Wikipedia
cleanup-date June 2005 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 (signals (biology)signals) 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). The term electrophysiology is used to describe both a scientific discipline (the study of the electrical properties of biological cells) and also a methodological approach (the means to study the electrical properties of biological cells). Each of these aspects of electrophysiology will be dealt with here, in turn.
Electrophysiology - the field of study -
Electrophysiology - the techniques - 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''.
Intracellular recording - Intracellular recording, sometimes known as transmembrane recording, is any technique that involves measuring voltage and/or current across the membrane of a cell. Operationally, this requires the insertion of a recording electrode into a cell, so that the intracellular potential can be measured against the extracellular potential. The recording of this transmembrane voltage is the basic measurement made in all intracellular recording. The electrode employed can come in variable configurations. The properties of the electrode are varied according to the requirements of a particular recording. In all cases the fabrication of an electrode requires a trade-off between size (smaller is better) and resistance (lower is better). In general, the smaller an electrode, the higher its resistance, so an electrode is a compromise between being small enough to target a single cell, doing minimum damage, while being large enough to have a low-enough resistance that small neuronal signals can be discerned from thermal noise in the electrode tip. Higher resistance electrodes produce larger amplitude thermal noise.
Current-clamp techniques - "Current Clamp" is the term used to describe a simple recording of trans-membrane voltage with the ability to inject current into a biological cell through the lumen of the recording electrode. The term is a misnomer, and somewhat misleading in that there is nothing being "clamped" in this type of recording. The term ''current clamp'' arose from two sources. First, it is often perceived as the "opposite" of voltage-clamp; voltage and current being terms on opposite sides of the Ohm's Law equation (V=IR). Second during "current-clamp" recordings, an investigator has the opportunity to inject current into a cell through the recording electrode. In actuality "current clamp" is nothing more than passive recording of the cell's membrane potential. To explain further: in ''voltage-clamp'' the membrane potential is held, or "clamped" through use of a negative feedback circuit, at a level set by the experimenter, and currents induced by changes in this voltage are measured. In "current clamp", the membrane potential is free to vary unmolested by the amplifier. The amplifier simply records whatever voltage changes the cell generates on its own or as a result of stimulation. There is no feedback involved to hold the cell's current at a particular value. While current can be passed across the cell membrane, the total cell current is left free to vary. Nothing is clamped. Any applied current however is merely an offset. It does not prevent voltage or current from varying in the cell.Unlike voltage-clamp, which is an "active" recording mode (the amplifier actively clamps the cell membrane potential by passing metered current through a feedback circuit), current-clamp is a ''passive'' recording technique. Apart from passing current offsets (not feedback controlled), during current-clamp recording, one is simply ''watching'' the cell behave. The electrophysiologist, through the amplifier, is merely passively eavesdropping on the cell's voltage behavior. While one might choose to inject a depolarizing current offset to make the cell fire action potentialaction potentials, these action potentials themselves are uncontrolled. An apt analogy might be that you come upon a person sitting quietly. You can watch the person's behavior. You can perturb the person in a particular way (insult him, poke him, throw water on him), but you can't control the behavior that follows your perturbation. You are not "clamping" that person.While on the subject of debunking nomenclature, everyone refers to their recording device as an "amplifier". In most cases it is not. Most current-clamp amplifiers provide little or no amplification of the voltage changes recorded from the cell. More accurately, the "amplifier" is actually an electrometer, sometimes also referred to as a "unity gain amplifier" (i.e. an amplifier that doesn't amplify). So what good is an amplifier that doesn't amplify? The main job of the electrometer is to change the nature of the small signals (in the mV range) produced by cells so that they can be accurately recorded by low-impedance electronics. What the "amplifier" actually does is to "impedance match" the signal on input and the output of the amplifier. In something closer to English, the unity-gain amplifier, increases the current behind the signal while simultaneously decreasing the resistance over which that current passes. Consider this example based on Ohm's Law (Voltage = (current)(resistance); V=IR). Say you have a voltage of 10 mV that is generated by passing 10 nano (10-8) amperes of current across 1 million Ohms of resistance. The electrometer changes this "high impedance signal" to a "low impedance signal" by converting it to 10-5 micro ampere across 1000 Ohms. Do the math. The current and resistance over which the current passes have both changed by orders of magnitude, yet the signal is still accurately recorded as 10 mV. This is what a unity gain current clamp amplifier does. It processes the signal from a high to a low impedance signal. Why is this necessary?The typical whole cell electrode has a resistance across its tip of 1-10 mega Ohms. A typical sharp micro electrode's resistance is more like 100 mega Ohms (100 Million Ohms). Add the membrane resistance that is in series with the electrode (typically 50-100 mega Ohms). In the most extreme (but not uncommon) of these conditions, your 10 mV signal is being measured dropping across 50-200ish mega Ohms. Typically, your run of the mill recording apparati have a standard input resistance of 1 mega Ohm. Again, do the math (V=IR). If you take a signal that is 10 mv dropping over 200 million Ohms, that will be 50 femto amperes (5 X !10-11) .? If you don't impedance match the signal, but input it directly into into a device with a 1 mega Ohm resistance , the voltage signal will be reduced to only 50 microvolts (5 X 10-11 amperes X 1 X 106 Ohms). This is under the detection threshold of even the best equipment.So, in simpler terms, what a unity gain current clamp amplifier does is to take the tiny voltage signals generated by cells, and "put more current behind them" to maintain the voltage when it is handed off to a lower impedance instrument. The amplifier accomplishes this by using a voltage follower circuit. A voltage follower reads the voltage on the input (caused by a small current across a big resistor. It then instructs a parallel circuit that has a large current source behind it (the electrical mains) and adjusts the resistance of that parallel circuit to give the same output voltage, but across a lower resistance. Think of it this way. You have a garden hose and a fire hose next to each other. The firehose can carry much more water/per second (current) than can the garden hose. Let's say further that there is a valve on the nozzle of the firehose. This valve is operated by the water pressure in the garden hose. If the garden hose has no pressure, the firehose valve is closed. If the garden hose has a medium pressure, the firehose valve opens more. Lot's of pressure in the garden hose, valve opened wide in the firehose. If you calibrate the relationship between the garden hose and the valve, you can produce a system where a small stream of water at pressure x in the garden hose is converted into a large stream of water, still at pressure x. So, why go to all this trouble? Why not just make all the equipment high impedance and not bother with this impedance matching stuff? The reason is economic. Electronics built to the less exacting standards that produces 1 M Ohm input resistance are ''much'' cheaper than instruments built to the very exacting standards of high-impedance circuitry. A DVD player is probably more complicated than a current-clamp amplifier. Yet you can buy a top of the line DVD player for a few hundred dollars. The best selling current clamp amplifier costs upwards of $9000. You pay up front, but then every piece of equipment downstream from the amplifier (e.g. oscilloscope, step up amp, A to D converter, PC computer) costs a fraction of what it would if it were operating in the Gigaohm range.There are three main advantages of using current clamp instead of voltage clamp. First, in current clamp you are watching the cell respond in something close to its "normal" manner. The cell is allowed to "behave" as the neural circuits and internal currents drive it. Second, sometimes you are more interested in what the cell's voltage is doing than you are what its current is doing. Third, it is a heck of a lot easier. Since there are no feedback circuits in a current clamp, there are fewer amplifier adjustments to be made, and the output is much more stable (harder to ring) than a voltage clamp.
Variations on "current clamp" recording - The recording of a cell's voltage falls into one of two broad categories: Extracellular recording and intracellular recording. As one might guess, in extracellular recording, the electrode is outside the cell, while in intracellular recording it is inside.
