Electrophysiological Recording Techniques

Javier Cuevas USF College of Medicine, Tampa, USA

ã 2007 Elsevier Inc. All rights reserved.

The electrical properties of cells and the cellular components that contribute to them
are key to the function of all living cells, from paramecia to plant cells to vertebrate
neurons. There are a variety of recording techniques to study cellular electrophysiology.
Outlined below is a general overview of the most commonly used methods.

Extracellular Recordings

Extracellular recordings measure the field potential outside of cells. One of the most
common uses of this approach is the measurement of electrical activity in brain slices.
Extracellular recordings may be conducted from single or multiple cells. Multiple-cell
recordings utilize microelectrode arrays to measure simultaneously the electrical activity
of many neurons to study their concerted activity. The extracellular signals recorded arise
from the flow of ionic current through the extracellular fluid (Fig. 1). The current flow is
measured as the potential difference (V) across the two electrodes, and is defined by
Ohm’s Law (V=IR), where I is the current flowing between parts of the cell and R is the
resistivity of the saline solution. As I fluctuates, a change in V is observed. As a result of
I and R being small, the recorded signal is very small, usually on the order of 10–500 mV.
Microelectrodes for these studies may be constructed from either metal (e.g., glass
insulated platinum) or may be saline-filled glass micropipettes.
A variation on this technique is carbon fiber microelectrode amperometry, which is
used to measure neurotransmitter release. This method employs a carbon fiber micro-
electrode that is set at a potential of 600–900 m V. At this voltage, various neurotrans-
mitters, including catecholamines and indolamines, are oxidized. Electrons are
transferred onto the carbon filament during this process, producing a measurable current
that is proportional to the amount of neurotransmitter released. Small peptide neuro-
transmitters containing cysteine, tryptophan, and/or tyrosine may also be detected using
this approach.

Fig. 1. Extracellular recording configuration.

1
2 Electrophysiological Recording Techniques

Patch-Clamp Recording Techniques

Patch-clamp recording techniques achieving ’gigaseals’ were first described by Neher and
Sackmann Hamill et al (1981). This technique radically improved the quality of recording
beyond that of classical electrophysiology. The three main advances provided by patch-
clamp techniques were that it facilitated the control of the membrane potential, it
permitted the study of isolated membrane patches, and that it dramatically decreased
electrical noise. The variance of the current noise (A2) through a resistor (R, O) is
predicted by the Johnson Noise Equation: A2 = 4kTfc/R, where k is Boltzmann’s constant,
T is absolute temperature ( K), and fc is the low-pass filter setting (Hz). Therefore, the
100 GO seals achieved with this technique result in noise that is 30-fold less than that
observed using classical electrophysiology, thereby permitting the resolution of unitary
currents with amplitudes less than 10 pA.
Patch-clamp recording techniques have been employed to study membrane responses
in a variety of excitable and nonexcitable cell types, ranging from neurons to lymphocytes.
These seals, which dramatically improve the signal-to-noise ratio, are achieved in an all-
or-nothing manner. Various conditions must be met to facilitate the establishment of
gigaohm seals: (1) The cell membrane surface must be free of extracellular matrix,
connective tissue and or support cells (e.g., Schwann cells). (2) Bath and pipette solutions
must be free of debris and are ideally filtered with a detergent-free 0.2 mM filter. (3) The
pipette tip must be smooth and symmetrical. Fire-polishing the electrode, or using a
multi-step puller, will result in suitable pipette tips. (4) Each electrode should be used for
only a single cell, and should be discarded if contact occurs with material other than the
cell to be studied. (5) A small amount of positive pressure should be applied to the patch
pipette prior to breaking the air-solution interface. This positive pressure must be
maintained until the seal is established. Once the pipette comes in contact with the target
cell, the positive pressure can be relieved and a small amount of negative pressure applied.
Depending on the conditions of the cell surface, solutions, and pipette tip, a gigaohm seal
may form instantaneously after a few minutes of negative pressure.
Patch-clamp recordings may be conducted under two distinct conventions, voltage-
clamp, and current-clamp. Under voltage-clamp, the membrane potential is held constant
by the patch-clamp amplifier, and membrane currents recorded. Conversely, under
current-clamp, current through the membrane is held constant and the membrane
potential is permitted to fluctuate. Current-clamp is used primarily to study the active
membrane properties of cells, although passive membrane properties, such as input
resistance, may also be studied.

