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10Single electrodevoltage-clamp (SEVC)

Harold A Coleman and Helena C ParkingtonPhysiology, Monash University, Australia

10.1 Basic ‘how-to-do’ and ‘why-do’ sectionThe great power of the voltage-clamp technique in elucidating ionic mechanisms,

particularly voltage- and time-dependence, was well demonstrated by the landmark

studies of Cole, and Hodgkin & Huxley and colleagues in the 1940s and 1950s.

During those and subsequent studies on animal and plant cells over the following

decades, the voltage-clamp was applied using two electrodes, one measuring

membrane potential while the other passed current across the membrane. The

requirement to insert two electrodes into a cell limited the use of this technique to

relatively large cells.

The eventual development of the patch-clamp technique in the early 1980s

overcame a number of the difficulties, enabling many types of cells, over a large

range of size, to be voltage-clamped with a single electrode. The relatively large

tip size (�1mm diameter) and the resulting very low access resistance (typically

�2–5MV), compared with the input resistances of cells (100–1,000MV), meant

that a single electrode could be used both to measure membrane potential and also to

pass current.

Prior to the development of the patch-clamp technique, a different approach,

based on a switching amplifier, was developed by Brennecke & Lindemann (1974a,

1974b) to enable voltage-clamping of small cells with a single microelectrode.

This approach is variously referred to as single-electrode voltage-clamp (SEVC),

switching voltage-clamp, or single microelectrode voltage-clamp.

Essential Guide to Reading Biomedical Papers: Recognising and Interpreting Best Practice, First Edition.

Edited by Phil Langton.

� 2013 by John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

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10.1.1 Advantages of SEVC

The SEVC technique combines the advantages of microelectrodes, listed in

Primer 9, with the power of the voltage-clamp technique to study ionic mechanisms

in small cells or in electrically short syncytial tissues. It can also enable the voltage-

clamping of cells that cannot be accessed with patch-electrodes, such as neurons

within deep layers of the brain (Richter et al., 1996). The flexibility of the micro-

electrode also enables contractile tissues, such as smooth muscle, to be voltage-

clamped despite contraction movements of the cells (Figure 10.1).

In other situations, there can be too much connective tissue to enable patch-clamp

recordings from the cells, and/or it is undesirable to use enzymes to clean the cells.

In these cases, SEVC can come to the rescue. Importantly, SEVC recordings cause

minimal, if any, disruption to intracellular messenger systems that may be critical

for the regulation of ionic mechanisms in cells.

10.1.2 Limitations of SEVC

The high resistance of microelectrodes and the discontinuous, switching nature of

SEVC limits the amount of current that can be passed by the electrode, thereby

preventing the clamping of large currents. Furthermore, the capacitance of the

microelectrode (see Primer 9) limits the speed of the SEVC, making it difficult to

impossible to clamp very fast events.

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Figure 10.1 Membrane currents in electrically short segments of guinea-pig submucosal arteriolesrecorded under voltage-clamp with single intracellular microelectrodes (SEVC). Acetylcholine acti-vated receptors on the endothelium to evoke endothelium-derived hyperpolarizing factor (EDHF).The underlying current involves intermediate- and small-conductance Ca2þ-activated Kþ channels,blocked by charybdotoxin and apamin, respectively, and whose I/V relationships, obtained fromperiodic ramps, arewell describedby theGoldman-Hodgkin-Katz equation for apureKþ current. Theresults show that no other currents in the endothelium or smooth muscle cells contributed to theoutward hyperpolarizing current. The recording of the EDHF current required that the endotheliumbe intact and electrically coupled to the outer, single layer of smooth muscle cells. This was onlyachievable by the use of microelectrode SEVC. Coleman HA, Tare M & Parkington HC. (2001). Kþ

currentsunderlying theactionofendothelium-derivedhyperpolarizing factor inguinea-pig, rat andhuman blood vessels. J. Physiol. 531: 359–373. # Physiological Society.

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10.1.3 Circumventing the limitations

Voltage-clamping of all kinds generally involves various compromises, and SEVC

is no exception. The essential point is to focus on the critical physiology of interest.

For example, the very wide range of potentials, over which the membrane is often

varied with the patch-clamp technique (e.g. �100 to þ100mV), usually results in

currents that are so large at the extreme potentials that it is difficult to discern what

happens at the physiologically relevant potentials (which lie between�80 and 0mV

for many cells). SEVC can often provide very important information over this

physiologically relevant range of potentials. SEVC control can be improved by the

judicious use of ion channel blockers, and ion substitution can be used to reduce the

concentrations of ions carrying currents that are not the focus of study.

