Axon Instruments Axoclamp 2A User Manual

DR. HARVEV UNIVERSITY DEPARTMENT 9500 GIU^AN DRIVE

KARTEN,

CALIFORNIA,

NEUROSCIENCES, 0608

M.D.

SAN

DIEGO

LA JOLLA, CA 92093-0608 February 1990

AXOCLAMP-2A MICROELECTRODE CLAMP

THEORY AND OPERATION

Written for Axon Instruments, Inc. by Alan Finkel, Ph.D.

Copyright 1988, 1990 Axon Instruments, Inc. No part of this manual may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from Axon Instruments, Inc.

QUESTIONS? Call (415) 571-9400

Part Number 2500-000 REV B PriMcd in

U.S.A.

OUT i(r..;.,) • • St^f 4<i-.^^

Ill

THE CIRCUITS AND INFORMATION IN THIS MANUAL ARE COPYRIGHTED AND MUST NOT BE REPRODUCED IN ANY FORM WHATSOEVER WITHOUT WRITTEN PERMISSION FROM AXON INSTRUMENTS, INC.

VERIFICATION

THIS INSTRUMENT IS EXTENSIVELY TESTED AND THOROUGHLY CALIBRATED BEFORE LEAVING THE FACTORY. NEVERTHELESS, RESEARCHERS SHOULD INDEPENDENTLY VERIFY THE BASIC ACCURACY OF THE CONTROLS USING RESISTOR/CAPACITOR MODELS OF THEIR ELECTRODES AND CELL MEMBRANES.

DISCLAIMER

THIS EQUIPMENT IS NOT INTENDED TO BE USED AND SHOULD NOT BE USED IN HUMAN EXPERIMENTATION OR APPLIED TO HUMANS IN ANY WAY.

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Illustrations of the rear-panel view of the AX0CLAMP-2A are shown on the fold-out page at the rear of the manual.

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

9500 GILMAN DR^VE '"'''^''' °^°^

LA JOLLA, CA 92093-0608

TABLE OF CONTENTS

Page

INTRODUCTION 1

FEATURES 3

FEATURES ..3

GLOSSARY 9

QUICK GUIDE TO OPERATIONS 11

DETAILED GUIDE TO OPERATIONS 15

ANTI-ALIAS FILTER 15

BATH PROBE i, 16

Bath Potential Measurement 16 Grounding 16

BLANKING 16

BRIDGE MODE 17

Description 17 Suggested Use 17 Intracellular Balancing 18

BUZZ 20

Remote Buzz 20

CALIBRATION SIGNAL 21

CAPACITANCE NEUTRALIZATION AND INPUT CAPACITANCE 21

Primary 21 Secondary 21

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Page

CLEAR 22

COMMAND GENERATORS 22

Step Conunand Generator 22 DC Command Generators 23 Extemal Conunand Inputs 23 Mixing Conunands. 23

CURRENT MEASUREMENT 25

DCC MODE 25

GROUNDING AND HUM 31

HEADSTAGES 32

The Meaning Of H 32 Which Headstage To Use .32 Capacitance Neutralization Range 34

7 Headstage Connectors 34

Tip Potentials - Detection 36 Tip Potentials - Prevention 37 Interchangeability 37

• Cleaning 37 Input Leakage Current And How To Trim It To Zero 37 Warning 38 DC Removal 38 Input Resistance ; 39

HOLDERS : .39

Features 39 Parts 40 Use 40

IONOPHORESIS

LINK-UP

AXOCLAMP-2A THEORY & OPERATION. COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Vll

Page

MICROELECTRODES FOR FAST SETTLING 43

Microelectrode Capacitance 43 Microelectrode Resistance 44

Filling Solutions.... 44

Recommended Reading 44

MODEL CELLS 45

The CLAMP-1 Model CeU 46

MONITOR

NOISE IN DCC AND dSEVC MODES 49

OFFSET CONTROLS 50

OUTPUT

FILTER 50

High-Order Lowpass Filters For Low-Noise Recordings 51 Rise Time Of High-Order Filters 51

Note On Ultimate Rise Time ;.... 51

OUTPUT IMPEDANCE AND PROTECTION 51

PANEL METERS 51

V„(mV) 51 V2(mV) 52 I(nA) 52

PHASE • 52

POWER-SUPPLY GLITCHES 53

POWER SUPPLY VOLTAGE SELECTION & FUSE CHANGING 54

Supply Voltage 54

Changing The Fuse 54

REMOTE 55

RMP BALANCE 57

AXOCLAMP-2A THEORY & OPERATION,

FEBRUARY

1990,

AXON

INSTRUMENTS.

INC.

