Electrophysiology

Electrophysiology is the science and branch of physiology that pertains to the flow of ions in biological tissues and, in particular, to the electrical recording techniques that enable the measurement of this flow and the potential changes related to them. In almost all cases, electrophysiological techniques record the voltage maintained across a cell membrane (i.e. the electrical potential difference between the inside and outside of a cell) or the ion currents that flow across a cell membrane (i.e. the movement of ions from the inside of the cell to the outside or vice versa).

An Introduction to Electrophysiological Technique

The technical goal of the electrophysiologist is simple: to record the voltage across a cell's membrane, the current flowing across that membrane, or (with extracellular recording) to record changes in current density. There are two major divisions of electrophysiological technique: intracellular recording and extracellular recordings. Within these two divisions are many variations. Extracellular recording includes single unit recording, field potential recording, single channel recording and amperometry (which is a special case). Intracellular recording techniques encompass two major subdivisions: Voltage clamp and so-called current clamp.

Voltage clamp techniques

The development of the concept and design of the voltage clamp apparatus is due to the pioneering work of Cole and Marmount [1] (http://neuron.duke.edu/userman/2/pioneer.html) in the 1940's. The purpose of the voltage clamp is to allow an electrophysiologist to measure the ion currents flowing across a cells membrane. Cole had the brilliant idea to use two electrodes and a feedback circuit to achieve this ability. Both electrodes are placed inside a cell. Transmembrane voltage is recorded through one of these electrodes (the "voltage electrode")relative to an outside reference (ground). The second electrode is used for passing current into the cell the "current electrode". Briefly, the experimenter specifies a "holding voltage" that s/he wishes the cell to maintain across it's membrane. Anytime the cell makes a deviation from this holding voltage say, by passing an ion current across its membrane, the operational amplifier generates an "error signal". The error signal is the difference between the holding voltage specified by the experimenter and the actual voltage of the cell. The feedback circuit of the voltage clamp passes current into the cell (via the current electrode) in the polarity needed to reduce the error signal to zero. Thus,the current is applied in the polarity opposite the current that the cell is passing across its membrane, and the clamp circuit provides a current that is the mirror image of the cellular current. This mirror or "clamp current" can be easily measured, giving an accurate reproduction of the currents flowing across the cell's membrane (albeit in the opposite polarity).

Cole developed his voltage clamp before the era of microelectrodes, so his two clamp electrodes were constructed from two fine wires twisted around an insulating rod. This construction could be inserted into only the largest biological cells. This difficulty accounts for the nearly exclusive use of the squid as the animal of choice for early electrophysiological experiments. Squid squirt jets of water when they need to move quickly, as when escaping a predator. To make this escape system as fast as possible, squid evolved an axon upwards of 1 mm in diameter. This squid giant axon was the first preparation that could be used to voltage clamp any biological transmembrane current, and along with Cole's voltage clamp, served as the basis of the experiments that defined the properties of the action potential by Hodgkin and Huxley.

Variations of the voltage clamp technique

As might be expected, several variations exist to the voltage clamp technique. These variations arose mostly to accommodate biological cells that were to small to accept giant electrode assemblies used in squid giant axon. These variations are:

1) Two-electrode voltage clamp using microelectrodes. This technique works on the exact same principal as Cole's "squid clamp" except the two electrodes are glass pipettes with very fine (< 1 micron) tips. A smaller cell such as a muscle cell are still large enough to accommodate the double impalement required for this technique. This technique has the advantage of allowing for voltage clamping of cells smaller than the squid axon, but also has significant disadvantages. Chief among these is that microelectrodes are much less ideal conductors than the much larger wires used by Cole. Because their tips are so small the sometimes cannot pass enough current fast enough to fully compensate for cellular current. Thus the voltage clamp may produce a distorted image of the cell's current. In general, the faster the kinetics of the current (onset and offset), the more likely it is that the voltage clamp won't be able to faithfully "follow" it. Another disadvantage involves "space clamp" issues. Cole's voltage clamp used a long wire that clamped the squid axon uniformly along its entire length. Microelectrodes can provide only a spacial point source of current that may not uniformaly affect different parts of an irregularly shaped cell.