Whole-cell patch clamp

Conventional dialyzing configuration. For conventional dialyzing, whole-cell recordings, the

membrane under the patch pipette is ruptured after the gigaohm seal is established. Two
methods are used to destroy this membrane. The first employs pronounced suction,
whereas the second involves applying a strong voltage across the membrane. Many
patch-clamp amplifiers come equipped with a ‘‘zap-button’’ for electrical disruption of
the patch membrane. While the most suitable method for a given experiment depends on
the cell-type to be studied, most scientists use the negative pressure technique. Under
dialyzing conditions, the patch pipette solution is the intracellular solution, and thus must
contain the appropriate composition. A typical conventional whole-cell patch-clamp
patch pipette solution contains (in mM): 140 KCl, 2 MgATP, 0.1 guanosine 5’-triphos-
phate sodium salt (GTP), K4BAPTA, 10 HEPES-KOH, pH 7.2. The composition of this
 Electrophysiological Recording Techniques 3

solution may be manipulated to add to or deplete the cytoplasm of specific reagents,

facilitating pharmacological characterization of signal transduction cascades, and/or
effector targets. The dialyzing whole-cell configuration is particularly useful when the
drugs under study act intracellularly but do not cross the cell membrane readily because
of charge or size (e.g., local anesthetic containing a quaternary amine, such as QX-222 or a
monoclonal antibody).
Perforated-patch configuration. The major advantage provided by perforated patch record-
ing techniques is that the intracellular milieu is preserved to a greater extent than by the
conventional dialyzing whole-cell method. Lindau and Fernandez (1986) were the first to
employ this technique, using elevated concentrations of ATP in the patch pipette to
increase the permeability of mast cell plasma membranes. The perforated-patch tech-
nique was further improved with the use of the polyene antibiotics nystatin and ampho-
tericinRae et al (1991). While these compounds partition into cholesterol containing
lipids and form ion channels that permit the flow of monovalent cations, they are
impermeable to multivalent cations and nonelectrolytes the size of glucose or greater.
Thus, macroscopic currents may be recorded for extended periods from ion channels that
‘‘run-down’’ or that exhibit altered biophysical properties as a result of cell dialysis. This
configuration also permits the study of ion channel modulation by diffusible cytosolic
second messengers that would be lost as a result of cell dialysis. For example, muscarinic
responses in autonomic neurons will ‘‘run-down’’ within 10 mins of cell dialysis. How-
ever, these responses are preserved for > 1 hr when the perforated patch method is used
Cuevas et al (1997). As in the case of conventional whole-cell patch-clamp, the patch
pipette solution for perforated-patch recordings is the intracellular solution. A typical
perforated patch pipette solution contains (mM): 75 K2SO4, 55 KCl, 5 MgSO4, 10 HEPES,
360 mg/ml amphotericin B, and 0.6% (v/w) DMSO (pH to 7.2 with N-methyl-d-gluca-
mine). Note that SO4 is substituted for Cl. This substitution limits the effects of voltage
errors produced by Donnan potentials because both amphotericin and nystatin channels
are selective for monovalent cations (Na+:Cl-, 10:1). For the same reason, N-methyl-d-
glucamine, rather than HCl, is used for titration.

Cell-attached and excised membrane patch

Cell-attached patch: Fig. 2 (left panel) dipicts a cell-attached patch. Cell-attached patches
are formed while establishing the gigaohm seal in patch-clamp recording techniques. The

Fig. 2. Various configurations used for membrane patch recordings.

