Intracellular recording - Scholarpedia. Intracellular recordings form a group of techniques used to measure with precision the voltage across, or electrical currents passing through, neuronal or other cellular membranes by inserting an electrode inside the neuron. ![]() That was preceded by a large body of work using other methods for larger cells. The most successful of those was the axial wire technique by Marmont (published in 1. Cole, Hodgkin and Huxley, ultimately leading to the development of voltage- clamp (Hodgkin et al., 1. The microelectrode method was later expanded by the invention of patch clamp by Neher and Sakmann (1. Brennecke and Lindeman 1. Although in some cases the use of other methods, like whole- cell patch clamp, would be possible, the intracellular recording technique remains the method of choice for this purpose. Erwin Neher and Bert Sakmann developed the patch clamp technique and were awarded the. Patch clamp technique is a technique in electrophysiology that allows the study of individual ion channels in. View history; Actions. Intracellular recording technique. ![]() Certainly, extracellular recording techniques are possible to use, and if multiple cell recordings are desired, they probably are a necessity. However, extracellular recordings can normally only pick up fast electrical events, like action potentials, but fail in case of slower, graded voltages, such as receptor potentials or synaptic potentials. The basic idea is to insert a conductive medium (the electrolyte filling the pipette, e. M KCl) through the cell membrane with minimal damage to the cell. This makes it possible to record the potential difference between the intracellular space (at the point of insertion) and some extracellular reference point. Simple as this sounds, the use of the technique is full of minor problems, although it remains the method of choice for most neuroscience investigations involving neural signaling. Although historically this was also done manually, special devices, microelectrode pullers, have been used since 1. In all pullers the glass is heated to a melting point, subsequently pulled and cooled quickly, either passively or with the help of additional air flow. In this process the electrode tip is formed when the two halves are separated. The sharpness of the resulting electrodes depends on the glass type (borosilicate, aluminosilicate or quartz), the manner of pulling, and the puller device technology, but the outer diameter of the tip is typically of the order of 5. The fabrication method crucially defines the usefulness and the properties of the microelectrode, and special attention has to be paid to the selection of the puller. Some pullers (because of the technological method used) may be more suitable for some specific purpose than another. Typical resistances of intracellular electrodes to DC- current are 1. Megaohms. The resistance depends not only on the length of the electrode shank and the size of the tip, but also on the nature of the conductive electrolyte inside (cf. Brown and Flaming, 1. As an example of the latter, a typical 1. Megaohm electrode (with 3 M KCl filling) with a shank of about 1. Megaohms or more when filled with the neuron- marking solution Lucifer Yellow (5 % LY with 0. Li. Cl), which has much lower mobility than potassium or chloride ions. The electrode resistance may also depend on the current used to measure the resistance. Typically glass microelectrodes have a very non- linear current- voltage relation, and often the response may saturate (i. The tip potential may be up to nearly - 1. V, and it is the larger, the sharper is the electrode. The tip potential is formed by two components, the liquid junction potential and the tip potential proper. The electrolyte in the pipette, when touching another electrolyte, will form a Nernst potential- like voltage that is defined by the composition of the solutions in question. However, the liquids are partially mixed in the tip, and the junction potential is not as large as it would be if calculated directly from the equation for the equilibrium potential (i. The tip potential proper is a more mysterious part of the phenomenon. It depends on the tip size and shape, but is abolished in symmetrical solutions (i. The tip potential is difficult to measure, but this is possible to estimate with a measurement of the recording electrode against a similar but broken- tipped specimen (Purves 1. Small differences in electrodes may result in disproportionally large changes in the tip potentials. In routine use the experimenter would use the amplifier's off- set control to remove the tip potentials, as well as to get rid of other similar voltages (like the voltage created by the dis- similarity between the recording and the indifferent electrode). It has to be noted that the exact recording of DC- voltage levels, most notably the cell resting potentials, may be biased by the tip potentials, although they have been partially offset from the recordings by electronics. Patch-Clamp Technique. Patch-clamp recording by revealing channel. The patch clamp technique is a laboratory technique in electrophysiology that allows the study of. Patch clamp recording