2) Single electrode Voltage Clamp

There are two basic variations of single electrode clamp. In this technique a single electrode is placed in contact with the intracellular compartment of a cell. That single electrode serves both the voltage-recording and current-passing duties that are performed by two separate electrodes in two-electrode clamp.

a) Continuous single electrode clamp (usually referred to as "patch clamp"*)

This technique utilizes an electrode with a relatively large diameter at its tip (> 1 micron), and made such that the tip forms a smooth surfaced circle (rather than a sharp tip; all tangents to this circle being perpendicular to the long axis of the electrode). This style of electrode is known as a "patch clamp electrode" (as distinct from a "sharp microelectrode" used to impale cells). This 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. This suction causes the cell to form a tight seal with the electrode (a so-called "giga-ohm seal", since the electrical resistance of that seal is in excess of a giga-ohm). From this point, the experimenter has 4 choices .

i) To leave the electrode sealed to this patch of membrane (so-called cell-attached patch). This allows for the recording of currents through single ion channels in that patch of membrane.

ii) To quickly withdraw the electrode from the cell, thus ripping the patch of membrane off the cell. This forms a so-called "inside-out" patch. This is useful when an experimenter wishes to manipulated the environment of the inside of ion channels.

iii) To slowly withdraw the electrode 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 part 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 (thus the name "outside out patch"). Outside out patching give 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 it's usual environment.

iv) To leave the electrode in place, but to apply harder suction to rupture the portion of the cell's membrane that is inside the electrode, thus providing access to the intracellular space of the cell. This is known as "whole-cell recording". It is sometimes called "whole cell patch", but that is a misnomer, precisely because you are now recording from the whole cell, not just a patch of membrane from that cell. The advantage of whole cell recording is that one can record the sum total current that flows across the cell's membrane. Whole cell recording has the advantage over sharp microelectrode recording in that the electrical access to the inside of the cell is at least an order of magnitude better (i.e. lower resistance) when using the patch clamp electrode. This is because it has a larger opening at it's tip than a sharp microelectrode.


The single-electrode voltage clamp can be used in any of these four electrode/cell configurations. However, as a recording system, single electrode voltage clamp has significant disadvantages compared to two-electrode voltage clamp, and only one advantage. But it's a huge advantage. The advantage is that you can record from small cells that would be impossible impale with two electrodes. The disadvantages are many. They are unavoidable, but they can be dealt with sufficiently in many case by a skilled electrophysiologist. Some of the disadvantages are:

1) Microelectrodes are imperfect conductors of ion current. They generally have a resistance in excess of a million ohms. They rectify (i.e. they change their resistance with voltage, often in an irregular manner), they sometimes have unstable resistance if clogged by cell contents, membrane, or general free-floating gunk. Thus, they will not faithfully record the voltage of the cell (especially when it's changing quickly) nor will they faithfully pass the current from the voltage-clamp.

2) Voltage and current errors: A major disadvantage of continuous single-electrode voltage clamp circuity is that it does not actually measure the voltage of the cell being clamped(as does two-electrode clamp). To put it as simply as I can, the patch-clamp amplifier is really identical in design to a two-electrode clamp, except that the voltage measuring and current passing circuits are connected directly to each other (in the two-electrode clamp, they are connected through the cell). The electrode is attached to a wire that contacts the current/voltage loop inside the amplifier. Thus, the electrode has only an indirect influence on the feedback circuit in the amplifier. The amplifier reads only the voltage at the top of the electrode, and feeds back current to compensate for that. But, if, as explaned above, the electrode is an imperfect conducter, the clamp circuity will have only a filtered and distorted view of the cell's membrane potential. Likewise, when the circuit passes back the current needed to compensate for that (distorted) voltage, the current will itself be distorted by the electrode before it reaches the cell. To compensate for this, the electrophysiologist uses the lowest resistance electrode possible, makes sure that the electrical characteristics of the electrode don't change during an experiment (so at least the error will be constant), and avoids recording currents that have kinetics likely to be too fast for the clamp to follow accurately. The accuracy of a continuous single electrode clamp goes up the slower and smaller are the voltage changes it is trying to clamp.

3) Series resistance errors: (This is hard to describe, and I will try to add a diagram to make it more clear). The currents passed to the cell through the electrode must go to ground to complete the circuit. Ground is outside the cell. The voltage recorded by the amplifier are recorded relative to ground (outside the cell). When a cell is clamped right at its natural resting potential, there is not problem. The clamp is not passing current and the voltage is being generated only by the cell. But, when attempting to clamp the cell at a potential different than it's natural resting potential, series resistance errors become a significant concern. When clamping away from normal resting potential, the cell will pass current across it's membrane in an attempt to get back to it's natural resting potential. The clamp amplifier will oppose this by passing current to keep the cell at the commanded holding potential. A problem arises because the electrode is located between the amplifier and the cell. Put another way, the resistor that is the electrode is in series with the resistor that is the cell's membrane. Thus, when passing current through the electrode and the cell, Ohm's Law tells us that this current will cause a voltage to form across both the cell's and the electrode's resistance. Since these two resistors are in series, the two voltage drops will add together. You are interested in only the voltage of the cell, but you are seeing the voltage of the cell + the electrode. For the sake of argument, lets say that the electrode and the cell membrane have equal resistances (they don't actually). If you command a 40 mV change from the cells resting potential, the amplifier will respond by passing enough current until it reads that it's achieved that 40 mV shift. However, in this example, half of that voltage drop is across the electrode, not the cell. You think you have moved the cell's voltage by 40 mV, but you've really only moved it by 20 mV. The difference between what you think you've done and what is actually done to the cell is called the "series resistance error". It is particularly troublesome when one is trying to accuarately asses the voltage-dependence of a particular ion current. If you don't know what the membrane potential of the cell really is, you can't make this measurment.