10.1.4 Applying the SEVC technique

The theory and operation of SEVC is well described by Finkel & Redman (1984).

However, a brief explanation is given here to enable a better understanding of its

limitations and possible pitfalls.

During SEVC, the switching amplifier alternately ‘samples’ the membrane

potential and then passes current that is in proportion to the difference between

the command potential and the sampled potential. The frequency of switching is

manually adjustable, but current is passed during about one-third of the total cycle

time. The current passes through both the microelectrode and the cell membrane,

resulting in potential differences from these two sources. Once the current passing

stops, the potential decays according to the membrane time constant and the

microelectrode time constant.

A major requirement of SEVC is that the potential on the microelectrode decays

much faster than that on the cell membrane. Thus, by the end of the 70 per cent non-

current passing period, the electrode potential needs to have decayed to essentially

zero, while there should only be a small change in the potential of the cell

membrane. At this time, the potential is sampled and compared with the command

potential in order to determine the amplitude of the next current passing phase. The

rate of switching has to be carefully adjusted to ensure that the potential on

the microelectrode has sufficient time in which to decay totally before the next

cycle starts. The quicker the potential decays off the microelectrode, the greater the

switching frequency that can be used, and therefore the faster the clamp response.

10.2 PitfallsThe greatest problem with SEVC is that it is very easy to voltage-clamp the

microelectrode but not the cell or preparation. Although published reports should

not contain data that is so empty of biological significance, you need to be on your

guard. You need to seek evidence that the authors recognize this risk and that they

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took appropriate steps to assure themselves (and you) that the data in their report

were reliably real.

Failure to clamp more than the electrode can result from a failure to recognize a

necessary compromise between the speed of the clamp (maxim ¼ ‘switching faster

is better’) and the requirement for the electrical potential difference of the electrode

to decay fully before the value of remaining potential (i.e. potential of the impaled

cell) is sampled and the switch is made to current passing (maxim¼ ‘switching too

fast is bad’).

In practice, the experimenter should monitor the voltage at the headstage of the

amplifier, using an oscilloscope at a fast sweep speed in order to observe the

characteristic shape of the decay of the potential from the microelectrode, and adjust

the switching frequency to ensure that the potential really does decay to zero. In any

event, the article should describe how the switching frequency was optimized so that

it was fast, but not too fast!

The crucial factors in determining the switching rate and quality of SEVC are the

electrical properties of the microelectrode, and authors need to state how these were

optimized. The use of microelectrodes with as low a resistance and as steep a taper

as possible for a particular tissue will reduce both electrode resistance and

capacitance. Electrode capacitance can be further reduced by minimizing the depth

of physiological solution in the bath, and by treating the outside electrode surface

with a hydrophobic compound.

There is a further complication. The risk of the problemdescribed above isminimal

if the electrode resistance is very low compared with the input resistance of the cells

under study.However, it is not uncommon to have electrode resistances and cell/tissue

input resistances that are comparable in magnitude and, in this situation, the issue

becomes critical. Journal articles will include in the data the values for electrode

resistance and estimated input resistance of the cell or preparation, but they will not

put them conveniently together unless they believe it important to do so.

10.2.1 Indications of poor clamping

There are several signs that indicate that the clamping is poor, which may result

from a switching frequency that is too high. In the more extreme case, the current

trace will be the inverse of the expected membrane potential response. For example,

depolarization may result in the current trace appearing like an upside-down action

potential. In the case of voltage-activated sodium and calcium channels, a good

indicator is the negative slope region of current-voltage (I/V) relationships

(Figure 10.2).

As the membrane is stepped to increasingly more depolarized potentials, there

should be a graded increase in the amplitude of these currents, as shown in

Figure 10.2. Under conditions of poor clamp control, there can be a sudden, all-

or-nothing-like increase in the amplitude of the inward current, and this will show

up as a very steep negative slope in the I/V relationship. It can also occur if the

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electrode cannot pass enough current to clamp the membrane, and/or the cell/tissue

is not sufficiently isopotential. That is, active responses may occur in electrically

distant cell processes that are too far from the electrode to be clamped.