Vlll

Page

SERIES RESISTANCE 57

Origin 57 Problem 57 Solutions 57

What is Uie True Membrane Potential Time Course? 58

SEVC MODE - CONTINUOUS.... 60

Important Note - Anti-Alias Filter 60 Suggested Use 60 cSEVC Compared WiUi Whole-Cell Patch Clamp 62

SEVC MODE - DISCONTINUOUS .....64

Description 64 Suggested Use 67 Important Note 70 Which SEVC to use witii a Suction Electrode 70 Minimum Sampling Rate and Maximum Gain 74 Clamp Error 74

Gain.... 74

SPACE CLAMP 75

TEN-TURN POTENTIOMETERS 75

TEVC MODE ; 75

Description 75 Suggested Use 76 Extremely Important Note - Coupling Capacitance 76 Saturation During The Capacitance Transient 79 Choosing the Microelectrode Resistances 79

TRIGGERED CLAMPING 79

TROUBLE SHOOTING 80

UNITY-GAIN RECORDING - THIRD POINT 80

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Page

VIRTUAL-GROUND CURRENT MEASUREMENT

10.V„

AND I^

SPECIFICATIONS

REFERENCES

WARRANTY

RMA FORM

B-1

C-1

POLICY STATEMENT

SERVICE

D-l

COMMENT FORM

OUTPUTS

A-1

D-l

E-1

FRONT AND REAR PANEL -

f^ir^yf'

iZ^^f ^Iffff^^P

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY

1990,

AXON INSTRUMENTS,

INC.

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AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

iNTRODUcrroN Page 1

INTRODUCTION

The AXOCLAMP-2A Microelectrode Clamp can be used as a dual channel microelectrode probe, or as a microelectrode voltage clamp.

Voltage clar; ping is a powerful technique for the control of membrane potential and for the investigation of processes alfecting membrane conductance. Voltage clamping has traditionally been performed using two intracellular microelectrodes and the AXOCLAMP-2A can be used for this purpose.

The AXOCLAMP-2A can also be used for discontinuous single-electrode voltage clamping (dSEVC) and for continuous single-electrode voltage clamping (cSEVC). A single-electrode voltage clamp (SEVC) is more convenient to use than a two-electrode voltage clamp (TEVC) in very small cells and cells which cannot be visualized. A particular advantage of a dSEVC is that the voltage drop due to current flow through the series component of cell membrane resistance (Rg) is not clamped. In addition, for both types of SEVC instabilities due to coupling capacitance and coupling resistance between two microelectrodes do not arise.

The disadvantages of a dSEVC compared with a TEVC are that the response speed is slower, the maximum achievable gain is lower, and the noise in the current and voltage records is greater. The design of the AX0CLAMP-2A reduces these disadvantages towards their theoretical minimums, thereby allowing singleelectrode voltage clamping to be performed in the many situations where conventional voltage clamping is not suitable.

A cSEVC i.s as low in noise as a TEVC but has a severe disadvantage in that the voltage drop across the microelectrode is clamped unless compensation is made. Since the required compensation is never perfect, tha rSF,V(;^ y^in nnly he. psfid wh^-ij thg e|ectrode resistance is very small compared with the cell input resistance. These favorable conditions can often be achieved by the whole-cell patch technique.

Because of the AXOCLAMP-2A's advanced design, it itself does not limit the achievable performance. Instead, the dominant factor affecting SEVC performance is the microelectrode. Users of the AXOCLAMP-2A in eidier of the SEVC modes should be quick to question, then adjust, the microelectrode and its placement.

TTie AXOCLAMP-2A is a sophisticated instrument. Even experienced researchers are advised to read this manual thoroughly and to familiarize themselves widi the instrument using model electrodes (i.e. resistors) and cells (e.g. parallel RC) before attempting experiments with real microelectrodes and cells.

We will be pleased to answer any questions regarding the theory and use of the AX0CLAMP-2A. Any conmients and suggestions on the use and design ofthe AX0CLAMP-2A will be much appreciated.

We would be most grateful for reprints of papers describing work performed with the AX0CLAMP-2A. Keeping abreast of research performed helps us to design our instruments to be of maximum usefulness to you who use them.

Axon Instruments, Inc.

AX0CLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Page 2 iNTRODUcnoN

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AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

FEATURES

Page 3

FEATURES

The AXOCLAMP-2A is a complete microelectrode current and voltage clamp for intracdiular investigations. It combines state-of-the-art single-electrode voltage clamping, two-electrode voltage clamping, and two complete bridge amplifiers into one instrument. Precision command voltages, meters, filters, offsets and many other features are built in to give you unprecedented flexibility.

4 discontinuous single-electrode voltage clamping 4 continuous single-electrode voltage clanqiing 4 two-electrode voltages clamping 4 discontinuous current clamping 4 two complete bridge amplifiers 4 high-speed headstages 4 low-noise low-hum operation 4 push-button selection of operating mode 4 computer selection of operating mode 4 two digital meters for voltage display 4 digital counter for display of sample rate 4 3-input digital meter for current display 4 separate current-measurement circuits for

each microelectrode

4 virtual-ground current measurement

VOLTAGE CLAMPING Voltage clamp with one or two microelectrodes — your choice is dictated

by the needs of your investigation; the AXOCLAMP-2A does both. Discontinuous Single-Electrode Voltage Clamping (dSEVC) is based on the technique of sampling the membrane potential while zero current flows and then retaining this sampled value while current is injected into the cell. This procedure is rapidly repeated to produce a smooth response. Continuous Single-Electrode Voltage Clamping uses a low resistance electrode to continuously record membrane potential and inject current. The error caused by voltage drop across the electrode resistance can be partially reduced by series resistance compensation. With Two-Electrode

