Current Clamp Recordings

with the EPC 10 Patch Clamp Amplifier

Dr. Christian Heinemann

Agenda

Part A: Current clamp circuitry

Part B: Sensing membrane potential: Parameters affecting the

shape of an action potential

Part C: Stimulation the cell by current injection: How much current

can be injected?
CC Circuitry of the EPC 10 amplifier

Voltage follower circuitry, direct

and fast measurement of the 1 pF C-fast comp (V-Ref)
membrane potential.

Current injection path via the

feedback resistor of the patch I-command (V-Ref)
clamp headstage. 500 MOhm

Current injection path via the - I-mon

injection capacitor.
+
- V-mon

4,7 pF +

V-Ref

Part A 1.2
Comparison of EPC 9 and EPC 10

The EPC 10 shows no deviations
from the command current pulse.
Good clamp performance.

The EPC 10 with its Voltage
Follower circuitry measures the
true action potential waveform.

The EPC 9 uses a slower current
feedback loop. Clamp
performance not perfect.

The EPC 9 gives only a
qualitative measurement of the
AP waveform.

Action potential recordings from DRG neurons. All recordings from cells within this
presentation are courtesy of the Center for Molecular Biomedicine (CMB), Department of
Biophysics, Friedrich-Schiller-University Jena.

Part A 1.3
Equivalent Circuit

Cm C-fast
Rs

Rm
Pipette
Cell

Part B 2.1
Stimulating in Voltage Clamp
i Rs =i Cm +i Rm
Cm = 22pF

Current charges the cell membrane

V com
capacitance, current transient. Rs = 5.1MOhm

During charging the membrane

potential time course is dominated by
the time constant: Rm = 500MOhm

tau Vm=R SC m=112s

The potential at the membrane drives Current Recording with C-Slow compensation active.
the current through the Rm. That is,
why the current we typically measure
also increases with exponential time
V com
course. i Rm

20mV voltage stimulus drive about

40pA through Rm.

Part B 2.1
Stimulating in Current Clamp
i Rs =i Cm +i Rm Cm = 22pF

Initially, the constant current mainly Rs = 5.1MOhm I com

charges the membrane capacitance.

With increasing potential the portion of

the current that flows through Rm Rm = 500MOhm
becomes dominating.

This equilibration happens with the time

constant given by:
tau=RmC m =11ms 40pA current injection result in a Vm of about 20mV.

The increase in Vm is typically much

slower in CC than VC recordings.

Part B 2.2
Sensing an action potential fired by a cell

If the cell fires an action potential, then

the equivalent circuit is reduced to a
combination of access resistance and
pipette capacitance.

The filtering element is given by:

measure
C-fast
tau=RSC fast =50s
1
BW eff = =3.18kHz 5,0
2pitau pF

Rs
Vm

Part B 2.3
CC Bandwidth: limited by C-fast?
stimulate 5.0pF measure

The time constant Rs * C-fast limits the 10MOhm

effective bandwidth of the voltage
recording in a current clamp
measurement.

C-fast compensation reduces the

contributing capacitance and therefore tau
increases the effective bandwidth.

rem. C- theor. Tau meas. Tau eff. BW

fast
5pF 50s 57s 2.8kHz

4pF 40s 47s 3.4kHz

3pF 30s 37s 4.3kHz

2pF 20s 27s 5.9kHz

Part B 2.4
AP shape Affected by C-fast Comp?

Rs = 3.3MOhm and rem. C-fast = 2pF the
eff. BW > 20kHz

Part B 2.5
Checking C-fast adjustment in CC mode

Power Spectrum

Voltage recording in CC mode with
1E-12
100kHz sampling frequency and V-Bessel [V^2/Hz]
5.78 pF
filter set to 15kHz

1E-13

pF
78
4.
1E-14

1E-15

1,000 10,000 100,000Hz

Part B 2.8
CC Bandwidth: limited by Voltage Filter?
stimulate 5.0pF measure

The maximum slope is effected

10MOhm
significantly by the voltage filter. But in a
speed range much faster than the rising
phase of an AP.

V-Bessel max. % slope

slope reduction max. slope
15kHz 2.156V/s

10kHz 1.781V/s 17%

7kHz 1.531V/s 29%

5kHz 1.187V/s 45%

Part B 2.6
AP shape Affected by Voltage Filter?
A D

The maximum slope of the AP is not
significantly affected at V-Filter settings
above 1 kHz.

Part B 2.7
Stimulation Artifact: Bridge Compensation Cm = 47.0 pF
stimulate - I

The initial fast voltage step over Rs can be Rs = 100 MOhm

compensated using the Bridge
Compensation. measure - V

100% of the Rs value can be

Rm = 500 MOhm
compensated. Adjust Rs or % value for
fine tuning.

Part C 3.1
Stimulation Artifact: Bridge Compensation Cm = 47.0 pF
stimulate - I

A fast enough time constant of Bridge Rs = 100 MOhm

Compensation eliminates the voltage step
completely. measure - V

Rm = 500 MOhm

100s

10s

Part C 3.3
AP Recordings with high access Resistance

estimated Rs about 70-80 MOhm, 80 pA

current injection induce about 6 mV
voltage drop.