Extracellular recording - At first, the idea of extracellular voltage recording might seem to make little sense. Everything that has been discussed up to now has rested on the principals of recording voltage across the resistor of the cell's membrane. In extracellular recording, the recording electrode is outside the cell which is the same location as the reference (ground). Voltages cannot develop over zero resistance. The resistance of salt water is negligible, so where is the resistive barrier? In extracellular recording, the resistor is the tip of the electrode itself. Extracellular electrodes can record transient changes in the local balance of positive and negative charges. Since the inside of the electrode is electroneutral, and the tip has a resistance, a voltage can develop across the electrode tip between the electroneutral interior and the exterior local change in charge balance. To fully understand this, one must first understand the concept of current sources and sinks.Essentially, what an extracellular electrode does is to detect local current sources and sinks. If a sink is nearby, a voltage is generated across the tip of the electrode of negative polarity. The opposite is true if the electrode is near a source. In the case of biological membranes, sinks and souces are caused by ion currents across cellular membranes through ion channel ion channels. The flux of, say, sodium ions into a cell during an action potential, leaves behind a net negativity outside the membrane. If an electrode is nearby, that negativity is detected. However, because that negativity is generated in an aqueous environment without any barriers to diffusion, this negativity will be very short lived as negative charges diffuse away and positive charges diffuse in. Therefore, for an electrode to record a source or sink with any appreciable time course, the flux of ions must continue for some time to maintain the local negativity or positivity. Thus, while the electrode is actually measuring voltage (across its tip), because of the special case of recording where there are no diffusion barrier, what is essentially being measured, is current across the membrane. More precisely, the voltage at the tip of the electrode at any instant is proportional to the summated current across local membranes. and postsynaptic neuron. This is meant to represent a large population of syapses and neurons. When the synapse releases glutamate onto the postsynaptic cell, it opens ionotropic glutamate receptor channels. The net flow of currentis inward, so a current sink is generated. A nearby electrode (#2) detects this as a negativity. A second extracellular ''intracellular'' electrode placed inside the cell body (#1) records the change in membrane potential that the incoming current causes.]]One of the most common uses for this technique is to record what are known as "extracellular field potentials". Extracellular field potentials are local current sinks or sources that are generated by the synchronous activity of a large population of cells. Usually this synchronous activation is achieved by the simultaneous activation of many neurons by synaptic transmission. The diagram to the right shows actual hippocampal synaptic field potentials. At the right, the lower trace shows a negative wave that corresponds to a current sink caused by positive charges entering cells through postsynaptic glutamate receptor glutamate receptors, while the upper trace shows a positive wave that is generated by the current that leaves the cell (at the cell body) to complete the circuit. For more specific information, see neuronal field potentials.
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 neuron.duke.edu 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 its 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 Andrew HuxleyHuxley.
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 micrometer) 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. Furthermore, microelectrodes can provide only a spacial point source of current that may not uniformly 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 (SEVC-c) (usually referred to as "patch !clamp"*) - =This technique utilizes an electrode with a relatively large diameter at its tip (> 1 micrometer), 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 manipulate 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 its 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 its 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 is 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 is 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 possible, 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 explained 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 errors 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 no 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 its natural resting potential, series resistance errors become a significant concern. When clamping away from normal resting potential, the cell will pass current across its membrane in an attempt to get back to its 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. The experimenter is interested only in the voltage of the cell, but is 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 usually don't). 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 has achieved that 40 mV voltage change. However, in this example, half of that voltage drop is across the electrode, not the cell. The experimenter thinks s/he has moved the cell's voltage by 40 mV, but has, in fact, moved it only by 20 mV. The difference between what the experimenter thinks has been done and what has actually been done to the cell is called the "series resistance error". It is particularly troublesome when one is trying to accurately asses the voltage-dependence of a particular ion current. If one doesn't know what the membrane potential of the cell really is, one can't make this measurement.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 measurement. 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 capacitors 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 soluble contents of the cell's interior 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. 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. A good example of this is that long-term potentiation can be induced during the first 10 minutes of a whole cell recording, but not after that.Dialysis of intracellular contents can be minimized by using a variation of the whole-cell configuration called a "perforated patch recording". In this variation, the experimenter includes small amounts of an antibiotic (usually Amphothericin-B or Gramicidin) into the electrode solution. The experimenter then makes a gigaseal onto the exterior of the cell membrane and waits for the antibiotic to generate small pores, or perforations, on the small bit of membrane between the inside of the electrode and the interior of the cell. This will allow small ions to flow through the patch allowing electrical access to the inside of the cell, but preventing larger soluble molecules from dialyzing out. While perforated patch is an excellent method when whole cell dialysis needs to be prevented, it also has some disadvantages. First, the access 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. The duration of an electrophysiolgical experiment is generally a function of how long the seal can be maintained between the electrode and the cell (an hour is typical). Thus, the average length of an experiment is shortened by the length of time it takes to form the perforations. Third, electrodes will generally not form seals with a cell if there is antibiotic present at the tip of the electrode. Thus one must take the additional step of filling the tip of the electrode with antibiotic-free solution and then backfilling the electrode with antibiotic containing solution (much of the time it takes to perforate the membrane can be accounted for by the time it takes the antibiotic to diffuse to the tip of the electrode). Fourth, perforated patches have the tendency to "break down". The membrane under the electrode tip is weakened by the perforations formed by the antibiotic and tends to rupture. When the patch ruptures, one 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. 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 clamp" referres specifically to the configuration of that electrode on the cell.