Voltage Clamping (TEVC) one microelectrode is used to continuously record membrane potential while the other is used to inject current. ~^

4 bath potential measurement and compensation 4 intemally generated precision command voltages 4 automatic clamping at resting membrane potential 4 offset compensation 4 rejection of stimulus artifacts 4 output bandwidth selection 4 calbration signal on outputs 4 electrode buzz 4 electrode clear 4 hands-free operation of buzz and clear

4 anti-alias filter 4 phase control 4 sampling clock synchronization 4 model cell

Gain of the voltage-clamp amplifier is quickly set on a smooth-acting nonlinear control. The phase response of the amplifier is altered from lead to lag by a Phase Shift potentiometer with a Center Frequency switch to select the range.

A unique variable Anti-Alias Filter helps reduce noise towards the theoretical minimum during dSEVC by slowing the response of the

sampling circuit to suit the sample rate and the microelectrode response. The Sample Rate can be continuously altered from a low value of 500 Hz to a high of in noise and response times occurring when faster sampling rates are used.

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

kHz. This enables you to take advantage of the decrease

Page 4

FEATURES

The sample clocks of two AXOCLAMP-2A's can be synchronized in a

'Master-Slave' configuration. This is useful in experiments in which two cells in the same preparations are independently voltage clamped using dSEVC. Linking the two clocks prevents the generation of spurious signals which would otherwise appear at harmonics of the difference in the two clocks firequencies.

Output compliance in TEVC mode is ±30 V. This reduces the chance of saturation while the membrane capacitance is charging after a step change in voltage. To further minimize the chance of saturation during TEVC a relay-switched headstage (HS-4) is available to automatically bypass the current-sensing resistor inside the headstage. The HS-4 headstage must therefore be used in conjunction with a virtual-ground current monitor (VG-2).

The HS-4 headstage is recommended only when large, ultra-fast

voltage steps in big cells must be established.

Another unique control is a Resting Membrane Potential (RMP) Balance

Indicator which enables you to preset the clamp offset so that when you switch into voltage-clamp mode the cell membrane will automatically be clamped at its resting value, irrespective of the clamp gain.

A remarkable "BLANK" facility can be used to force the voltage clamp system to ignore stimulus artifacts that would otherwise be picked up by the voltage-recording circuit and result in large current artifacts which could damage the cell under clamp.

A "Monitor" output enables the input to the sampling circuit to be observed. It is essential to observe this signal during dSEVC to ensure that the microelectrode voltage due to current passing has time to adequately decay at the end of each cycle. An oscilloscope trigger signal at the sample rate is provided for use with the Monitor signal.

The AX0CLAMP-2A allows very fast discontinuous single-electrode voltage clamping. In a test cell (see specifications) the 10% to 90% rise time is only 100 /ts. In a real setup the response speed is limited by the microelectrode characteristics, but membrane potential rise times (without overshoot) of less than 1 ms have been regularly achieved in a variety of cell types. Two-electrode voltage clamping is much faster.

CURRENT CLAMPING Two controls for each microelectrode are devoted to clearing blocked

microelectrode tips and assisting cell penetration. One is a "Clear" switch which can be used to force large hyperpolarizing or depolarizing currents through the microelectrode. The other is a "BUZZ" switch which causes the mocroelectrode voltage to oscillate. Depending on the microelectrode and the preparation, one of these two methods will often succeed in lowering the resistance of blocked microelectrode tips. When used while the tip of the microelectrode is pressing against the membrane, Buzz and Clear may also cause the microelectrode to penetrate the cell.

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

FEATURES

Page 5

HEADSTAGES

Unity-voltage-gain HS-2 headstages are available in several current gains. These cover the range of cell input impedances from less than 1 MO to greater than 1 GO. Ultrahigh-input impedance versions are also available for ion-sensitive electrodes.

High speed and low noise are achieved by using bootstrapped power supplies for the input circuit of each headstage. These bootstrapped power supplies are derived from special high-voltage circuits so that the headstages will not be saturated by the large voltages that may occur during the passage of ciurent through high-resistance microelectrodes. Capacitance Neutralization is also derived from high-voltage circuits so that fast responses are not degraded during large input signals.

Current in each microelectrode is continuously measured during both voltage clamp and current clamp. This measurement does not include currents from sources other than the microelectrode (e.g. hum, ionophoresis, the other microelectrode) and indicates zero if the microelectrode blocks.

Headstages have a gold-plated 2 mm (0.08") input socket to directly accept standard microelectrode holders. 2 mm plugs are supplied with the headstages to connect wire leads, if used.

COMMAND GENERATORS

In any mode, level and step commands can be generated intemally. Level Commands (one for voltage clamp and one for each microelectrode for a total of 3) are set on precision ten-tum potentiometers. The Step Command is set on a 3'/i-digit thumbwheel switch and can be directed to either one of the microelectrodes or to the voltage clamp. An indicator light for each microelectrode illuminates during current commands. Extemal command sources can be used simultaneously with the intemal command sources.