All modern patch clamp amplifiers have built in circuity that tries to compensate for the series resistance error. These circuits are helpful, but they compensate only 70-80% of the error, leaving significant error in the measurment. The electrophysiologist can further decrease the influence of series resistance error by recording at or near the cell's natural resting potential, and by using as low a resistance electrode as possible.

4) Capacitance errors. All microelectrodes act as capacitors as well as resistors. They are particularly troublesome capitors because they are non-linear. The capacitance of an electrode arises because the ion containing solution inside the electrode is separated by an insulator (glass)from the ion-containing solution outside the electrode. This is, by definition and function a capacitor. Worse, since the thickness of the glass changes the farther you get from the tip, the time constant of the capacitor will vary over many values. The main problem caused by this electrode capacitance is that it produces a distorted record of the membrane voltage or current any time they are changing. Amplifiers have means of compensating for this electrode capacitance, but can't entirely because the capacitance has many time-constants. The experimenter can reduce this problem by keeping the cell's bathing solution as shallow as possible (thus exposing less glass surface to liquid) and by thickening the walls of the electrode. This is accomplished by coating the electrode with silicone, resin, paint, or another substance that will cling to the glass and make the distance between the inside and outside solutions larger.

5) Space clamp errors. Your single electrode is but a point source of current. In distant parts of the cell, the current passed through the electrode will be less influential than nearby parts of the cell. This will particularly a problem when recording from cells like neurons that have elaborate dendritic structures. There is basically not a damned thing you can do about space clamp errors except to temper the conclusions of your experiment to account for them.

6) Whole cell dialysis. This applies only to whole cell configuration. The size of the opening at the end of the electrode is large and the volume of the salt solution inside the electrode is huge compared to the volume of the cell. Thus, the soluable contents of the cells insides are replaced by the contents of the electrode (a much simpler brew to be sure!). This is referred to as the electrode "dialyzing" the cell. Thus, any properties of the cell that depend on these lost soluble contents will be altered.



Note: "Continuous single electrode voltage clamp" and "patch clamp" are not competing nomenclatures. The former refers to the fact that one is using only a single electrode to voltage clamp. "Patch clam" referres spcifically to the configuration of that electrode on the cell.


At the cellular level, these techniques include so-called passive recordings, sometimes referred to as "current clamp" as well as the active (voltage clamp) techniques, that "clamp" or maintain the cell potential at a value the experimenter specifies. Voltage control is established using feedback through an operational amplifier circuit. The main value of voltage-clamp techniques is that they allow one to measure the amount of ionic current crossing a cell's membrane at any given voltage at a given time. This is most obviously of value in the study of voltage-gated ion channels, but also aids in characterizing conductance. Current clamp, on the other hand, is used to record a cell's membrane potential. "Current clamp" is something of a misnomer, because nothing is "clamped" while using this technique. Unlike voltage clamp recording where the cell's membrane voltage is held, or "clamped", at a particular value, in current clamp recording, the current flow across the cell's membrane is not controlled. The misnaming derives from two sources. First current clamp is perceived as the "opposite" of voltage clamp, and second, when using current clamp, the experimenter has the opportunity to inject specificed current offsets into a cell. However, even with such offsets, the cell is still free to vary it's membrane current in response to other stimuli. The experimenter has no direct control over the current flow across the cell's membrane. Current clamp is nothing more than a method of passively recording a cell's trans-membrane voltage with the added ability to produce voltage offsets by injecting current into the cell through the recording electrode. Current clamp is useful anytime the experimenter needs to record the voltage across a cell's membrane such as during studies of cell excitability by analyzing the action potentials under conditions more consistent with the cell's natural environment. Though most scientists understand that "current clamp" involves no clamping of anything, they still use the term as it has become the vernacular to describe voltametry.