Discontinuous single-electrode voltage-clamp (SEVC-d) - SEVC-d (single-electrode voltage clamp – discontinuous) is one of the most interesting and underutilized techniques in electrophysiology and has some striking advantages over continuous SEVC (SEVC-c) when doing “whole-cell” recording. In this technique a completely different electronic approach is taken for passing current and recording voltage through the same electrode. Rather than doing so simultaneously as in SEVC-c, the electrode is time-shared so the current is passed and voltage is recorded at different times. Basically, an SEVC-d amplifier oscillates between passing current and measuring voltage. One such oscillation of the amplifier is known as its “duty cycle”. During one duty cycle, the amplifier measures the membrane potential of the cell. It compares that membrane potential to the experimenter specified “holding potential” (or command potential). An operational amplifier measures the difference between these two potentials and generates an error signal that specifies how much current needs to be passed into the cell to bring it to the command potential. This clamp current is then measured, and will be a mirror image of the current generated by the cell. In these ways SEVC-d is identical to all other forms of voltage clamping. How SEVC-d differs is that it measures the voltage and passes current each at different times during the duty cycle, and each time only briefly. The amplifier outputs feature sample and hold circuits, so that, for instance, each briefly recorded (sampled) voltage is then held on the output until the next measurement is made in the next duty cycle. More specifically, the amplifier measures voltage in the first few milliseconds of the duty cycle, generates the error signal, and then spends approximately the last 2/3 of each duty cycle passing current into the cell to reduce that error. At the beginning of the next duty cycle, voltage is measured again, a new error signal generated, current passed, and so on…The experimenter sets the length of the duty cycle (or put another way, the frequency at which the amplifier oscillates). In a perfect world, one would set the frequency very high (say 100 kHz). This would give excellent time resolution of both the cells voltage and the amount of current passed (at 100 kHz – samples would be taken every 10 micro-seconds). In practice, one must set the oscillation much slower than that, usually in the 2-3 kHz range (giving a sample every 500-333 micro seconds; i.e. time resolution of about half a milli second). So why can’t you cycle faster? Why does this work at all? This technique takes advantage of the fact that the capacitance of the electrode is usually lower than the capacitance of the cell being recorded. Capacitance has the effect of slowing the kinetics (the rise and fall times) of currents. If the capacitance of the electrode is significantly lower than that of the cell, then when current is passed through the electrode, the electrode voltage will change faster than the cell voltage. Thus when you inject current into the cell through the electrode and then turn it off (at the end of a duty cycle), the electrode voltage will decay faster than the cell voltage. As soon as the electrode voltage asymptotes to the cell’s voltage, the voltage sample can be taken (again) and the next bolus of current applied. Thus the frequency of the duty cycle is thus limited to the speed at which the electrode voltage rises and decays while passing current. The lower the electrode capacitance, the faster one can cycle.The reason that this technique works at all is because the electrode can change it’s voltage much faster than can the cell (or maybe better put: it is a requirement of this technique that an electrode can change it’s voltage faster than the cell). This way, the electrode can get a sample, pass current to move the cell’s membrane potential, but when the current is turned off at the end of the duty cycle (to allow for the next voltage measurement), the cell cannot recover from the previous cycles current injection before the next cycles current injection beings. In order for this to work, the cell capacitance must be higher than the electrode capacitance by at least an order of magnitude. Better still, 2 orders of magnitude.Say you are standing at the bottom of a hill holding a soccer ball. You have a friend standing up on the hill who wants the ball. The friend can move up and down the hill at will. Your friend, or more accurately, the altitude of your friend, is the command potential. The altitude of the soccer ball is the cell’s membrane potential. The difference between the altitude of your friend and the altitude of the ball is the error signal. You, are the electrode (Be the electrode!). In discontinuous single-electrode ball movement, you move the ball up the hill with your feet. You can’t judge the distance to your friend and see the ball at the same time (say you have to look down to see the ball, and up to see your friend). So you judge the distance to your friend (measure the voltage difference) then kick the ball toward your friend (an epoch of current passing). The distance is too great to make it to your friend in one kick, so you run up the hill after the ball, (entering the next duty cycle) judge the distance again and kick the ball up the hill again. You repeat this until the ball reaches your friend (and thus, the error signal is zero). Now, if the speed at which you can run up the hill is too slow compared to the ball’s speed at rolling back down the hill, you won’t make any progress. But if you are significantly faster than the ball, you can run