OUTPUTS Two dedicated Digital Voltmeters continuously display the

microelectrode voltages while a third displays the currmt in the selected microelectrode or in a virtual-ground circuit, if used. Front-panel controls for each microelectrode and the virtual ground set the scaling of the current meter to suit the gain of your headstage.

A Digital Counter lets you know precisely what sampling rate you are using during single-electrode voltage clamp or discontinuous current clamp.

Offset Controls are provided for each microelectrode, and a variable Lowpass Filter is provided for the microelectrode used in single-electrode voltage clamping. As well, an intemally generated Calibration Signal can be superimposed onto each of the outputs. Hence, the output signals in many cases can be wholly conditioned within the AXOCLAMP-2A to suit your recording apparatus.

AXOCLAMP;2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Page 6

FEATURES

Six outputs are conveniently located at the front panel for connectmg to your oscilloscope. These outputs are repeated at the rear panel, where the other outputs, the inputs and the headstage connectors are also located.

REMOTE CONTROL

Hands-free operation of Buzz is possible using the footswitches supplied with every AXOCLAMP-2A. Selection of the operating mode can be made remotely for computer sequencing of experiments.

All AXOCLAMP-2AS have a Buzz oscillator to assist in cell penetration. The duration of the Buzz oscillation is normally equal to the time that the front-panel switch is pressed. Practically, the shortest duration that this switch can be pressed is about 100 ms. For small cells, 100 ms Buzz oscillation sometimes damages the cells immediately after penetration.

The Remote Buzz Duration Control supplied with the AX0CLAMP-2A is a hand held control that contains a trigger switch to buzz either electrode, and a duration control for setting the Buzz duration in the range 1-50 ms. An appropriate duration can be fotmd for most cells that is sufficiently long to allow penetration of the membrane but short enough that the cell is not damaged after penetration.

MODEL CELL Every AXOCLAMP-2A is supplied with a CLAMP-1 model cell. This

model cell plugs directly into the input sockets of the headstages. A switch allows the —

two 50 MQ electrodes to ground, or (b) CELL mode — two electrodes

CLAMP-1

model cell to be configured as (a) BATH mode

connected to a 50 MO // 500 pF cell.

The CLAMP-1 model cell can be used to test and practice using bridge current clamp, discontinuous current clamp, single-electrode voltage clamp and two-electrode voltage clamp. It is a useful tool to use while leaming the operation of the AX0CLAMP-2A and subsequently to verify the correct operation of the AXOCLAMP-2A and the recording pathway.

GENERAL A third HS-2 headstage can be used extracellulariy to record bath

potential. The bath potential is then subtracted from the potentials recorded by the two intracellular microelectrodes to compensate for shifts in bath potential due to changing of solutions or temperature.

A VG-2 Virtual-Ground headstage may be used to measure total bath current. Generally, the built-in current monitors are more useful since they yield the microelectrode currents separately without any interfering currents (e.g. from ionophoresis). Since both microelectrode amplifiers are complete, one microelectrode can be used for ionophoresis while the

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

FEATURES Page?

other is used intracellularly. Internally generated hum due to the built-in power supply has been prevented by using a specially constmcted lowradiation transformer, by placing the supply well away from the rest of the circuitry, and by using intemal shielding. The incoming power is filtered to remove radio-frequency interference (RFI).

QUALITY The excellence of the components and constmction will be obvious to you

from the high quality of the cabinet and controls. Precision ten-tura potentiometers and reliable switches abound. But the high qualify is more than "skin deep' gold plated connectors ar^ used throughout, ultralowdrift operational amplifiers are used in all critical positions, I.C.s are

socketed for easy maintenance, and the circuit designs and operation have been well tested in laboratories throughout the world. All this adds up to low-noise, low-drift, reliable and accurate operation. And the excellence does not stop with the hardware. We also provide a detailed operator's manual that serves as a handbook of procedures for microelectrode users. A separate service manual is also supplied.

FURTHER INFORMATION

AND ORDERING

The AXOCLAMP specification sheet contains complete technical details and ordering information. Please call the factory for answers to any questions you may have.

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Pages

FEATURES

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AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

GLOSSARY

Page 9

GLOSSARY

AXOCLAMP and AX0CLAMP-2A are used interchangeably.