The most common electrophysiological recording techniques establish electrical contact with the inside of a cell or tissue with a "glass electrode." Such an electrode is fashioned by the experimenter from a fine capillary glass tube, which is then pulled to an even finer (but still hollow of about 1 micrometer diameter for patch-clamp, 0.1 micron for intracellular "sharp electrode" recording) tip under heat and allowed to cool. This glass "micropipette" is then filled with a salt solution, and a silver chloride-coated silver wire is inserted to establish an electrochemical junction with the pipet fluid and the tissue or cell into which the pipet is inserted (typically with the aid of a microscope and finely adjustable pipet holders, known as micromanipulators). This salt electrode filling solution varies widely depending on the planned experiment. For sharp microelectrode intracellular recording, high concentration (2-3 molar) salt is used. Potassium chloride, potassium acetate, potassium methylsulfate are salts commonly used to fill shart microelectrode. Note that all contain potassium to match the predominant intracellular ion (although in special circumstance, cesium salts may be used instead of potassium). While a filling solution of potassium chloride gives the smallest and most stable electrochemical "junction potential" when in contact with silver chloride, care must be taken when using chloride as the counter ion to potassium. Injection of chloride ions into the cell will raise the chloride concentration and thus reverse the direction of the cell's choride currents (this characteristic is often used as an easy way to identify chloride currents). The chloride-coated silver wire connects back to the amplifier. Classically, electrophysiologists watched biological currents/voltages on an oscilloscope and recorded them onto chart paper/screen, but now the vast majority use computers. Other requirements are an air or sand table to reduce vibration, and a Faraday cage to eliminate outside interference from the tiny measured currents.

Where experiments require low impedance measurements and no ionic contribution from the microelectrode, the chloride solution is replaced with cerralow, a low melting temperature alloy. The tip is electroplated with soft gold and platinum black, from chloroplatinic acid. Electrodes of this type are used to measure electrical pulses in unmyelinated axons down to 100 nm.

There are four main types of cellular electrophysiological recordings:

1. Intracellular recording. This technique entails impaling a cell, usually a neuron, with a sharp glass electrode and recording either the voltage (current-clamp) or the current (voltage-clamp) across the membrane. This technique is widely used when recording from brain slices or when performing "in-vivo" recording from live animals. While sharp electrode recordings are typically used for recording voltage, voltage-clamp recordings can be performed by impaling larger cells with two sharp electrodes. This is the original voltage-clamp method, which has been superceeded by the superior patch-clamp recording (see below). The two-electrode voltage clamp was used by Alan Lloyd Hodgkin and Andrew Fielding Huxley to describe the ionic-basis of the action potential. This work won them the Nobel Prize in 1963.

2. Extracellular recording. In this technique an electrode is placed on the extracellular medium and field-potentials contributed by the action potentials of many neurons are recorded. Some popular clinical applications of extracellular recording are the electrocardiogram (ECG) and the electroencephalogram (EEG).

3. The patch-clamp technique. With this technique it is possible to clamp the cell potential (voltage-clamp) or the cell current (current-clamp) using a glass micropipette as explained previously. Current-clamp recordings allow the detection and measurement of action potentials in excitable cells such as neurons and the beta cells of the pancreas. Voltage-clamp recordings are very popular for measuring macroscopic currents in which the activity of many ion channels is occurring at the same time. However with this powerful technique it is also possible to measure the current flowing through a single ion channel and study its behavior. There are different modalities of the patch-clamp technique. This technique was developed by Erwin Neher and Bert Sakmann who received the Nobel Prize in 1991.

  • Cell attached mode.
Advantages: Single channels can be recorded and channel properties are not changed.
Disadvantages: Poor pharmacology.
  • Whole cell mode.
Advantages: Good pharmacology, large current is recorded because it is the whole cell.
Disadvantages: The cell is perforated so some cell contents are diluted.
  • Excised patch (inside-out or outside-out patch).
Advantages: Recordings can be taken from individual channels, good pharmacology and the inside/outside solutions can be changed.
Disadvantages: Risk that channel properties are changed.
  • Perforated patch.
Advantages: It is possible to obtain large, whole cell currents without washing out the intracellular medium.
Disadvantages: Impossible to record from single channels.

4. Axon recording.

Advantages: Chemistry of cell is unchanged, axon pulses are discriminated from the less frequent retrograde cell action potentials.
Disadvantages: Experimental protocol requirements are strict.


