# Intracellular recording

Post-publication activity

Curator: Matti Weckstrom

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.

## 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 larger 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

Figure 1: Schematic representation of the main cell-level methods, with which the neural signals can be recorded

When the investigator desires to record neuronal membrane signals reliably with high signal-to-noise ratio in situ, that is, in tissue, the intracellular recording technique has to be employed. 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. 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.

Intracellular recordings can be done only with glass micropipettes (or microelectrodes) at present. The basic idea is to insert a conductive medium (the electrolyte filling the pipette, e.g. 1-3 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.

## 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), 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. 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.

Figure 2: A sharp glass capillary electrode used for intracellular recordings as seen through light microscopy (courtesy of R. Sabattini)
Figure 3: A microelectrode electrographed with SEM (courtesy of A. Piironen)

### 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 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, 1986). 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 the neuron-marking solution Lucifer Yellow (5 % LY with 0.1 % LiCl), 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.e. no larger currents can be pushed through) with currents of only a few nA's; this of course depends on the resistance and the other properties of the electrode tip.

A glass microelectrode will generate a voltage called a tip potential, when in contact with any solution, be it intra- or extracellular, artificial or in tissue. The tip potential may be up to nearly -100 mV, 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.e. it is smaller than $$E_{\rm Y}=RT/(F z_{\rm Y}) \ln({\rm [Y]_o/[Y]_i})\ ,$$ where $$[Y]_o$$ and $$[Y]_i$$ are the concentrations of any single ion in the electrode and the outside solution; here we are assuming a bi-ionic case, where the voltage formed by them would be equal). 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.e. when the filling solution and the outside liquid have the same components). 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 1981). 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.

The glass electrode has also a (distributed) capacitance, which is high enough (in the range of picoFarads) to define, with the electrode resistance, a large time constant for the electrode (the time constant $$=R C$$). 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 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 capacitance is not grossly overcompensated, causing "ringing" (fast oscillatory behavior that is purely artefactual). However, 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 glass microelectrodes. It is a DC-amplifier with a large input resistance (of the order of $$10^{11}$$ 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. 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 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 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.

### Current injection

A straight-forward manipulation of the recorded cell could be done by injecting an 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 compensated with newer methods requiring the use of time-sharing (switched) techniques.

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 record intracellular voltage and an additional electrode injects current. 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.

### 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), in which one intracellular electrode alternates between recording voltage and injecting current. This can be realized with high frequency (in the kHz 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. An advanced application of the single-electrode clamp is to use current or voltage clamping to probe the input impedance (or in case of voltage clamping, the input admittance) of the neuron. 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 (Weckström et al, 1991).

## 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.
• Weckström M, Kouvalainen E. and Juusola M. (1992) Measurement of cell impedance in frequency domain using discontinuous current clamp and white-noise-modulated current injection. Pfluegers Arch 421:469-472.

General references

• Glass microelectrodes (1969), eds M. Lavellee, O.F. Schanne and N.C. Hebert. Wiley: New York etc.
• Purves R.D. (1981) Microelectrode methods for intracellular recording and ionophoresis. Academic Press: London etc.
• Brown K.T. and Flaming D.G. (1986) Advanced micropipette techniques for cell physiology. Wiley: Chichester, New York etc.
• The axon guide [1], R. Sherman-Gold (ed.).

Internal references

• Erwin Neher and Frederick J. Sigworth. Patch-Clamp. Scholarpedia (in preparation).
• Rodolfo Llinas (2008) Neuron. Scholarpedia, 3(8):1490.