PATCH CLAMPING AN INTRODUCTORY GUIDE TO PATCH CLAMP ELECTROPHYSIOLOGY PDF

Set-up[ edit ] Classical patch clamp setup, with microscope , antivibration table, and micromanipulators During a patch clamp recording, a hollow glass tube known as a micropipette or patch pipette filled with an electrolyte solution and a recording electrode connected to an amplifier is brought into contact with the membrane of an isolated cell. Another electrode is placed in a bath surrounding the cell or tissue as a reference ground electrode. An electrical circuit can be formed between the recording and reference electrode with the cell of interest in between. Schematic depiction of a pipette puller device used to prepare micropipettes for patch clamp and other recordings Circuit formed during whole-cell or perforated patch clamp The solution filling the patch pipette might match the ionic composition of the bath solution, as in the case of cell-attached recording, or match the cytoplasm , for whole-cell recording. The solution in the bath solution may match the physiological extracellular solution, the cytoplasm, or be entirely non-physiological, depending on the experiment to be performed.

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Set-up[ edit ] Classical patch clamp setup, with microscope , antivibration table, and micromanipulators During a patch clamp recording, a hollow glass tube known as a micropipette or patch pipette filled with an electrolyte solution and a recording electrode connected to an amplifier is brought into contact with the membrane of an isolated cell. Another electrode is placed in a bath surrounding the cell or tissue as a reference ground electrode.

An electrical circuit can be formed between the recording and reference electrode with the cell of interest in between. Schematic depiction of a pipette puller device used to prepare micropipettes for patch clamp and other recordings Circuit formed during whole-cell or perforated patch clamp The solution filling the patch pipette might match the ionic composition of the bath solution, as in the case of cell-attached recording, or match the cytoplasm , for whole-cell recording.

The solution in the bath solution may match the physiological extracellular solution, the cytoplasm, or be entirely non-physiological, depending on the experiment to be performed.

The researcher can also change the content of the bath solution or less commonly the pipette solution by adding ions or drugs to study the ion channels under different conditions. Depending on what the researcher is trying to measure, the diameter of the pipette tip used may vary, but it is usually in the micrometer range. Typical equipment used during classical patch clamp recording 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.

To obtain this high resistance seal, the micropipette is pressed against a cell membrane and suction is applied. A portion of the cell membrane is suctioned into the pipette, creating an omega -shaped area of membrane which, if formed properly, creates a resistance in the 10— gigaohms range, called a "gigaohm seal" or "gigaseal".

The pipette in the photograph has been marked with a slight blue color. 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. This allows a researcher to keep the voltage constant while observing changes in current. To make these recordings, the patch pipette is compared to the ground electrode.

Current is then injected into the system to maintain a constant, set voltage. The current that is needed to clamp the voltage is opposite in sign and equal in magnitude to the current through the membrane.

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 enables 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.

Cell-attached patch[ edit ] Cell-attached patch configuration For this method, the pipette is sealed onto the cell membrane to obtain a gigaseal, while ensuring that the cell membrane remains intact. This allows the recording of currents through single, or a few, ion channels contained in the patch of membrane captured by the pipette. By only attaching to the exterior of the cell membrane, there is very little disturbance of the cell structure.

The resulting channel activity can be attributed to the drug being used, although it is usually not possible to then change the drug concentration inside the pipette. The technique is thus limited to one point in a dose response curve per patch. Therefore, the dose response is accomplished using several cells and patches. However, voltage-gated ion channels can be clamped successively at different membrane potentials in a single patch.

This results in channel activation as a function of voltage, and a complete I-V current-voltage curve can be established in only one patch. Another potential drawback of this technique is that, just as the intracellular pathways of the cell are not disturbed, they cannot be directly modified either. This is useful when an experimenter wishes to manipulate the environment at the intracellular surface of single ion channels.

For example, channels that are activated by intracellular ligands can then be studied through a range of ligand concentrations. To achieve the inside-out configuration, the pipette is attached to the cell membrane as in the cell-attached mode, forming a gigaseal, and is then retracted to break off a patch of membrane from the rest of the cell.

