Intracellular recording
From Scholarpedia
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This article is still unfinished, but will be completed (hopefully) before the end of 2007
Contents |
Basic principles of intracellular recording
When the investigator desires to record neuronal signals in situ, intracellular recording technique has to be employed, although in some cases whole-cell patch clamp is also possible. It can be done only with glass micropipettes (or electrodes) 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 signaling.
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. In this process the electrode tip is formed. The sharpness 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 usually 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 a shank of about 15 mm and with a tip outer diameter of about 70 nm has a resistance of about 600 Megaohms when filled with neuron marking Lucifer Yellow solution (5 % with 0.1 % LiCl).
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 surreounding the cells, like the extracellular fluid, but some of the capacitance may form between the electrode filling liquid and nearby grounded equipments. This all means that the electrode is inherently slow, if nothing is done about the problem. Most of the capacitance in the electrode is distributed between the conductive interior and conductive surfaces nearby, like those of other parts of the recording system. The electrode time constant (uncompensated) can be up to seconds, but may be brought down to the microsecond range with a proper capacitance compensation even in a 100 Megaohm electrode.
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 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 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.
Advanced techniques
Current injection
A straight-forward manipulation of the recorded cell is injection of electrical current through the recording electrode. This causes a displacement of the cell’s membrane voltage, but also a voltage change in the electrode resistance. Various techniques exist to avoid the electrode artifact. The oldest is realized by a bridge-balance circuit in the amplifier, but this can only compensate for the resistive artifact, not the effects of the capacitance. Newer methods require the time-sharing (switched) technique.
Current injection can also be used to introduce substances into the cell, carried with the current (iontophoresis). This is utilized in numerous cell marking techniques, e.g. with Lucifer Yellow, cobalt.
Voltage clamp
Classical voltage clamp with two (or more) electrodes 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 the feedback circuit, with which the cell membrane voltage may be controlled and the membrane current recorded as the feedback current injection required.
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 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 circuits. The core of the idea is that while recording membrane voltage the electrode voltage has time to relax nearly completely. In the end of this period the membrane voltage can be sampled with only a residual, small electrode artifact. 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.