4 Electrophysiological Recording Techniques

membrane under the electrode is not ruptured or physically separated from the cell, thus
preserving its intracellular integrity. This technique is frequently used to study unitary
conductances of ion channels that ‘‘run-down’’ (e.g., sodium channels, nicotinic recep-
tors) as a result of the loss of a cytosolic component or intracellular ligand such as ATP
(black filled triangle). The tight seal between the patch pipette and the cell membrane
serves as a barrier that restricts the movement of large transmembrane proteins (e.g.,
membrane receptors, ion channels). Such proteins do not migrate into or out of the
membrane under the patch pipette. Therefore, only diffusible cytosolic second messen-
gers (black filled circle) or membrane components under the patch pipette influence the
electrical activity of ion channels within the patch. For this reason, the cell-attached patch
configuration is frequently used to determine if a diffusible cytosolic second messenger is
modulating an ion channel.
For cell-attached patches, the patch pipette solution is usually similar to the extracel-
lular solution. Agonists, such as acetylcholine, may be added to the pipette solution
(black filled diamond) to activate ion channels. Ion channel blockers may also be added to
the patch pipette solution to isolate the channels of interest. For example, the chloride
channel blockers, 4-acetamido-4’-isothiocyanato-2,2’-stilbenedisulfonic acid disodium
salt (SITS), and 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid disodium salt (DIDS),
are frequently added to the patch pipette solution to study sodium channels in cell
attached-patches. Patch pipettes may also be back-filled with pipette solution containing
a channel blocker such as tetrodotoxin, for Na+ channels, or tetraethyl ammonium, for K+
channels. This solution will slowly diffuse onto the membrane and eventually blocks the
activity of the targeted channel.
One of the difficulties in working with cell-attached patches is determining the
potential of the membrane patch. The membrane potential of the cell-attached patch
(Vpatch) is a function of the membrane potential of the cell (Vcell) and the potential applied
through the patch pipette (Vpipette), and is defined by the formula: (Vpatch) = (Vcell)-
(Vpipette). As the membrane potential of the cell can only be estimated, the Vpatch is also an
estimate. However, the membrane under the pipette may be ruptured at the end of an
experiment to obtain a measurement of (Vcell). Also, in the cell-attached patch configura-
tion, inward currents appear as outward currents. As some software and hardware auto-
matically compensate for this configuration, care must be exercised to ensure correct
voltage polarity and orientation of the signal.
Outside-out patch: Fig. 2 (center panel) is a depiction of an outside-out membrane patch.
Outside-out membrane patches are formed by first establishing a gigaohm seal, then
rupturing the membrane under the patch by applying negative pressure (identical to
conventional whole-cell configuration), and, finally, by slowly pulling the patch pipette
away from the cell until the membrane detaches. The membrane then folds onto itself and
the seal, forming an excised outside-out membrane patch. In some instances, the mem-
brane may fold incorrectly, resulting in an inside-out membrane patch. This patch
configuration is generally used to study ligand-gated ion channels, with the ligands
applied onto the patch through the bath solution (black filled diamond). In this manner,
the orientation of the patch may be confirmed. Under this configuration, the patch
solution is the intracellular solution, and therefore must contain appropriate intracellular
components (black filled triangle, i.e., ATP, GTP, cyclic AMP) that may be required for
proper function of the ion channel and/or signaling mechanism under investigation.
Inside-out patch: Fig. 2 (right panel) is a depiction of an inside-out membrane patch.
Inside-out membrane patches are formed by first establishing a gigaohm seal, and then
slowly pulling the patch pipette away from the cell until the membrane detaches. The
membrane then folds onto itself forming a vesicle. The outer face of the membrane vesicle
is then ruptured by rapidly passing the vesicle through the bath solution-air interface,
 Electrophysiological Recording Techniques 5

exposing the vesicle to low Ca++ solution, or momentarily making contact with a droplet
of paraffin or cured Sylgard.
The patch-pipette in the inside-out patch configuration usually contains extracellular
solution. This recording configuration is used to study ion channels activated by extracel-
lular ligands when the ligands are included in the patch pipette (black filled diamond).
The solution in the chamber bathes the cytoplasmic membrane face, permitting intracel-
lular modulators or activators of the ion channels in the patch to be studied through bath
application (black filled triangle, e.g., Ca++, spermidine, cyclic GMP). Thus, this configu-
ration is ideal for studying channels that are activated or modulated by intracellular
components. It is important to note that the membrane potential in this configuration is
equal and opposite to the patch pipette potential. Also, as in the case of the cell-attached
patch, inward currents appear as outward currents.

Loose-patch clamp

Patches with low-seal resistance are useful in specific situations. The ‘‘loose-patch’’ clamp
has been used to record currents from macroscopic patches containing numerous chan-
nels. Electrodes for these studies have tip diameters of 5–10 mM, in contrast to the smaller
tips of conventional patch-clamp electrodes, which have diameters in the range of 0.5–2 m
M. As these electrode tips can also be reused and repositioned, they can be employed to
map the spatial distribution of ion channels on a cell. The loose-patch clamp can also be
used to measure currents from neuronal processes that may not be studied with conven-
tional patch-clamp techniques because of their small diameter.