Recordings with Bridge Comp On and Off.

Note change in AP amplitude. Slope is not
affected.

Part C 3.2
How much current can be injected into a cell?
max. compliance voltage = appr. 12 V

Voltage Measurement Limit:

The maximum voltage that can
Cm = 22.0 pF
drive the current through the
input resistance is 1V in the Rs = 5.1 MOhm Rf = 500 MOhm
standard EPC 10 operating mode
and 5V with Extended Stimulus
Range turned on.
Rm = 500 MOhm

Compliance Limit: The overall

voltage that can drive current
through the circuitry is appr. 12V. input resist. = Rm + Rs
voltage measuring limit

Part C 3.4
Example 1: 10nA injection via 500M
max. compliance voltage = appr. 12 V
CC-Gain I max

0.1 pA/mV 1 nA Cm = 22.0 pF

Rf = 500 M
Rs = 5.1 MOhm Rf = 500 MOhm
1.0 pA/mV 10 nA
Rf = 500 M i i

10 pA/mV 100 nA i
Rf = 5.1 M
Rm = 500 MOhm
100 pA/mV 1 A
Rf = 5.1 M
input resist. = Rm + Rs
voltage measuring limit

U Rf =i Rf =10nA500MOhm=5V
1V extended Stimulus Range:
Rinput = =100MOhm U Rf =i Rf =10nA500MOhm=5V
10nA
1V 5V
i limit = =2nA Rinput = =500MOhm
500MOhm 10nA
U compl =1V+5V=6V U compl =5V+5V=10V

Part C 3.5
Example 2: 1A injection via 5.1M
max. compliance voltage = appr. 12 V
CC-Gain I max

0.1 pA/mV 1 nA Cm = 22.0 pF

Rf = 500 M
Rs = 5.1 MOhm Rf = 5.1 MOhm
1.0 pA/mV 10 nA
Rf = 500 M i i

10 pA/mV 100 nA i
Rf = 5.1 M
Rm = 500 MOhm
100 pA/mV 1 A
Rf = 5.1 M
input resist. = Rm + Rs
voltage measuring limit

U Rf =iR f =1A5.1MOhm=5.1V extended Stimulus Range:

1V U Rf =iR f =1A5.1MOhm=5.1V
R input = =1MOhm 5V
1A R input = =5MOhm
1V 1A
i limit = =2nA 5V
500MOhm i limit = =10nA
U compl =1V+5.1V=6.1V 500MOhm
U compl =5V+5.1V=10.1V

Part C 3.6
Selection of the CC Gain Range
CC-Gain I max

0.1 pA/mV 1 nA
Rf = 500 M
high input
1.0 pA/mV 10 nA resistance
Rf = 500 M

10 pA/mV 100 nA
Rf = 5.1 M
low input
100 pA/mV 1 A resistance
Rf = 5.1 M

Part C 3.6
Attention: High CC Gain might cause Voltage
offsets!
CC-Gain I max Current stimulus is generated by 16bit DAC with a typical
resolution of about 0.3mV/bit.
0.1 pA/mV 1 nA
Rf = 500 M
What happens if we are just off by one bit?

1.0 pA/mV 10 nA
Rf = 500 M
100pA
10 pA/mV 100 nA 0.3mV =30pA
Rf = 5.1 M mV
100 pA/mV 1 A U offset =500MOhm30pA=15mV , model cell WholeCell Mode
Rf = 5.1 M
U offset =10MOhm30pA=0.3mV , model cell PipetteBath Mode

Note:
High CC-gains can not be used or tested with high input resistances, e.g.
model cell in Whole Cell Mode!

Part C 3.7
 Ext. Stimulus Range: 5 x stronger injection

5x more current at given input
resistance

current injection possible at 5x higher
input resistance

CC-Gain I max (R input), I max (R input),

standard range extended range
0.1 pA/mV 1 nA (1 G) 1 nA (1 G)
Rf = 500 M
1.0 pA/mV 10 nA (0.1 G) 10 nA (0.5 G)
Rf = 500 M
10 pA/mV 100 nA (10 M) 100 nA (50 M)
Rf = 5.1 M
100 pA/mV 1 A (1 M) 1 A (5 M)
Rf = 5.1 M

Part C 3.8
Summary - CC related EPC 10 Features

Voltage Follower Circuitry: True

Current Clamp

small Rs makes C-fast

C-fast Compensation: increase setting less critical

bandwidth of CC recording

filtering with 5kHz or more

Voltage Filter: analog filter for the

typically does not affect
voltage signal
the AP shape

4 different CC Stim Gains: for

amount of injected current
injection of large currents depends on input
resistance

Bridge Compensation: remove

with Rs lower than
current injection artifact 10MOhm artifact typically
less than 1mV

5x more current at given
input resistance

Extended Stimulus Range: 5x

stronger current injection
given current at 5x higher
input resistance
Thank you!

Questions ?