| Homepage | Imprint |
|
||
 |
ElectrophysiologyElectrophysiology is the science and branch of physiologythat pertains to the flow of ionsin biological tissues. In particular, it encompasses the electrical recording techniques that enable the measurement of this flow and the potential changes (signals) related to them.
Definition and scopeIn almost all cases, electrophysiological techniques record the voltagemaintained across a cell membrane(i.e. the electrical potentialdifference between the inside and outside of a cell) or the ioncurrentsthat 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). The technical goal of the electrophysiologist is 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 recordingand amperometry(which is a special case). Intracellular recording techniques encompass two major subdivisions: The voltage clamp and the current clamp. Many particular electrophysiological readings have specific names:
Intracellular recordingIntracellular 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 electrodeinto a cell, so that the intracellular potentialcan 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 and electrical resistance: 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 of thermal noise. In the 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. Current clamp"Current Clamp" is the term used to describe a simple recording of trans-membrane voltagewith the ability to inject current into a biological cell through the lumenof 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 Lawequation (Voltage = (current)x(resistance); V = IR). Second during "current-clamp" recordings, an investigator has the opportunity to inject current into a cell through the recording electrode. In actuality, the "current clamp" is nothing more than passive recording of the cell's membrane potential. Unlike in the voltage clamp, which holds the membrane potential at a level determined by the experimenter, 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, so nothing is clamped. Any applied current is merely an offset. It does not prevent voltage or current from varying in the cell. Also unlike the 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 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. AmplifiersAnother misleading term is "amplifier", which is commonly used to refer to the recording device. 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-impedanceelectronics. What the "amplifier" actually does is to "impedance match" the signal on input and the output of the amplifier. In other words, 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: A voltage of 10 mV is generated by passing 10 nanoamperesof current across 1MΩof resistance. The electrometer changes this "high impedance signal" to a "low impedance signal" by converting it to 10-5 microampere across 1000Ω. 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. The typical whole cell electrode has a resistance across its tip of 1-10 MΩ. A typical sharp micro electrode's resistance is more like 100 MΩ (100 million Ω). Add the membrane resistance that is in series with the electrode (typically 50-100 MΩ). In the most extreme (but not uncommon) of these conditions, a 10 mV signal is being measured dropping across around 50-200 MΩ. Typically, an average recording apparatus has a standard input resistance of 1 MΩ. A signal that is 10 mV dropping over 200 MΩ will be 50 femtoamperes (5×10</sup>-11</sup>). If you don't impedance-match the signal, but input it directly into a device with a 1 MΩ resistance, the voltage signal will be reduced to only 50 μV (5×10</sup>-11</sup> amperes × 1×106 Ω). 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 followercircuit. 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. For example, a fire hose can carry much more water per second (current) than can a garden hose. Assume that there is a valve on the nozzle of the firehose that is operated by the water pressure in the garden hose. If the garden hose has no pressure, the fire hose valve is closed. If the garden hose has a medium pressure, the fire hose valve opens more. Lots of pressure in the garden hose opens the valve wide in the fire hose. 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. Why not just make all the equipment high impedance and not bother with impedance matching? 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 G&Omega 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. Voltage clampImage:Voltage clamp.jpg The voltage clamp, an "active" technique, is a means by which an experimenter can "clamp" or maintain the cell potentialat a value he or she specifies. Voltage control is established using feedbackthrough an operational amplifiercircuit. 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. Voltage clamp measurements of ionic current are made possible by the near-simultaneous, digital subtraction of transient capacitive currents that pass as the recording electrode and cell membrane are charged to alter the cell's potential. Limits to voltage clamp measurements come into play when ionic currents are large, when recording pipette (series) resistance is high, or when the cell membrane area is large. Excesses in any of these three parameters alone or in combination will cause a significant drop in the applied voltage as the current passes through the pipette. Cells with a large capacitance cannot be accurately voltage clamped due to the excessively slow time constant of their membranes, which can only be charged as fast as current can pass into the cell through the series resistance. That is, if the channels gate faster than the spread of depolarization across the membrane (easy for sodium channels), then there is a non-uniform activation of the channels in the cell, making measurements of channel kinetics and voltage dependence into messy approximations at best. The drop in voltage due to series resistance leads to an error in the depolarizing direction for inward currents that can shift current-voltage relationships significantly to the left (early activation) without proper compensation. Voltage error due to series resistance can be compensated to about 90-95% of the set voltage with a relatively stable feed-forward circuit that is integrated into most patch clamp devices. Extracellular recordingSingle unit and field potentialsImage:Field potential schematic.jpg 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 sources are caused by ion currents across cellular membranes through 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. 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 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. AmperometryAmperometryis 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 monoamine neurotransmitters, e.g., norepinephrine (noradrenalin), dopamine, serotonin (5-HT), are oxidizable. The method is also applicable to cells that do not secrete oxidizable neurotransmitters by loading 5-HT or dopamine. External links
fr:Canal ionique lt:Elektrofiziologija nl:Ionkanaal ja:???????? ru:????????????????? fr:électrophysiologie
|