up the hill, and kick it again before it’s had much of a chance to roll back down the hill. This is basically what is happening during SEVC-d. The electrode is kicking the cells membrane potential toward a goal, pausing to measure the voltage, and then kicking it again before it can decay significantly. Of course, such analogies can go only so far in explaining. Here for instance, it doesn’t really need to be your friend on the hill. It could just as well be your enemy (although why you’d want to give your enemy a soccer ball is beyond me). But your enemy might want to torment you by constantly changing the command potential, so maybe its better if you stick with a friend.Just to carry this analogy to its logical extreme, two electrode voltage clamping would be the same, except you could just carry the ball up the hill without ever taking your eyes off of your friend. SEVC-c would be like giving the ball to a little kid who you ‘’think’’ is reliable, but you’re not completely sure, and asking the kid to carry the soccer ball to your friend. You lost your glasses, so you can only kind of see the kid, so you think he’s doing the job, but you are not really sure ;-).So why use SEVC-d? SEVC-d has the major advantage over SEVC-c in that you can ''actually measure the membrane potential of the cell''. Furthermore, since you are never passing current and measuring voltage at the same time, there is never a series resistance error. The main disadvantages of this technique are that the time resolution is limited, and it takes considerably more skill to perform than SEVC-c. The main reason that SEVC-d is more difficult is that the amplifier, being an oscillator within a feedback loop, is inherently unstable. If the amplifier passes too much current such that the goal voltage is over-shot, it will reverse the polarity of the current in the next duty cycle. This will cause you to undershoot the target voltage so the next duty cycle reverses the polarity of the injected current again. This error can grow larger with each duty cycle until the amplifier is changing current polarities with each duty cycle at its maximum ability to pass current. In more understandable terms, the amplifier just oscillates out of control. This is known as “ringing” the amplifier. While “ringing” does no harm to the amplifier, it almost always results in the destruction of the cell being recorded.The investigator is faced with two competing interests. First s/he wants to make the duty cycle as fast as possible to improve temporal resolution. The amplifier has a number of adjustable compensators that will make the electrode voltage decay faster. So to improve the temporal resolution as much as possible, the investigator sets these compensators at their maximum level. The trouble is, setting these compensators too high causes the amplifier to ring. So the investigator is always trying to “tune” the amplifier as close to the edge of uncontrolled oscillation as possible. The trouble with this strategy is that small changes in the recording conditions (say, a small disturbance in the depth of the solution bathing your cells) can cause the amplifier to ring if it’s already teetering on the edge of ringing. There are two solutions to this problem. First one could “back off” the amplifier settings into a safe range. This would be analogous to asking a NASCAR driver to drive slower during a race to preserve the car. The second solution is hyper vigilance. The investigator must sit there eyes raptly glued to the oscilloscope screen looking for warnings that the amplifier is about to ring. One often gets a half second or so of warning before ringing becomes uncontrollable, so the investigator must also sit there with his/her hands actually ‘’on the knobs’’ in order to be able to react fast enough to save the cell. Because of this, it is one of the most mentally intense electrophysiology experiments, but also the most !adrenaline-producing._________ ____________________Text? below here is "old" and has not yet been incorperated into the new structure. Feel free to do so, I will get to it soon if someone else doesn't User:SynaptidudeSynaptidude 23:02, 15 August 2005 !(UTC)_________________________ ____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 its 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 micrometer 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 studying 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.Category:physiologyCa tegory:Neuroscienceru:Элек трофизиологияfr: lectrophysiologie
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Websites
North American Society of Pacing and Electrophysiology
Details about NASPE who is dedicated to the study and management of cardiac rhythm disorders. Includes find a specialist, a library, products and courses, scientific sessions, news and information about the heart.
http://www.naspe.org
Electrophysiology and the Molecular Basis of Excitability
Simulations of ion channels and excitable membranes including diffusion, membrane potentials, nerve voltage clamp, and propagated action potentials.
http://pb010.anes.ucla.edu/
Futura
A multi-media medical educational organization dedicated to disseminating specialized, clinical information to the medical community through publications, electronic media, conferences, courses and symposia.
http://www.futuraco.com/
Indian Pacing and Electrophysiology Journal
Free full text access, peer-reviewed online journal devoted to cardiac pacing and electrophysiology, published quarterly. Manuscript submission and peer review are entirely through electronic media.
http://www.ipej.org
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