Amperometry is another technique of electrophysiology, which uses a carbon electrode and is typically used to detect and record changes in the chemical composition of the oxidized components inside of biological solution being studied. It has typically employed for studing the exocytoses in the neural and endocrine systems. Many monamine neurotransmitters, e.g., norepinephrene (noradrenaline), dopamine, serotonin (5-HT), are oxidizable. The method is also applicable to cells that do not secrete oxidizable neurotramsmitters by loading 5-HT or dopamine.ru:Электрофизиология

Navigation

  • Art and Cultures
    • Art (https://academickids.com/encyclopedia/index.php/Art)
    • Architecture (https://academickids.com/encyclopedia/index.php/Architecture)
    • Cultures (https://www.academickids.com/encyclopedia/index.php/Cultures)
    • Music (https://www.academickids.com/encyclopedia/index.php/Music)
    • Musical Instruments (http://academickids.com/encyclopedia/index.php/List_of_musical_instruments)
  • Biographies (http://www.academickids.com/encyclopedia/index.php/Biographies)
  • Clipart (http://www.academickids.com/encyclopedia/index.php/Clipart)
  • Geography (http://www.academickids.com/encyclopedia/index.php/Geography)
    • Countries of the World (http://www.academickids.com/encyclopedia/index.php/Countries)
    • Maps (http://www.academickids.com/encyclopedia/index.php/Maps)
    • Flags (http://www.academickids.com/encyclopedia/index.php/Flags)
    • Continents (http://www.academickids.com/encyclopedia/index.php/Continents)
  • History (http://www.academickids.com/encyclopedia/index.php/History)
    • Ancient Civilizations (http://www.academickids.com/encyclopedia/index.php/Ancient_Civilizations)
    • Industrial Revolution (http://www.academickids.com/encyclopedia/index.php/Industrial_Revolution)
    • Middle Ages (http://www.academickids.com/encyclopedia/index.php/Middle_Ages)
    • Prehistory (http://www.academickids.com/encyclopedia/index.php/Prehistory)
    • Renaissance (http://www.academickids.com/encyclopedia/index.php/Renaissance)
    • Timelines (http://www.academickids.com/encyclopedia/index.php/Timelines)
    • United States (http://www.academickids.com/encyclopedia/index.php/United_States)
    • Wars (http://www.academickids.com/encyclopedia/index.php/Wars)
    • World History (http://www.academickids.com/encyclopedia/index.php/History_of_the_world)
  • Human Body (http://www.academickids.com/encyclopedia/index.php/Human_Body)
  • Mathematics (http://www.academickids.com/encyclopedia/index.php/Mathematics)
  • Reference (http://www.academickids.com/encyclopedia/index.php/Reference)
  • Science (http://www.academickids.com/encyclopedia/index.php/Science)
    • Animals (http://www.academickids.com/encyclopedia/index.php/Animals)
    • Aviation (http://www.academickids.com/encyclopedia/index.php/Aviation)
    • Dinosaurs (http://www.academickids.com/encyclopedia/index.php/Dinosaurs)
    • Earth (http://www.academickids.com/encyclopedia/index.php/Earth)
    • Inventions (http://www.academickids.com/encyclopedia/index.php/Inventions)
    • Physical Science (http://www.academickids.com/encyclopedia/index.php/Physical_Science)
    • Plants (http://www.academickids.com/encyclopedia/index.php/Plants)
    • Scientists (http://www.academickids.com/encyclopedia/index.php/Scientists)
  • Social Studies (http://www.academickids.com/encyclopedia/index.php/Social_Studies)
    • Anthropology (http://www.academickids.com/encyclopedia/index.php/Anthropology)
    • Economics (http://www.academickids.com/encyclopedia/index.php/Economics)
    • Government (http://www.academickids.com/encyclopedia/index.php/Government)
    • Religion (http://www.academickids.com/encyclopedia/index.php/Religion)
    • Holidays (http://www.academickids.com/encyclopedia/index.php/Holidays)
  • Space and Astronomy
    • Solar System (http://www.academickids.com/encyclopedia/index.php/Solar_System)
    • Planets (http://www.academickids.com/encyclopedia/index.php/Planets)
  • Sports (http://www.academickids.com/encyclopedia/index.php/Sports)
  • Timelines (http://www.academickids.com/encyclopedia/index.php/Timelines)
  • Weather (http://www.academickids.com/encyclopedia/index.php/Weather)
  • US States (http://www.academickids.com/encyclopedia/index.php/US_States)

Information

  • Home Page (http://academickids.com/encyclopedia/index.php)
  • Contact Us (http://www.academickids.com/encyclopedia/index.php/Contactus)

  • Clip Art (http://classroomclipart.com)
Toolbox
Personal tools