Single Electrode Voltage Clamping

Transcription

1 Plymouth Microelectrode Techniques Workshop Single Electrode Voltage Clamping Alasdair Gibb Research Department of Neuroscience, Physiology & Pharmacology, University College London ciona intestinalis (sea squirt) eggs Latin name means, literally, "pillar of intestines",

2 Why is voltage-clamping useful? The membrane current, I m is given by: R m C m I m V R m m C m dv dt Ionic current Capacitative current Cell

3 Voltage recording + - A1 V m VE CELL BATH Ref The membrane voltage (V m ) is recorded by a unity-gain voltage follower (A1), which receives inputs from the voltage-recording electrode (VE) and the bath Ag/AgCl reference electrode (Ref). A1 records differentially between these two points to give output V m.

4 Squid electrophysiology at the Marine Biological Association Laboratory, Plymouth, England. Common squid, Loligo

5 Single electrode voltage clamp Key references: Brennecke & Lindemann (1974). Theory of a membrane-voltage clamp with discontinuous feedback through a pulsed current clamp. Rev. Sci. Instrum. 45: Wilson & Goldner (1975). Voltage clamping with a single microelectrode. J. Neurobiology. 6: ** Finkel & Redman (1984). Theory and operation of a single microelectrode voltage clamp. J. Neurosci. Meth. 11: Axoclamp-2B manual available online at Alan Finkel (2007), Scholarpedia, 2(8):3528.

6 Axoclamp 2B Amplifier Headstage Dr Alan Finkel, PhD Electrical Engineering (1981), Chancellor of Monash University (2008)

7 Single electrode voltage clamping in principle Use electronic feedback to inject current into cell via the electrode + - A1 Compare V m with command potential, V cmd V m V cmd VE Ref CELL BATH

8 Just one big problem Current passing down the microelectrode generates an error voltage due to the electrode resistance, according to Ohms Law: V error = IR e current, I + - A1 V m R e e.g. I = 10 na, R e = 10 M V error = 10-8 A x 10 7 = 10-1 V CELL = 100 mv

9 Solution. Electronically switch between periods of current injection, and voltage recording.. A switched single electrode voltage clamp (SEVC)

10 SEVC modes of operation (i) Bridge recording mode record membrane potential (ii) Discontinuous Single Electrode Current Clamp - dsecc (iii) Discontinuous Single Electrode Voltage Clamp - dsevc Bridge dsecc dsevc

11 Bridge mode V 1 +V C Current command, V C } Bridge balance potentiometer, R bridge Current step, I o R E voltage drop = R E I o R E when R bridge = R E Bridge and electrode voltage cancel R o voltage drop = (V 1 +V C ) V 1 = V C Axoclamp 2B manual. Axon Instruments Inc. Current through R o = V C /R o = I o

12 Bridge balance Capacitance compensation Voltage, V 1 A. Bridge not balanced Electrode capacitance transients B. Bridge balanced Axoclamp 2B manual. Axon Instruments Inc.

13 Discontinuous Single Electrode Current Clamp - dsecc R e C e t e = R e C e Axoclamp 2B manual. Axon Instruments Inc.

14 Discontinuous Single Electrode Voltage Clamp - dsevc Switching frequency fs = 1/T Axoclamp 2B manual. Axon Instruments Inc.

15 Axoclamp 900A Software Control

16 NPI Electronic

17 SEVC - Advantages Requires only a single electrode some cells are easily damaged with two electrodes Penetration can be done blind : useful in brain slices or in vivo CNS Series resistance errors are avoided Can clamp large cells (100pF) and large currents (~10nA)

18 SEVC - Disadvantages Clamp has more noise, slower response and lower fidelity than TEVC or patch Clamp has low gain/instability with small cells -: better to use patch clamp Practical aspects of circuit design and construction, electrodes and recording setup are all more difficult than for TEVC or for patch clamp

19 Comparing patch, SEVC and TEVC Patch SEVC TEVC C m 1pF 20pF 50pF 100pF 200pF 200nF I m 1pA 100pA 1.0nA 1.0nA 10nA 10mA

20 Practical considerations 1. Electrodes: aim for lowest resistance and capacitance compatible with stable recordings R e C e Electrode time constant limits switching frequency, which limits maximum gain with clamp t e = R e C e

21 Practical considerations Capacitance compensation 1) Underutilized 2) Optimum 3) Overutilized t e = R e C e Bridge mode R e C e e.g. C e = 1.0 pf, R e = 10 M t e = F x 10 7 = 10-5 s = 10 ms dsecc

22 Capacitance compensation G C in + - A1 V m VE CELL BATH Ref The signal from the headstage voltage follower (A1), is amplified (G) and fed back to the amplifier input, rapidly charging the input capacitance, C in, so that charging of C in no longer slows the signal from the cell.

