corrected for, however, which offers the opportunity to compare and contrast recordings made from different areas on the cell of interest.

Automatic patch clamping Edit

Automated patch clamp systems have recently been developed, in order to inexpensively collect large amounts of data in a shorter period of time. Such systems typically include a single-use microfluidic device, either an injection molded or a PDMS cast chip, to capture a cell or cells, and an integrated electrode.

See alsoEdit

ReferencesEdit

External linksEdit

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