Cia Total input capacitance of the headstage due mainly to the microelectrode and any

connecting cable Cm Input capacitance of cell cSEVC Continuous single-electrode voltage clamp DCC Discontinuous current clamp dSEVC Discontinuous single-electrode voltage clamp

fg Sampling rate; rate for switching from current passing to voltage recording in DCC

and dSEVC modes G The average gain during dSEVC GT

The instantaneous gain of the controlled current source during dSEVC H Headstage current gain 11 Continuous current flow in microelectrode 1 12 Current flow in microelectrode 2 Im Membrane current flow Lag High-frequency cut Lead High-frequency boost MEI Microelectrode 1 ME2 Microelectrode 2 Re Electrode resistance Rg Resistance in series with membrane RMP Resting membrane potential Rm.Rin Input resistance of cell membrane SEVC Single-electrode voltage clamp TEVC Two-electrode voltage clamp Vl Continuous voltage recorded by microelectrode 1 Vj Voltage recorded by microelectrode 2 VC Voltage Clamp VG Virtual-ground output attenuation Vm Membrane potential recorded by microelectrode 1 Vmon Voltage at the input of the sample-and-hold amplifier (SHI)

AX0CLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Page 10 GLOSSARY

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AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

QUICK

GUIDE TO OPERATIONS Page 11

QUICK GUIDE TO OPERATIONS

The controls and operation of die AXOCLAMP-2A are very briefly described in this section. Detailed explanations are given in the alphabetically organized Section E of this manual.

Dl.

HEADSTAGES

(1) HS-2 Series HS-2 series headstages are standard. Two supplied with AX0CLAMP-2A.

All HS-2 headstages record voltage at unity gain.

i .^. K [J> CC ^^^-M- j

Available in several headstage current gains (H). Front-panel controls read direcdy in indicated units when H = xl. All H values are powers of 10. Small H values used widi high-resistance cells and electrodes. Large H values used to pass large curretits.

H= xlO, xl, xO.l, xO.Ol for recording and clamping. H = 0.0001 for ion-sensitive electrodes. Headstages normally supplied in L version (low-noise, low capacitance-neutralization range). M version

can be supplied to compensate large capacitance of grounded shield. Red connector: Microelectrode input

Gold Connector: Driven shield; case Yellow connector: Ground output

(2) HS-4 Series Optional for current-passing electrode (ME2) in two-electrode voltage clamp. (Requires VG-2 for current measurement.) Bypasses internal current-setting resistor during two-electrode voltage clamp so output

voltage appliad directly to electrode. Supplied in L or M versions only. When AX0CLAMP-2A is not in two-electrode voltage clamp mode HS-4 operates same as HS-2.

(3) VG-2 Series Optional virtual-ground headstage measures total badi current. Not required for normal operation. Required in two-electrode voltage clamp if HS-4 headstage used. Virtual Ground output attenuation (VG) specifies the sensitivity. Smaller VG is more sensitive; used for low currents.

AXOCLAMP-2A

THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

Page 12

D2.

MODE GROUP

QUICK GUEIE TO OPERATIONS

Illuminated pushbuttons reconfigure AXOCLAMP-2A for different operating modes. BRIDGE: Two conventional microelectrode amplifiers.

DCC:

Discontinuous current clamp on microelectrode 1.

SEVC: Single-electrode voltage clamp on microelectrode 1.

Discontinuous SEVC (dSEVC) uses time-sharing technique (electrode switches repetitively from voltage recording to current-passing).

Continuous SEVC (cSEVC) is .analogous to whole-cell patch clamp (electrode simultaneously does voltage recording and current passing).

TEVC: Two-electrode voltage clamp. Microelectrode 1 does voltage recording. Microelectrode

^2 does current passing. -——- - . - •

Cont./Discont.: Switch and lamps operate only in SEVC mode.

D3.

MICROELECTRODE 1 (MEI) GROUP

Complete intracellular/extracellular electrometer. Capacitance Neutralization:

Neutralizes electrode input capacitance. Clockwise rotation reduces effective input capacitance and speeds response. Overutilization oscillates headstage.

Buzz:

Deliberate overutilization of capacitance neutralization. Oscillation helps cell penetration. Footswitches supplied as standard accessories.

Bridge:

Compensates electrode voltage drop during current passing. Resistance (scaled by H) read on ten-tum dial. Range automatically reduced tenfold during cSEVC.

Input Offset:

Adds ±500 mV DC to electrode voltage at early stage, electrode voltage while extracellular.

DC Current Command:

For injection of j^nsjant current. Magnitude set on ten-tum dial. Polarity set on switch. LED indicates when current injection activated.

Clear:

Passes large hyperpolarizing and depolarizing current to clear blocked electrodes or help cell impalement.

Voltmeter:

Indicates membrane potential (Vm) in mV.

Use to zero

D4.

MICROELECTRODE 2 (ME2) GROUP

An independent intracellular/extracellular electrometer similar to MEI. Differences are:

Potential is labelled V2. Output offset adds ±500 mV to electrode voltage in output stage.

AXOCLAMP-2A THEORY & OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

QUICK GUIDE TO OPERATIONS

D5.

VOLTAGE-CLAMP GROUP

Page 13

Gain:

Sets open-loop gain during voltage clamp. In SEVC modes output is current source. Therefore gain is nA/mV. In TEVC mode ou^ut is

voltage source. Therefore gain is V/V. Holding Position: RMP Balance Lamps:

Sets holding potential during voltage clamp. Range ±200 mV.

Null during Bridge or DCC so that when activated, voltage clamp will be

at resting membrane potential.

Phase shift:

Modifies frequency response of voltage-clamp amplifier. Compensates for

nonideal phase shifts of membrane. Potentiometer adds phase advance

Oead) or phase delay Gag). Switch selects range.

