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Using the Patch Clamp

be essential if you will be giving voltage pulses in your experiment. It is a good idea to start out with

V-membrane

set to zero, as we specified above; an alternative is to start with

V-membrane

set to the holding potential you

desire (e.g., -70 mV). If no voltage jumps are required, turn the stimulus off to avoid introducing artifacts. If
voltage jumps are to be applied, switch the

Gain

and

Filters

to the values you will be using (the settings may be

programmed under

SEAL

). Be sure to use

Gain

settings of 50 mV/pA or above for lower noise in single-channel

recordings. Keep the

Filter 1

switch set at 10 kHz unless you actually will need the 100 kHz full bandwidth for some

reason; otherwise you might drive the current monitor output or your recorder’s input amplifiers into saturation
with the very large amount of high-frequency noise. Should you use the full bandwidth, you should avoid

Gain

settings above 100 mV/pA for the same reason. If you are applying voltage pulses to the patch membrane, you
probably will want to subtract control traces from the traces containing the channels of interest in order to remove
the capacitive transients. Nevertheless, it is important to try to cancel the capacitive transients as well as you can
in order to avoid saturating any amplifiers, the recording medium or the AD converter. It is a good idea to set
the

C-fast

and

-fast

controls while you observe the signal without any filtering beyond the internal 10 kHz filter.

Then, during the recording, watch to see if the clipping light blinks. When it does, it means that internal amplifiers
in the

EPC 10 USB

are about to saturate, and/or that the current monitor output voltage is going above 10 V

peak, on the peaks of the transients, and you should readjust the transient cancellation controls. Otherwise, it is
likely that the recording will be non-linear and subtraction will not work correctly. The fast transient cancellation
is not sufficient to cancel all of the capacitive transients in a patch recording. This is partly because the pipette
capacitance is distributed along the length of the pipette; therefore, each element of capacitance has a different
amount of resistance in series with it, so that a single value of

-fast

will not provide perfect cancellation. The time

course of the transients also reflects dielectric relaxation in the plastic of the pipette holder and in the pipette glass.
These relaxations are not simple exponentials, but occur on time scales of about 1 ms. If you use pipette glass
with low dielectric loss (e.g., aluminosilicate glass) or if you are careful to coat the pipette with a thick coating and
near to the tip, the relaxations will be smaller. You can cancel part of these slow relaxations by using the

C-slow

controls, with the

C-slow Range

set to 3 pF.

Note:

For cell-attached or inside out patch configuration, positive pipette voltages correspond to a

hyperpolarization of the patch membrane, and inward membrane currents appear as positive signals
at the current monitor outputs. The

Patchmaster

program compensates for this by inverting digital

stimulus and sampled values in these recording configurations such that the stimulation protocols, holding
voltages, and displays of current records in the

Oscilloscope

all follow the standard electrophysiological

convention. In this convention, outward currents are positive and positive voltages are depolarized.
However, the analog current and voltage monitor outputs are not inverted in these recording modes.

10.3

Whole-Cell Recording

10.3.1

Breaking the Patch

After a gigaseal is formed, the patch membrane can be broken by additional suction or, in some cells, by high
voltage pulses (600-800 mV, see

Zap

function). Electrical access to the cell’s interior is indicated by a sudden

increase in the capacitive transients from the

Test Pulse

and, depending on the cell’s input resistance, a shift in

the current level. Additional suction sometimes lowers the access resistance, causing the capacitive transients to
become larger in amplitude but shorter. Low values of the access (series) resistance are desirable and, when Rs-
compensation is in use, it is important that the resistance be stable as well. A high level of Ca

buffering capacity

in the pipette solution (e.g., with 10 mM EGTA) helps prevent spontaneous increases in the access resistance due
to partial resealing of the patch membrane.

10.3.2

Capacitive Transient Cancellation

If the fast capacitance cancellation was adjusted (as described above) before breaking the patch, then all of the
additional capacitance transient will be due to the cell capacitance. Canceling this transient using the

C-slow

and

R-series

controls will then give estimates of the membrane capacitance and the series resistance. The easiest way

of cancellation is provided by the

Auto C-slow

function which may be activated by selecting the

Auto

button or

the

Whole Cell

button (if included in the protocol function). In cases where the