Pulling off a membrane patch often results initially in the formation of a vesicle of membrane in the pipette tip, because the ends of the patch membrane fuse together quickly after excision. The electrode is left in place on the cell, as in cell-attached recordings, but more suction is applied to rupture the membrane patch, thus providing access from the interior of the pipette to the intracellular space of the cell.

This provides a means to administer and study how treatments ex. The first is by applying more suction. The amount and duration of this suction depends on the type of cell and size of the pipette. The other method requires a large current pulse to be sent through the pipette. How much current is applied and the duration of the pulse also depend on the type of cell. The advantage of whole-cell patch clamp recording over sharp electrode technique 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 pipette solution used usually approximates the high- potassium environment of the interior of the cell to minimize any changes this may cause. There is often a period at the beginning of a whole-cell recording when one can take measurements before the cell has been dialyzed.

After the whole-cell configuration is formed, the electrode is 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.

While multiple channels can exist in a bleb of membrane, single channel recordings are also possible in this conformation if the bleb of detached membrane is small and only contains one channel. The experimenter can perfuse the same patch with a variety of solutions in a relatively short amount of time, and if the channel is activated by a neurotransmitter or drug from the extracellular face, a dose-response curve can then be obtained. On the other hand, it is more difficult to accomplish.

The longer formation process involves more steps that could fail and results in a lower frequency of usable patches. Perforated patch[ edit ] Perforated patch technique This variation of the patch clamp method is very similar to the whole-cell configuration.

The main difference lies in the fact that when the experimenter forms the gigaohm seal, suction is not used 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 , which diffuses into the membrane patch and forms small pores in the membrane, providing electrical access to the cell interior.

The perforated patch can be likened to a screen door that only allows the exchange of certain molecules from the pipette solution to the cytoplasm of the cell. Advantages of the perforated patch method, relative to whole-cell recordings, include the properties of the antibiotic pores, that allow equilibration only of small monovalent ions between the patch pipette and the cytosol, but not of larger molecules that cannot permeate through the pores.

Consequently, one can have recordings of the entire cell, as in whole-cell patch clamping, while retaining most intracellular signaling mechanisms, as in cell-attached recordings. As a result, there is reduced current rundown, and stable perforated patch recordings can last longer than one hour. This may decrease current resolution and increase recording noise.

It can also take a significant amount of time for the antibiotic to perforate the membrane about 15 minutes for amphothericin-B, and even longer for gramicidin and nystatin. 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. To achieve a loose patch clamp on a cell membrane, the pipette is moved slowly towards the cell, until the electrical resistance of the contact between the cell and the pipette increases to a few times greater resistance than that of the electrode alone.

The closer the pipette gets to the membrane, the greater the resistance of the pipette tip becomes, but if too close a seal is formed, and it could become difficult to remove the pipette without damaging the cell. For the loose patch technique, the pipette does not get close enough to the membrane to form a gigaseal or a permanent connection, nor to pierce the cell membrane.

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 repeated measurements in a variety of locations on the same cell without destroying the integrity of the membrane. This flexibility has been especially useful to researchers for studying muscle cells as they contract under real physiological conditions, obtaining recordings quickly, and doing so without resorting to drastic measures to stop the muscle fibers from contracting.

This leakage can be partially corrected for, however, which offers the opportunity to compare and contrast recordings made from different areas on the cell of interest. Such systems typically include a single-use microfluidic device, either an injection molded or a polydimethylsiloxane PDMS cast chip, to capture a cell or cells, and an integrated electrode.

In one form of such an automated system, a pressure differential is used to force the cells being studied to be drawn towards the pipette opening until they form a gigaseal. Then, by briefly exposing the pipette tip to the atmosphere, the portion of the membrane protruding from the pipette bursts, and the membrane is now in the inside-out conformation, at the tip of the pipette. In a completely automated system, the pipette and the membrane patch can then be rapidly moved through a series of different test solutions, allowing different test compounds to be applied to the intracellular side of the membrane during recording.

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