23 Practical considerations Clamp phase shift In a parallel RC circuit, at high frequencies C m will introduce a 90 o phase shift between membrane current and membrane voltage. Real cell membranes often show less than 90 o phase shift so phase lead (achieved by boosting the high frequency gain of the clamp amplifier) could be used to compensate however tends to cause instability. Phase lag is generally used effect is to decrease the high frequency gain of the clamp, so improves stability. In some situations, phase lag may reduce clamp noise, but may also tend to cause ringing.

24 Correctly set up SEVC I m R m = 100 M, C m = 33 pf R e = 100 M f s = 7 khz, G T = 1nA/mV V m True V m Headstage voltage

25 Incorrectly set up SEVC ( false clamp ) I m V m True V m Headstage voltage Capacitance neutralisation under-utilised Phase shift: maximum lag, t = 2 ms

26 SEVC using a suction (patch) electrode R m = 300 M, C m = 33 pf R e = 3 M f s = 50 khz, G T = 0.7nA/mV Phase lag, t = 0.2 ms

27 SEVC of action potential currents KCl pipette CsCl pipette R m = 500 M, C m = 13 pf R e = 3 M Sah, Gibb & Gage (1987) J.Gen.Physiol. f s = 40 khz, G T = 5nA/mV

28 Loss of voltage control Breakthrough currents on voltage steps Sah, Gibb & Gage (1987) J.Gen.Physiol.

29 SEVC of hippocampal neurone sodium current Sah, Gibb & Gage (1987) J.Gen.Physiol.

30 SEVC experiment Preparation: ciona intestinalis (sea squirt) eggs Aims: 1. Basic sharp electrode intracellular recording 2. Bridge mode and SECC capacitance compensation 3. SEVC clamp gain and phase shift

31 Two-microelectrode Voltage Clamp I A2 + - e V cmd R 0 - A3 + V I + - A1 V m IE CELL VE I m BATH RE The membrane voltage (V m ) is recorded by a unity-gain buffer preamplifier (A1), which receives inputs from the voltage-recording electrode (VE) and the bath Ag/AgCl pellet and agar bridge reference electrode (RE). The preamplifier records differentially between these two points to give output V m. This output is compared to the command potential (V cmd ) in a high-gain differential clamping amplifier (A2). The output of the clamping amplifier is proportional to the difference (e) between V m and V cmd and the voltage at its output forces current to flow through the current-passing microelectrode (IE) into the cell. The polarity of the gain in the clamping amplifier is such that the current in IE reduces e. A third differential amplifier (A3) measures the voltage drop across the current setting resistor, R o. This voltage drop (V I ) is proportional to the total current (I) flowing through R o and so by Ohms Law, V I, is a good measure of the membrane current (I m ).

32 Patch-clamp recording R f (500 M or 50 G ) Patch pipette - + A1 - A2 + I m V cmd CELL BATH RE The membrane current (I m ) is measured by a currentvoltage converter operational amplifier (A1) and a differential amplifier (A2). The inputs to A1 are from the patch pipette and the bath Ag/AgCl reference electrode (RE). The current-voltage converter configuration means that the two inputs to A1 are forced to be equal by passing current through R f. The current is measured as the difference between the output and the + input of A1 by the amplifier A2 to give the current which is proportional to the voltage drop across R f.

33 R1 OPERATIONAL AMPLIFIERS NON INVERTING AND INVERTING SUMMING CONFIGERATIONS R5 Operational amplifier U2 V in - R3 U1 V a + R4 R2 V b RV1 +15V -15V D. C. Offset C1 - U2 + R6 V out Inverting summing R5 R5 Gain R3 R4 R5 R5 Vout Va Vb R3 R4 Select values of R5 and R3 for a gain of 5 and a value of R4 to give an offset capability of +/- 150 mv approx. at the input of U1. Suggested value for R5 is 100K R6 = R5 in parallel with R3. Operational amplifier U1 Non inverting R1 Gain 1 R 2 Va Vin 1 R1 R 2 Select values of R1 and R2 for a gain of 2 R5 and C1 make up a time constant in the feedback and gives a frequency response where : 1 f 2 RC C in farads and R in ohms. Select various values for C1. Note 1 Xc 2 fc R. Xc Z R Xc Circuits from Chris Courtice (School of Pharmacy)