Anti-Alias Filter:

D6.

STEP-COMMAND GROUP

Used in DCC or dSEVC modes to reduce noise of electrodes that have fast

and slow setding characteristics.

Uses D/A converter to generate precision command voltage. Destination Switch:

Selects voltage clamp or either microelectrode as target for command. Commands are mV or nA respectively.

Thumbwheel Switch:

Sets magnitude widi 0.05% resolution.

Ext./Cont./Off Switch:

Cont. position activates step command. Ext. position thumbwheel switch is off unless logic level HIGH applied to rear-panel Step Activate input. Off position overrides logic input.

Indication:

When destination is a microelectrode and step command is activated, lamp in microelectrode DC Current Command Section illuminates.

D7.

RATE GROUP

Counter indicates sampling rate (cycling rate) in DCC and dSEVC modes. Potentiometer adjusts rate from 500 Hz to 50 kHz.

AXOCLAMP-2A THEORY St OPERATION, COPYRIGHT FEBRUARY 1990, AXON INSTRUMENTS, INC.

•v7

Page

D8.

INPUTS

QUICK GUIDE TO OPERATIONS

AND

OUTPUTS

Vm, Im Output Bandwiddi switch selects Current

used All BNC inputs

Im 11 12

voltmeter displays

select meter input. Decimal point

and

outputs located

output: Membrane current recorded

Cont. Output:

MEI

current (equals Im

output: ME2 current.

-3 dB

frequency

single-pole lowpass

current from eidier microelectrode

set on Hi, H2 or

rear panel. Frequendy used outputs repeated

VG switches.

by MEI.

Bridge, cSEVC

IviRT output: Virtual-ground current.

10.Vm output: Membrane potential recorded Vl Cont. output: Instantaneous Monitor output: Input

MEI

potential.

sample-and-hold amplifier. Should

oscilloscope during DCC

by MEI;

Bridge Balance.

and

dSEVC modes.

gain

V2 output: ME2 potential. Includes Bridge Balance. Sample Clock output: Logic-level pulses

VBATH

output: Potential recorded

Cal. Activate input:. Logic HIGH

at the

sample rate; used

bath electrode.

this input puts calibration voltage proportional

and

10.

Vm outputs.

virtual ground

and

TEVC modes).

used. Switch

front panel.

of 10.

observed

trigger monitor oscilloscope.

thumbwheel

second

setting onto voltage

and

current outputs. Step Activate input: Logic HIGH activates Step Command. Blank Activate input: Logic HIGH activates Blank. During Blank,

Thus stimulus artifacts

Ext.

Command input: Voltage

Ext.

MEI

Command input: Voltage Ext. ME2 Command input: Voltage R» Comp. input: Used

normally required.

VBATH

input: Bath potential recorded

connected

D9.

REMOTE

Allows certain functions

to be

remotely activated

this input converted into voltage-clamp command.

this input converted into

this input converted into ME2 current command.

compensate voltage drop across membrane

this input.

are

See

service manual

computer

rejected.

MEI

current command.

for

suggested circuit.

other equipment subtracted from

switches. These

Clear.

DIG. CLOCK LINK-UP Allows sampling clocks from

talk when

two

AXOCLAMP-2As

two

AX0CLAMP-2AS

dSEVC mode used

to be

synchronized. This eliminates electrode cross-

clamp

two

cells

LU-1 link-up cable.

prevented from updating.

during TEVC.

Not

Vi and V2 if

are

Mode, Buzz

same preparation. Requires

and

AXOCI-AMP-2A

THEORY & OPERATION, COPYRIGHT FEBRUARY

1990,

AXON INSTRUMENTS,

INC.

DETAILED GUIDE TO OPERATIONS Page 15

DETAILED GUIDE TO OPERATIONS

ANTI-ALIAS FILTER A property of all digital sampling systems is that noise in the input signal at frequencies greater than 0.5 of

die sample rate (fg) is folded down to appear as extra noise in the bandwidth from zero to 0.5 of fg (see section on noise). This phenomenon is known as aliasing.

Aliasing can be overcome by filtering die input signal before sampling, thereby reducing die highfrequency noise content. However, this filtering procedure degrades the dynamic response of the input signal and when used with an ideal microelectrode worsens the clamp performance.

The voltage across a real microelectrode often has a two-phase decay after the end of a current pulse, either because of redistribution of ions in the tip, or because of the distributed nature of the capacitance through the wall of the microelectrode (see Fig. 1). The final stages of the decay may often be so slow that additional delay introduced by a filter us^ to prevent aliasing (an Anti-Alias Filter) causes insignificant worsening of the dynamic response. The Anti-Alias Filter can be used by the experimenter to trade off the noise recorded in DCC and dSEVC modes against the dynamic response. That is,

increasing the Anti-Alias Filter setting decreases the noise but can lead to instability in dSEVC and can

make it more difficult in DCC to balance the response to a current step. The Anti-Alias Filter also has an effect in the continuous modes. It acts as a lowpass filter on the voltage

recorded by MEI. Thus the effects during TEVC and cSEVC are the same as those due to a slow voltagerecording microelectrode. Using the Anti-Alias Filter in these modes is not recommended.

