Intracellular recording
From Scholarpedia
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Author: Prof. Matti Weckstrom, University of Oulu, Finland, Department of Physics, Division of Biophysics
Contents |
History
Intracellular recording technique as we know it today became possible after the use of glass microelectrodes for this purpose was invented by Gerard and Ling (1949). That was preceded by a large body of work using other methods for large cells. The most successful of those was the axial wire technique by Marmont (published in 1949), further developed by Cole, Hodgkin and Huxley, ultimately leading to the development of voltage-clamp (Hodgkin et al., 1952). The microelectrode method was later expanded by the invention of patch clamp by Neher and Sakmann (1976) and the adaptation of electronics for single electrode voltage clamp (Brennecke and Lindeman 1974).
Basic principles of intracellular recording
When the investigator desires to record high signal-to-noise neuronal membrane signals reliably in situ, that is, in tissue, intracellular recording technique has to be employed. Although in some cases the use other methods, like whole-cell patch clamp has been shown to be possible, intracellular recording technique remains the method of choice. Certainly, extracellular recording techniques are possible to use, and in case of multiple recordings they often are a necessity.
Intracellular recordings can be done only with glass micropipettes (or microelectrodes) at present. The basic idea is to have a conductive medium (the electrolyte filling the pipette, e.g. 1-3 M KCl) inserted through the cell membrane with minimal damage. 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 technique is full of problems, although it remains the method of choice for most neuroscience investigations involving neural signalling.
Fabrication and properties of glass microelectrodes
Fabrication
Intracellular electrodes are made of thin glass pipettes that are pulled to a very fine and sharp ending or tip. Although historically this was also done manually, special devices, Microelectrode pullers, have been used since 1950s, and microprocessor controlled pullers since the end of 1980s. 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) and the manner of pulling and the puller device technology, but the outer diameter of the tip is typically of the order of 50-500 nanometers.
Electrical properties
The electrolyte inside creates a (distributed) resistor, but normally most (probably more than half) of the resistance resides in the tip. Typical resistances of intracellular electrodes to DC-current are 10-500 Megaohms. The resistance depends on the length of the electrode shank, the size of the tip, but also on the nature of the conductive electrolyte inside. As an example of the latter, a typical 100 Megaohm electrode (with 3 M KCl filling) with a shank of about 15 mm and with a tip outer diameter of about 70 nm will have a resistance of about 600 Megaohms or more when filled with neuron marking solution with Lucifer Yellow (5 % LY with 0.1 % LiCl) that has much lower mobility than potassium or chloride ions.
The glass electrode has also a (distributed) capacitance, which is high enough to define, with the electrode resistance, a large time constant for the electrode. 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 equipments. 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 100 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.e. the electrode does not appreciably low-pass filter the signals to be recorded), or as long as the capacatitance is not grossly overcompensated, causing "ringing" (fast oscillatory behaviour that is purely artefactual). However, the capacitance compensation is critical with single-electrode clamping techniques.
Electrode in the cell - voltage recording
When the electrode is advanced through the cell membrane in a measurement set-up, a recording circuit is formed. A voltage-follower based amplifier (an “Intracellular amplifier”) is normally used with the glass microelectrodes. It is a DC-amplifier with a large input resistance (of the order of 
ohms. 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 for. 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 voltage follower in the amplifier sends reliably forward the voltage at the tip (if all is in order with the recording set-up otherwise), to be stored or processed further. The voltage recorded also contains noise from the cell membrane, but also 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 50 Hz (or 60 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.
Advanced techniques
Current injection
A straight-forward manipulation of the recorded cell is injection of electrical current through the recording electrode. This is normally done to probe the passive or active properties of the cell membrane. The current injection causes a displacement of the cell’s membrane voltage, but also a voltage change in the electrode resistance. 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 done with newer methods requiring the use of time-sharing (switched) technique.
Current injection can also be used to introduce substances into the cell, carried with the current (iontophoresis), in case these substances are charged. For uncharged molecules something else is needed, like pressure injection. Iontophoresis is utilized in numerous cell marking techniques, e.g. with Lucifer Yellow or cobalt.
Voltage clamp
Classical voltage clamp with two or more electrodes (TEVC) requires the use of intracellular techniques, whereby one or two electrodes act like in the intracellular voltage recording and an additional as a current injecting electrode. 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 include 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, that 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.
Single-electrode clamp (switched clamp)
Resistive and capacitive properties of the intracellular glass microelectrode can be (ideally) completely compensated with the help of a time-sharing technique (single electrode voltage clamp), where one intracellular electrode acts alternatingly as a voltage recording and as a current injecting electrode. This can be realized with high frequency (in kHz range) with digital swithching 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.
References
- Hodgkin A.L., Huxley A.F., and Katz B. (1952) Measurement of current-voltage relations in the membrane of the giant axon of Loligo. J. Physiol. 115(4):424-48.
- Hodgkin A.L. and Huxley A.F. (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117(4):500-44
- Ling, G. and Gerard, R.W. (1949). The normal potential of frog sartorius fibres.
- Marmont G. (1949) Studies on the axon Membrane; a new method. J Cell Physiol. 34:351-82.
- Brennecke, R. & Lindemann, B. Theory of membrane voltage clamp with discontinuous feedback through a pulsed current clamp. Rev. Sci. Instrum. 45: 184-188, 1974.
Internal references
- Alan Finkel (2007), Single electrode voltage clamp. Scholarpedia, 2(8):3528.
- John W. Moore (2007) Voltage clamp. Scholarpedia, 2(9):3060.
- Erwin Neher and Frederick J. Sigworth. Patch-Clamp. Scholarpedia (in press).