The time course of tail currents can also indicate problems with poor clamp

control. Usually, tail currents decay with an exponential time course. However, in

the presence of a large series resistance, the current can almost ‘linger’ at a large

amplitude before declining to zero in a non-exponential-like manner. These issues

also apply to all voltage-clamp techniques, including patch-clamp, and are dis-

cussed in detail in a number of papers dating back to the earlier days of voltage-

clamping (Attwell & Cohen, 1978; Ram�on et al., 1975).

In summary, SEVC has some significant advantages. So long as it is applied

within its limitations, and particular care is taken with adjusting the clamp controls

(such as clamp gain, negative capacitance, phase, filtering and switching fre-

quency), SEVC can provide important information on ionic mechanisms in small

cells and/or tissues that are not amenable to other forms of voltage-clamping. Thus,

SEVC should be seen as a complementary voltage-clamp technique that adds to an

electrophysiologist’s armamentarium.

10.3 Alternative techniques10.3.1 Gigaseal electrodes

This technique utilizes glass electrodes with tips of much larger diameter and

having a steep taper. AGigaohm seal forms between the electrode tip and the plasma

membrane, in contrast with the insertion of the tip of a sharp microelectrode through

the plasma membrane into the cell (see Primer 11).

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Figure 10.2 Current-voltage (I/V) relation typical of voltage gated calcium current. The dashedline indicates the part of the current voltage relation that is referred to in the text as a ‘region ofnegative slope’. This simply means that, as the voltage becomes more positive, the conductancebecomes increasing negative (i.e. the slope of the relation of current as a function of voltage isnegative). Courtesy of Dr. Helena Parkington.

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Advantages and disadvantages of Gigaseal over sharp microelectrodes The

larger tip size of the Gigaseal electrode permits greater current flow and, hence,

greater control in voltage-clamp experiments. However, in whole-cell mode, cyto-

plasmic constituents can be lost into the verymuch larger volume of the pipette unless

steps are taken (see Primer 11). To facilitate Gigaseal formation, the plasma

membrane of the cell may need to be cleaned, necessitating isolation of the cell

from its tissue. This places constraints on the questions that can be addressed, but cells

within tissues may be studied using Gigaseal electrodes in some tissues (e.g. brain

slices) to considerable effect.

10.3.2 Potential sensitive dyes

The loading of cells with potential-sensitive dyes can permit recording of membrane

potential changes in vivo and in large contracting tissues, such as heart (Efimov

et al., 2010).

Advantages and disadvantages of optical methods over sharp microelec-trodes Whereas optical methods can provide information regarding the spread

of membrane potential change within complex tissues, problems with interpreta-

tion require considerable skill and experience in the experimenters (Efimov

et al., 2010).

10.4 Comparison between sharp microelectrodeversus patch electrode recordings

In a detailed and elegant study, Li et al. (2004) made a direct comparison between

sharp microelectrode and patch electrode recordings (in current clamp mode) in

spinal neurons (�20mm in diameter) in frog tadpoles.

The results showed essentially few differences between the two techniques.

However, two differences were apparent:

� First, the action potentials recorded with sharp microelectrodes were attenu-

ated, compared with action potentials recorded via the patch electrode (see our

recordings in Figure 10.3). This was most likely due to the greater capacitance

of the sharp intracellular microelectrodes (see above).

� Second, there appeared to be some evidence of 1damage (i.e. injury) upon

microelectrode penetration, which largely, but not totally, recovered over

the following minutes. It is possible that, with recording for longer than the

3–15 minutes in this study, the recovery may have been more complete.

1 Damage tends to result in depolarization of the membrane potential.

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Additionally in this study, the microelectrode was buzzed in, and it is likely that

gentle tapping to make the impalement would have produced less damage. The tip

potentials in this study,�14mV, are relatively large, perhaps due to the nature of the

preparation. In many preparations, removal of the microelectrode from the cell

under study results in the potential returning to within a few mVof zero potential.

10.5 Issues in the literatureFrom the foregoing discussion, several issues require careful scrutiny when absorb-

ing information on intracellular recordings from the literature.

1. Breaking into the cytoplasm, whether with a sharp microelectrode or a patch

electrode, inflicts some damage to the cell, and recovery requires a longer time

when using sharp microelectrodes. Thus, data from impalements at less than

about 15 minutes must be viewed with some caution.

