HEKA Electrophysiology UpdateRa/Rs, Rm and Cm measurements

Telly GaliatsatosGeneral Manager HEKA instruments Inc.

telly.galiatsatos@heka.com

Overview

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Measuring Cm and Ra parameters during voltage clamp experiments is important to determine the integrity of your recording.

In whole cell voltage clamp:

• Cm values are often used to normalize whole-cell currents• Ra values, when too high, can lead to voltage errors and distorted

currents

EPC 10

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• zero the current signal (pipette offset)

Typical voltage clamp recording

• optimize the electrode (or stray) capacitance compensation on the amplifier (C-fast) following Giga Ohm seal formation

• optimize C-slow compensation - the capacitive transients are completely compensated

• start an acquisition – i.e. IV • monitor R-membrane, R-Series

and Membrane capacitance after each series

Discussion

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• How to measure these parameters with an EPC 10 USB.

• How to measure these parameters with a classical amplifier.

EPC 10

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A background process is running, updating I-mon, V-mon and R-membrane at all times.R-membrane is computed in one of two Ways:

Rmemb

Rmemb

• With test pulse: determined from the current sampled during the baseline and the second half of the Test Pulse.

• In-between sweep / series acquisition: by using the pipette current only.

EPC 10

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Cm and R-Series are measured by selecting Auto C-slow, which performs an automatic compensation of C-slow and R-series.

This procedure does the following:• applies short trains of square-wave

pulses (number and amplitude of these pulses can be specified)

• averages the resulting currents• fits an exponential to deduce the

compensation values required to cancel the current

Further information can be found in: Sigworth FJ, Affolter H, Neher E(1995). Design of the EPC-9, a computer-controlled patch-clamp amplifier.2. Software. J Neuroscience Methods 56, 203-215.

EPC 10

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Testing with the model cell

• Provides three positions for simulating:• “open” pipette with a resistance of 10 MΩ• a pipette attached to the cell membrane after the Giga-Ohm

seal formation ~6 pF capacitance• whole cell patch-clamp configuration

• access resistance ~ 5.1MΩ• Membrane resistance ~ 500 MΩ• Membrane capacitance ~22 pF

EPC 10

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• Switch model cell to 10M position to simulate a 10 MΩ pipette open to the bath solution

• Click on the SETUP protocol• Reset the EPC 10USB• Set recording mode to “Whole Cell”• Change Gain to 5.0 mV/pA• Apply a 5 ms 5 mV test pulse• Perform an auto-zero to cancel any voltage

offset

EPC 10

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ResultRectangular current of ~500 pA (I =U/R = 5 mV/10 MΩ)R-membrane ~ 10 MΩ

EPC 10

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• Switch model cell to middle position to simulate a pipette attached to the cell membrane after the Giga-seal formation

EPC 10

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• Click on the SEAL protocol• Set recording mode to “Whole Cell”• Change Gain to 20 mV/pA• Perform an auto C-fast to cancel any “fast

capacitance” transients

EPC 10

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ResultFast transients are neutralized

EPC 10

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• Switch model cell to 0.5G position to simulate a “model cell”

EPC 10

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• Click on the WHOLE-CELL protocol• Set recording mode to “Whole Cell”• Change Gain to 10 mV/pA• Set initial C-Slow value to 50 pF• Set initial R-Series value to 20 MΩ• Perform an auto C-Slow to cancel

capacitance transients

EPC 10

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ResultCapacitance transients are neutralizedRectangular current ~ 10 pA R-membrane ~ 500 MΩC-slow (Cm) = 21.82 pF R-series = 5.1 MΩ

EPC 10

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Classic amplifier

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• perform an auto-zero• optimize the electrode (or stray) capacitance compensation on

the amplifier (C-fast) following Giga Ohm seal formation • optimize C-slow compensation - when the capacitive transients

are completely compensated

Note: optimizing the capacitance compensation is extremely important for the accuracy of the Cm measurement

Typical recording with membrane test

Classic amplifier

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• disable C-slow and series resistance compensation• set the lowpass filter to 3 kHz (medium bandwidth)• execute membrane test protocol to measure Rm, Cm and RS

Additional steps for using membrane test protocol

Membrane Test

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Q1 = Integral (above I1)∆V = V1 – V2∆I = ∆V / (Ra + Rm)Q2 = ∆I * tauQt = Q1 + Q2Rt = ∆V / ∆IRa = tau * ∆V / QtRm = Rt - RaCm = Qt * Rt / (∆V * Rm)

V1

V2

Required formulas

PGF

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Three segment pulse

log(|I_mon|) - to confirm that the decay is single exponentail

• 5 μs sampling (200 kHz)• Pulse segment #2 uses

parameters P1 for duration and P2 for amplitude to be able to change the pulse width / amplitude automatically

• Record voltage, current response and virtual trace

Analysis

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Analysis

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V1 - amplitude of pulsed segment

Standard functions

Analysis

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V2 - amplitude of segment before pulse

Standard functions

Analysis

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∆t - duration of pulsed segment

Standard functions

Analysis

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I1 - mean (80-100%) of pulsed segment

Standard functions

Analysis

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I2 - mean (50-100%) of segment before pulse

Standard functions

Analysis

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Max_t - time of peak current

Standard functions

Analysis

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Tau - cursors 1%-15% after time of peak

Standard functions

Analysis

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Q1 - Integral of segment

Standard functions

Analysis

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∆V = V1 - V2

Equations

Analysis

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Equations

∆I = I1 - I2

Analysis

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Equations

Qt = Q1 - (∆I* ∆t) + (∆I * tau)

Analysis

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Equations

Rt = ∆V / ∆ I

Analysis

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Equations

Ra = tau * ∆V / Qt

Analysis

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Equations

Rm = Rt – Ra

Analysis

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Equations

Rm = Rt – Ra

Analysis

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Equations

Cm = Qt * Rt / (∆V * Rm)

Analysis

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For estimation of optimal pulse length:

DownLevel (I_Max-I1)*0.2+I1: calculate the level at which current has decayed to 20%.

Equations

Analysis

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Equations

t_Thresh: time of crossing the Down Level, result is stored in value-2.

Results

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Black: current traceRed: voltage traceBlue: log(|current trace|)Cursor range 1%-15% of pulse length, starting at I_max