Intracellular Recording Techniques

The intracellular microelectrode, or sharp electrode, recording technique is one of the

classical methods for studying the electrical properties of cells. This technique involves
impaling cells with a fine glass microelectrode filled with a concentrated saline solution (e.
g., 3 M potassium chloride or 1 M potassium acetate). Electrodes used for these studies
have a high resistance (70–80 MO), limiting the diffusion of the salt solution into the cell.
However, the sharp tip is necessary to limit cell damage. These electrodes are sometimes
bevelled after they are pulled to decrease the tip resistance without increasing tip
diameter. Although sharp electrode studies may be used to study isolated cells or intact
tissue, they are primarily used for studies where patch-clamp recording techniques are not
possible due to the conditions of the cell surface. For example, intracellular microelec-
trodes are frequently used to study neurons in peripheral ganglionated plexi in situ. The
extracellular matrix of neuronal clusters of these plexi makes formation of a gigaseal
impossible, and mechanical or enzymatic cleaning of these plexi is impractical because it
often results in disruption of the circuits and/or compromises the integrity of the neurons.
Intracellular recordings may be conducted using current-clamp or voltage clamp
conventions. However, various problems associated with sharp electrode studies have
led to the decline in the use of this technique relative to the patch-clamp method: (1)
Access (series) resistance to the cell is high (~75 MO; <10 MO for patch-clamp), and
thus, errors in the measurement and/or loss of space-clamp may occur. Loss of space-
clamp refers to uneven clamping of the cell membrane (e.g., under voltage-clamp, one
region of the cell might be held at 60 mV and a different region at 55 mV).
Discontinuous single-electrode voltage clamp is often used to reduce measurement
errors. In discontinuous single-electrode voltage clamp there is multiplexing of the
electrode for current injection and recording. (2) Leak of solution through the
6 Electrophysiological Recording Techniques

membrane/microelectrode interface may result in recording artifact. For example, Ca++

entry through this gap often stimulates Ca++-activated K+ channels in neurons. (3)
Microelectrodes may produce significant damage to small cells and damage may be
exacerbated if there is cell movement (e.g., contracting cardiomyocyte).
One frequently used variation of this recording technique is the two-electrode voltage
clamp used for electrical studies in the Xenopus oocyte expression system. In two-elec-
trode voltage-clamp there is no need for multiplexing because one electrode is dedicated
to current injection and the second to recording. The use of two electrodes is necessary to
overcome the difficulties posed by clamping the large membrane area of the oocyte (>106
mM2) and recording the sizable currents (>10 mA) often encountered after injecting these
cells with cyclic RN A. The large size of these cells, however, does permit the use of
pipettes with larger tip size (0.5–2 MO), which decreases problems arising from series
resistance.

Ion Channel Reconstitution

Ion channel reconstitution involves the incorporation of intact membrane vesicles

containing proteins, or of water-soluble channels, into an artificial planar lipid bilayer
(Fig. 3). The most common configuration used for planar bilayer recordings is the open-
chamber configuration. In this case, the bilayer, also known as a black lipid membrane, is
formed by ‘‘painting’’ a partition separating two open bath chambers with phospholipids
(e.g., 75% phosphatidylethanolamine and 25% phosphatidylserine dissolved in a solvent
such as decane). In the case of proteins inserted into the membrane via a vesicle, the
proteins are oriented such that the face of the protein protruding from the vesicle faces
the chamber to which the vesicle was added (Fig. 3). Ion channel reconstitution techni-
ques using the open-chamber configuration have several benefits: (1) This method facil-
itates the recording of species of ion channels that are not amenable to the methods
described above, such as ion channels found in intracellular organelles (e.g., ryanodine
receptor). (2) It is possible to regulate the lipid composition of the membrane, making it
possible to study the influence of the lipid environment on channel function. (3) The

Fig. 3. Incorporation of vesicle and protein orientation in a lipid bilayer.

 Electrophysiological Recording Techniques 7

composition of the solution on both sides of the membrane may be readily controlled, thus
facilitating permeability and pharmacological studies.

Other Information – Web Sites

http://www.axon.com/manuals/Axon_Guide.pdf. This guide offers a comprehensive

description of electrophysiological recording methods.

Journal Citations

Rae, J., Cooper, K., Gates, P., Watsky, M., 1991. Low access resistance perforated patch recordings using
amphotericin B. J., 37, 15–26.
Cuevas, J., Harper, A.A., Trequattrini, C., Adams, D.J., 1997. Passive and active membrane properties of
isolated rat intracardiac neurons: regulation by H- and M-currents. J., 78, 1890–1902.
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for
high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch., 391,
85–100.

Further Reading

Ogden, D., Microelectrode Techniques. The Company of Biologists Limited, Cambridge, U.K., Edition 2,
1987
B. Hille, Classical biophysics of the squid giant axon. In Ion Channels of Excitable Membranes, Sinauer
Associates, Sunderland, MA, Edition 3, 2001