uses a glass micropipette called a patch. The patch-clamp technique. Much of the capacitance is formed in connection to the usually grounded liquid surrounding the cells, like the extracellular fluid, that is normally connected to recording ground reference. However, some of the capacitance is also formed between the electrode- filling electrolyte and nearby grounded equipment. Because the resistance and the capacitance form together a low- pass filter, the electrode is inherently very slow, if nothing is done about the problem. The electrode time constant (uncompensated) can be up to seconds, but may be brought down to the microsecond range with a proper capacitance compensation circuit in the recording amplifier, even in an electrode with a resistance of 1. Megaohms or more. The exactness of capacitance compensation is not a critical issue in routine voltage recordings, as long as the electrode time constant does not limit the time resolution (i. However, capacitance compensation is critical with single- electrode clamping techniques. A voltage- follower based amplifier (an . It is a DC- amplifier with a large input resistance (of the order of \(1. How fast signals can be recorded (how reliable is the high frequency end of the spectrum) is determined by how well the electrode capacitance can be compensated. Different manufacturers use different electronic designs for this, but with some it is possible to decrease the electrode time constant from the range of tens of milliseconds to some microseconds. The recorded voltage contains noise both from the cell membrane and from the electrode. This is because during recording a small (bias) current flows through the electrode tip. In addition to this, the glass electrode is especially prone to pick up 5. ![]() Hz (or 6. 0 Hz) hum. This requires some elaborate planning concerning the grounding of the recording set- up. All that can be done to reduce electrode capacitance is useful as well, although some theoretically- attractive techniques, like so- called driven shield (driving the recorded signal to the immediately surrounding grounded surface in order to eliminate the capacitance), do not help very much in practice. The Ion Channel Group, Methods, Electrophysiology. One of the advantages of the patch clamp technique is that it allows measurements on very small cells such.This is normally done to probe the passive or active properties of the cell membrane. The current injection causes a displacement of the cell. Various techniques exist to avoid this electrode artifact. The oldest is realized by a bridge- balance circuit in the amplifier, which can only compensate for the resistive artifact, not the effects of the capacitance. The latter can be compensated with newer methods requiring the use of time- sharing (switched) techniques. For uncharged molecules something else is needed, like pressure injection. Iontophoresis is utilized in numerous cell marking techniques, e. A voltage- clamp amplifier forms a feedback circuit between the recorded voltage and the injected current. With this the cell membrane voltage may be controlled and the membrane current recorded as the equivalent of the feedback current injection required to realized the desired current. Several errors are possible in voltage- clamping cells. Some are related to the speed with which the voltage of the neuronal membrane may be changed. Some are related to the absolute level of attained voltage as compared to the command voltage. Solutions, at least partial, for minimizing these errors are available, and they involve both amplifier design and practical solutions in the recording set- up. One grave error that is difficult to avoid in classical two- electrode voltage- clamp is the so- called series resistance error, which creates a voltage artifact, whereby the voltage of the neuronal membrane is not in reality what it appears to be. This is avoided, at least in principle, in single- electrode clamp. This can be realized with high frequency (in the k. Hz range) with digital switching circuits. The core of the idea is that the current injection - either for current clamping or for voltage clamping - is done with short regular pulses, between which voltage is recorded. While recording membrane voltage, the electrode voltage has time to relax nearly completely, until it is sampled in the end of each injection- recording cycle. If the relationship between the electrode and cell time- constants are such that at the sampling time a considerable fraction of the voltage change still remains in the cell membrane, the cell can be clamped. In the end of the cycle only a residual, small electrode voltage artifact remains. This requires a careful tuning of the amplifier and requires a good capacitance compensation circuit in the amplifier, to enable the use of high- resistance electrodes in tissue (Weckstr. Ling, G. The normal potential of frog sartorius fibres. Theory of membrane voltage clamp with discontinuous feedback through a pulsed current clamp. Pfluegers Arch 4. Academic Press: London etc. Wiley: Chichester, New York etc. Scholarpedia (in preparation). Scholarpedia, 3(8): 1.
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