Rotating the Anti-Alias Filter control clockwise logarithmically increases die amount of filtering. In the fully counterclockwise position the filter time constant is 0.2 ;xs and the discontinuous clamp responses are unaffected. In the fiiUy clockwise position the filter time constant is 100 /iS. There is a maximal reduction in noise but the maximum sampling rate which can be achieved is severely limited (to about 1 kHz or less).

FIGURE 1 - TWO-PHASE MICROELECTRODE DECAY

Page 16 DETAILED GUIDE TO OPERATIONS

BATH PROBE

Bath Potential Measurement

In certain experimental circumstances it is desirable to make all voltage measurements relative to a reference point in the bathing solution radier than relative to ground. (These conditions may include

precision measurements during changes of temperature or ion content of the saline, or cases of restricted

access from the extracellular space to the grounding point.) All measurements are normally made relative to the system ground. However, if an HS-2 headstage is

plugged into the rear-panel Bath Probe connector, measurements by both MEI and ME2 are automatically

made relative to the potential recorded by this headstage. For optimum voltage-clamp performance, the bandwidth of die bath potential is limited to 300 Hz before it is subtracted from the potentials recorded by MEI and ME2 (see Finkel & Gage, 1985). The bath microelectrode cannot be used for current passing.

The fiill-bandwiddi voltage recorded by die badi microelectrode is available at the VBATH OUT connector. If there is no HS-2 headstage plugged into the Bath Probe connector, a reference potential from an external

amplifier can be subtracted by connecting a reference source to the VBATH IN connector.

Grounding It is quite uncommon to measure the bath potential. Irrespective of whether or not the bath potential is

measured, the preparation bath should be grounded by direcdy connecting it to the yellow ground connector on the back ofthe MEI headstage (or to a virtual-ground headstage if used).

BLANKING

A common problem when using stimulating electrodes is that some of the stimulus is direcdy coupled into the recording microelectrode. This can saturate subsequent high-gain amplifiers and die coupling capacitors of AC circuits. The saturation effects may take tens or hundreds of milliseconds to subside. The best way to minimize or even eliminate this artifact is at the source, by using small stimuli, isolated stimiilators, placing an grounded shield between the stimulating electrodes and the microelectrodes, etc. Often, though, it is not possible to reduce the artifact to manageable levels.

The AXOCLAMP-2A can circumvent the effects of the stimulus artifact by Blanking. At the moment the logic level of the Blank Activate input goes HIGH the value of

is sampled and saved. For the duration

ofthe HIGH signal, this saved value is used instead ofthe actual potential. In voltage-clamp modes the voltage-clamp current during the Blanking period will be held at the level

which existed at the start of the period. A small deviation from the command potential may develop during the Blanking period as a result of comparing the command to the sampled value of the instantaneous value of

Vm.

This deviation will only be seen when the Blanking period ends. Usually

instead of

this deviation is preferable to the situation that can occur if Blanking is not^used. If Blanking is not used the artifact pick^ up by MEI is treated by the voltage-clamp circuit as an attempt by the cell to change its potential. Therefore, the voltage-clamp circuit causes a current to be passed into the cell to clamp this presumed membrane potential change. If

the

stimulus artifact is large, the consequent current artifact can

be large enough to damage the cell.

DETAILED GUIDE TO OPERATIONS Page 17

The width of the Blanking period should be no longer than the minimum width required to cover the period of the stimulus artifact. It is important not to Blank for longer than necessary since during Blanking no updating of minimize the artifact at the source.

BRIDGE MODE

Description

In Bridge mode the microelectrode voltages are monitored continuously, and continuous currents can be injected down MEI or ME2.

Associated with the current flow (I) in a microelectrode is a voltage drop across the microelectrode which depends on the product of the current and the microelectrode resistance (Re)- This unwanted IR voltage drop adds to the recorded potential. The Bridge Balance control can be used to balance out this voltage drop so that only membrane potential is recorded. The term "Bridge" refers to the original Wheatstone Bridge circuit used to balance the IR voltage drop and is retained by convention even though die circuitry

has been r^laced by operational amplifier techniques.

is allowed. Even when Blanking is used, attempts should still be made to

The particular setting required to balance the Bridge is aJmeasure^Qhe-microelectrodTiresistance. j =• /\ In cSEVC mode the Bridge potentiometer compensates electrode IR voltage drop at one-tenth sensitivity.

Suggested Use

Set die Destination switch to ME 1/2 and externally trigger die Step Command generator so that ^uls^ of current are repetitively injected into MEl/2. (Altematively, derive the command for injecting current pulses by connecting a signal source to the Ext. ME 1/2 Command input.) Start with the Bridge Balance control set to zero. Advance the dial until the fast voltage steps seen at die start and finish of the current step are just eliminated. The Bridge is correcdv balanc^. The residual transient at the start and finish of the current step is due to the finite response speed of'the ihicroelectrode. No attempt is made to balance this transient since it covers a very brief period only and it is a useful indication of the frequency response of the microelectrode. The transient can be minimized by correcdy setting the Capacitance Neutralization.