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Figure 10.3 Recordings made in separate experiments from NPY neurons in the arcuate nucleusin slices of mouse brain. Both cells had input resistances of�1.4 GV, and input (membrane) timeconstants of � 50ms. Fluctuations in baseline are due to a high frequency of spontaneoussynaptic activity that occasionally gave rise to action potentials. Recordings made with anintracellular microelectrode (left panels) had smaller action potentials due to electrode capaci-tance than those recorded with a patch electrode (right panels). Measures to reduce micro-electrode capacitance were not vigorously pursued, since the amplitude of the action potentialwas not a critical parameter in the experiment. Action potentials marked by � in the upper panelsare shown at an expanded scale in the lower panels. Activity recorded by the microelectroderemained reasonably constant for four hours, at which time the microelectrode was deliberatelyremoved from the cell and the potential went to�6mV. The membrane potential was corrected forthis tip potential. The recording with the patch electrode was for about half an hour, by whichtime there was evidence of some run-down in activity. Courtesy of Dr. Helena Parkington.

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2. Values of resting membrane potential in absolute terms may need to be

interpreted with caution when large tip potentials are apparent. It is good

practice to find that studies use the average of the potential differences

recorded upon impalement and withdrawal of the electrode. Certainly, authors

should not ignore the potential recorded after withdrawal, as it may be very far

from zero.

3. The amplitude of rapid changes in membrane potential, such as neuronal

action potentials, may not be faithfully recorded. One of the principal causes of

the attenuation that is observed is electrode capacitance.

4. Noisy traces must be viewed with suspicion. Fluctuations in membrane

potential occur, but these must be on a ‘clean’ trace, with a good signal-to-

noise ratio. The inherent noise in a well-designed and well-maintained

microelectrode setup is not much more than 1mV.

5. Although critical cytoplasmic components are unlikely to disappear up into a

microelectrode, perturbations in Kþ and Cl� levels can occur over time. The

authors should at least discuss this possibility and should use logic or

experimental controls to discount the impact on the interpretation of the data.

6. Sometimes electrode movement can produce artefactual changes in the

membrane potential trace.

10.6 Complementary and/or adjunct techniques� Primer 9: Microelectrode recording.

� Primer 11: Patch-clamp in either voltage- or current-clamp modes.

� Two electrode voltage-clamp for large cells (e.g. oocytes).

Further reading and resourcesAttwell, D. & Cohen, I. (1978). The voltage clamp of multicellular preparations. Progress In

Biophysics and Molecular Biology 31, 201–245.

Brennecke, R. & Lindemann, B. (1974a). Theory of a membrane-voltage clamp with

discontinuous feedback through a pulsed current clamp. Review of Scientific Instruments

45, 184–188.

Brennecke, R. & Lindemann, B. (1974b). Design of a fast voltage clamp for biological

membranes, using discontinuous feedback. Review of Scientific Instruments 45, 656–61.

Brown, K.T. & Flaming, D.G. (1977). New microelectrode techniques for intracellular work

in small cells. Neuroscience 2, 813–827.

Coleman, H.A., Tare, M. & Parkington, H.C. (2001). Kþ currents underlying the action of

endothelium-derived hyperpolarizing factor in guinea-pig, rat and human blood vessels.

Journal of Physiology 531, 359–373.

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Efimov, I.R., Fedorov, V.V., Joung, B. & Lin, S.F. (2010). Mapping cardiac pacemaker

circuits: methodological puzzles of the sinoatrial node optical mapping. Circulation

Research 106, 255–71.

Finkel, A.S. & Redman, S. (1984). Theory and operation of a single microelectrode voltage

clamp. Journal of Neuroscience Methods 11, 101–127.

Li, W.C., Soffe, S.R. & Roberts, A. (2004). A direct comparison of whole cell patch and

sharp electrodes by simultaneous recording from single spinal neurons in frog tadpoles.

Journal of Neurophysiology 92, 380–6.

Ram�on, F., Anderson, N., Joyner, R.W. & Moore, J.W. (1975). Axon voltage-clamp

simulations. A multicellular preparation. Biophysical Journal 15, 55–69.

Richter, D.W., Pierrefiche, O., Lalley, P.M. & Polder, H.R. (1996). Voltage-clamp analysis of

neurons within deep layers of the brain. Journal of Neuroscience Methods 67, 121–131.

Schanne, O.F., Lavallee, M., Laprade, R. & Gagne, S. (1968). Electrical properties of glass

microelectrodes. Proceedings of the IEEE 56, 1072–1082.

FURTHER READING AND RESOURCES 93