The Bridge balancing procedure is illustrated in Fig. 2. The trace in A was recorded in a_model cell when the Bridge Balance control was correctly set. In response to a positive current pulse the membrane potential began to charge up. Before the membrane potential reached its final value die current pulse was terminated and the membrane potential exponentially decayed to its

Hie traces in B were recorded at a sweep speed which was fast compared with the membrane time

constant, hence the membrane responses look like straight lines. The top trace shows the voltage recorded when no Bridge Balance was used. The response was dominated by the IR voltage drop across the electrode. In the middle trace the Bridge Balance was optimum and in the bottom trace it was slighdy overused.

final

value.

When the Bridge is correctly balanced the resistance of the microelectrode can be read directly from the dial. The sensitivity is 10 -^ H

per turn.

Page 18

DETAILED GUIDE TO OPERATIONS

The Bridge Balance controls operate on die 10.Vm output and on the

output. On the 10.Vm output the

Bridge Balance control satiirates when the IR voltage drop exceeds ±600 mV referred to the input.

Intracellular Balancing The traces in Fig 2. were all recorded with the electrode inside the cell. Since the electrode response and

the oscilloscope swe^ speed were fast compared with the membrane time constant (as in Fig. 2B), the correct Bridge Balance setting was easy to see, even through die electrode was inside the cell.

It is sometimes usefiil to inject a brief small current pulse at the start of each oscilloscope sweep in order to

continually check the Bridge Balance setting during the course of an experiment.

Figure 2 Illustration of Bridge balancing technique. All traces were recorded from the 10.Vm output. The model

cell was 10 M0//1 nF.

was 10 MO.

Recording bandwiddi: 30 kHz.

Vertical calibration: 20 mV referred to Vm-

A. Response.to.+5.nA.10.nis current pulse,. Bridge correctly balanced. Trace is membrane

response only"!

Cal. bar: 20 ms.

Response to -t-5 nA 1 ms pulse.

Cal. bar:

ms.

Top trace: No Bridge balance used.' Fast voltagesteps at start and finish of the current

pulse are the electrode IR voltage drop.

Middle

trace:

Bridge correcdy balanced. Trace is membrane response only. Transient

electrode response remains.

Bottom trace: Bridge balance overused. Negative going step is introduced by the Bridge

Balance circuit.

DETAILED OinDE TO OPERATIONS

Page 19

FIGURE 2 - BRIDGE BALANCING PROCEDURE

Page 20

DETAILED GUIDE TO OPERATIONS

BUZZ

When the Buzz switch or the footswitch is depressed, the amount of Capacitance Neutralization is

increased. If the Capacitance Neutralization control is within a few tums of optimuni, this extra compensation causes the headstage to go into high-frequency oscillation. If this is done while the tip of the microelectrode is pressing against the cell membrane the oscillation will often help the microelectrode impale the cell. The exact mechanism is unknown, but it may involve attraction between the charge at

the tip of

the

microelectrode and bound charges on the inside of

the

membrane.

To use the FS-3 footswitches, plug them into the 4 mm jacks on the back panel. The red jack labelled "+5 V" is shared by the two footswitches. There is one violet jack for each of the two footswitches.

Precise control of the duration of Buzz can be achieved by connecting a pulse generator to pin 15 of the Remote connector (see Remote Section). For some small cells a long duration Buzz can be deadly. In this case it may be helpful to use an external pulse generator connected to pin 15 of the Remote connector to gate the Buzz oscillation so that it is on for just a few milliseconds. The hand-held Remote Buzz generator (see next page) is designed to allow you to conveniently generate Buzz durations between 1 and 50 ms.

It is difficult to interpret the operation of Buzz by watching die 10.Vm trace. This is because the xlO gain and lowpass filter on the 10.Vm output strongly affect the amount of headstage oscillation seen. As a

quick guide, if the 10.Vm trace is unaffected dien Buzz did not succeed (so increase die Capacitance

Neutralization setting). If

the

10.Vm trace jumps then Buzz was successful. The Buzz oscillation can be clearly observed on the Vi Cont. output. If a grounded shield adds a lot of capacitance to ME2 the Capacitance Neutralization range may be

insufficient when an HS-2L headstage is used. In this case it will be necessary to use an HS-2M headstage

(see Headstage Section).

Ranote Buzz

Installation: Plug the Buzz control into the rear-panel ' remote' connector of the Axoclamp.

If you want to use some of the pins on the rear-panel remote connector to remotely select the operating mode or activate the Clear currents, you will have to remove the cover from the plug on the Remote Buzz unit and solder your inputs to the appropriate spare pins on this plug.

Use:

Set the desired Buzz duration on the Duration control of die Remote Buzz unit. Press the button corresponding to the electrode you want to buzz. Note that the Duration control is shared by the two electrodes.

For Buzz durations greater dian 50 ms, use the buttons on the front panel of the Axoclamp. Neither the buttons on the front panel of the Axoclamp nor the footswitches use the duration set on the Remote Buzz unit.

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Axon Instruments Axoclamp 2A User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual