Axon Instruments Axoprobe 1A User Manual

AX0PR0BE-1A MICROELECTRODE AMPLIFIER

OPERATOR'S MANUAL

November 1986

Written for Axon Instruments, Inc.

Alan

Finkel, Ph.D.

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

othenwise,

without written permission from Axon

Instruments,

Tnc.

QUESTIONS? Call (415) 340-9988

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.

CONTENTS

Page

INTRODUCTION .....;..........A-1

FEATURES AND SPECIFICATIONS ...B-1

GLOSSARY C-1

QUICK

DETAILED

GUIDE TO

OPERATIONS D-1

OPERATIONS E-1

Audio Monitor E-2

Bath Probe : E-3 Blanking E-4 Buzz E-5 Calibration Signal .E-6 Capacitance Neutralization and Input Capacitance E-7

Primary • Secondary

Clear E-9 Command Generators E-10

Step command generator • DC current command * Pulse current command • Extemal command inputs * Mixing commands

Current Measurement E-13 C^Comp

E-14

Enhancement * Neutralization • Transient coupling • DC coupling

Grounding and Hum E-17 Headstages E-19

The meaning of H • Which headstage to use • Capacitance neutralization range • Headstage connectors * Tip potentials • Interchangeability • Cleaning * Input leakage current and how to trim it to zero • Warning • DC removal * Input resistance

Holders E-28

Vll

Page in Use/Standby E-31 lonophoresis E-32

Ion Sensitive Electrodes - Special Considerations E-33

Buzz * Capacitance neutralization

Microelectordes For Fast Settling E-34

Microelectrode capacitance • Microelectrode resistance * Filling solutions * Recommended reading

Models.. E-36 Offset E-37 Output Filters , E-38

Order • Type • Risetime

Output Impedance and Protection E-40

Panel Meters E-41

Voltage • Current • Round-off error and zero error

Power Supply Glitches E-42

Power Supply Voltage Selection and Fuse Changing E-43

Supply voltage • Changing the fuse

Resistance Compensation E-45

Description • Suggested use * Intracellular balancing

Ten-Turn Potentiometers E-48

Test Current ....E-49

Cell resistance

Troubleshooting E-50

References E-51

GENERAL INFORMATION F-1

Warranty F-1 RMA form F-3 Policy statement F-5 Service F-5 Comment form • F-7

Vlll

A-1

INTRODUCTION

The AX0PR0BE-1A multipurpose microelectrode amplifier contains two microelectrode

amplifiers (ME1 and ME2). These amplifiers may be independently used for Intracellular and extracellular voltage recording with simultaneous current passing, or differentially for

recording with ion-sensitive electrodes. Many built-in features make the AX0PR0BE-1A

extremely convenient to use.

To learn how to make the most of these features, we advise first-time users of the AX0PR0BE-1A to read this manual thoroughly and to familiarize themselves with the

instrument using resistor/capacitor models of their electrodes and cells.

We will be pleased to answer any questions regarding the theory and use of the AX0PR0BE-1A. Any comments and suggestions on the use and design of the AXOPROBE-1A

We would be most grateful for reprints of papers describing work performed with the AXOPROBE-1 A. Keeping abreast of research performed helps us to design our instruments to be of maximum usefulness to you who use them.

will

be much appreciated.

Axon Instruments, Inc.

Axon Instruments.

Inc

ff'

AXOPROBE-1

Multipurpose Microelectrode Amplifier

CURRENT DISPLAY UtCROELECTROOE I (ME1

STEP COMMAND

^H \^4<ofT Imttunienii ^m^^ ^*^^^

AXOPROBE 1A

MULUPURPOSE UICRD

CLICTHOOF AMPttflfn

••:.*

•.**>•

MICROELECTRODE 2 |ME7|

7 0

••;:,*

^^•^'

viy ^wi^^ ^*^^ mu

® ifel

AUOK) UONITOn

f»>

.«.

Fl INPUT rilNPUT

^^i

'1 no FREO |H/) -3 ita FR£0 |Hf)

DUAL INTRACELLULAR AMPLIFrER

ION-SENSITIVE ELECTRODE AMPLIFIER

EXTRACELLULAR AMPLIFIER

lONOPHORESIS & CURRENT PASSING

The

AXOPROBE-1 A is a dual-channel microelectrode amplifier designed and differential recording make current passing through high-resistance electrodes reliable to enhance flexibility. Programmable "Buzz"

and

easy

operate. Command generators, digital voltmeters, lowpass filters

ideal

for

ion-sensitive electrode measurements. Thirty-volt output compliances enable

for dye

and an

injection

audio monitor simplify cell impalement.

plus

and

for

a variety

ionophoresis.

experiments. High input resistances

The

AXOPROBE-1

and

many other features

A is

fast, low-noise,

are

built

HEADSTAGES

The

AXOPROBE-1 full range of microelectrode resistances, from hundreds of kn to tens of Cft. To accomodate this range, a variety of interchangeable headstages are available. These all record at unity voltage gain but have different currentpassing gains (H).

Headstages also come with a choice of capacitance neutralization ranges. The range depends on the size of the capacitor used for neutralization. The high-frequency noise is also proportional to this capacitor. Usually the headstages are supplied with the smallest capacitor (L version) but other choices are available.

The actual current in each microelectrode is mea-

sured.

That is, the measurement falls to zero if the micro-

electrode blocks even though a current command is set

up.

During current passing, up to ±30 V can be applied across the microelectrode to enable control of current in high-resistance electrodes.

For ion-selective electrode measurements either one or both headstages can be ultrahigh input impedance types.

A is designed to be used with the

VOLTAGE RECORDING

and CURRENT PASSING

Voltage recording and current passing are always performed simultaneously. Passive voltage recording is

ply achieved by switching off the current commands.

MICROELECTRODE 2 (ME2)

RESISTANCE

COMPENSATION

(lOvH MU/TURNI

CAPITANCE

NEUTRALIZATION

sim-

During current passing the voltage drop across the microelectrode can be eliminated from the electrode voltage by setting the Resistance Compensation control (also known as Bridge Balance control). At this setting the microelectrode resistance can be read from the ternatively, the microelectrode resistance can be read on the digital panel meter by using the Test switch.

Capacitance Neutralization uses a 10-turn control for

maximum sensitivity.

To clear blocked tips, a Clear switch forces large hyperpolarizing and depolarizing currents through the electrode.

In some preparations, flicking the Clear switch

helps the microelectrode impale the

When using ultrahigh-impedance electrodes (e.g. ionselective electrodes), there can potentially be a DC error introduced by leakage through the capacitor used for capacitance neutralization. This is totally eliminated in the AXOPROBE-1 A by an original circuit that removes the DC voltage from across this capacitor, thus preventing any leakage.

A common problem when using stimulating electrodes is that some of the stimulus is directly coupled into the recording microelectrode. The AXOPROBE-1 A has special circuits to blank the stimulus artifact.

cell.

dial.

Al-

COMMAND GENERATORS

Several current commands can be generated internally.

Each amplifier has a DC Current Command and a Pulse Current Command. In addition, a Step Command can be directed to either amplifier. The Step Command generator uses a thumbwheel switch to set the output of a digital-to-analog converter. Thus a high degree of precision and repeatability is achieved. Timing is set externally, indicator LEDs light whenever an internal command is activated.

All commands add linearly. Externai command sources can be used simultaneously with the internal command sources.

STEP COMMAND

DC CURRENT

- COMMAND (10H nA TURN)

.t-OM

PULSE CURRENT

COMMAND

(100H nA/TURN)

W •^ii^

TEST

CURRENT

OFF

CONT

9 ME1

DESTINATION

(xH nA)

ME2 Q

DIFFERENTIAL MEASUREMENT

For ion-sensitive measurements and some current determinations in cylindrical cells it is necessary to measure

V^-Vj.

This signal is

provided as an output and can

^C^COMP_^^^

/ IN ENHANCE \

OUT NEUT

also be displayed on one of the

digital panel meters. A C, Compensation control enhances the cross capacitance (C^) between the two electrodes thereby en-

OFF

MAX

abling their responses to common-mode potentials to be exactly matched.

For double-barrel current-passing experiments the C^ Compensation control can be used to neutralize the cross capacitance. The coupling resistance can also be neutrallized.

DISPLAYS, OUTPUTS

and LOWPASS FILTERS

Two dedicated digital panel meters continuously display the microelectrode voltages and a third displays the current in the selected microelectrode. The decimal point of the current meter automatically shifts to comply with

the various headstage current gains (H).

The Vg outputs are the raw electrode voltages. The xlO

outputs contain the Offset voltage and Resistance Compensation. coupled outputs useful for extracellular measurements.

The xlOO outputs are ultralow-noise AC-

— FILTERED OUTPUTS —

40 dB/DECADE LOWPASS

Fl INPUT

10(V,-V2

F2 INPUT

VBATH

Internally or externally generated calibration voltages

can be added to all of the outputs except Vg.

Two second-order lowpass filters have twelve select-

able -3 dB frequencies. The filters have been designed for low noise and zero overshoot. The input to each filter can be switched to one of six signals. Thus all signals can be monitored on the two front-panel connectors without shuffling cables or cluttering up the equipment rack. The unfiltered and filtered signals are separately

available at output connectors on the rear panel.

BUZZ

The most crucial stage of an intracellular experiment is obtaining a good impalement. A commonly used method is to press the microelectrode tip against the membrane and then oscillate

(BUZZ) the microelectrode tage.

For unknown reasons (vibra-

vol-

tion of the tip and electrostatic attraction to bound charges on the inside membrane surface have

been postulated) this causes the tip to penetrate. Until now, deliberately over utilized capacitance neutralization has been used to establish the oscillation.

This produces widely variable results because the oscillation parameters are uncontrolled. In the AXOPROBE-1 A a revolution- i MIN

ary approach is used. The experi- \^^ menter is given complete control of the three essential Buzz

parameters — frequency, duration and amplitude — so that requirements for easy impalement can be optimized. Buzz can be activated by a front-panel switch or by the footswitches provided.

/— BUZZ

/^PARAMETERS

FREQUENCY

fkHz

MAX

(10H

mV/nA)

-3 dB FREO. (Hz)

100 200

50 \ / 500

BYPASS

ACTIVE

Fl OUT

:®i

(10H

mV/nA)

-3 dB FREQ. (Hz)

100 200

50 \ / SOO

10K

BYPASS

ACTIVE

F2 0UT

AUDIO MONITOR

So the experimenter can watch the preparation without interruption, an audio monitor indicates the voltage change following a successful cell impalement. The moment that the electrode tip first touches the solution can

also be clearly heard. The selected input (V, or Vj) determines the pitch of the monitor.

AUDIO MONITOR

VOLUME

^fl^

MIN

PHONE

MAX

SOURCE

OFF

GENERAL

A third electrode can be used extracellularly to record the bath potential. To compensate for potential shifts caused by changing the bath solution or temperature, the bath potential is subtracted from the potentials recorded by the two main electrodes.

A specially constructed low-radiation transformer eliminates the source of line-frequency noise (hum). The

incoming line voltage is filtered to remove radio-fre-

quency interference (RFI).

FURTHER INFORMATION

and ORDERING

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

Strong emphasis has been placed on quality. Precision ten-turn potentiometers, reliable switches and plated connectors are used throughout. Ultralow-drift operational amplifiers are used in all critical positions and ICs are socketted for easy maintenance. Detailed operator's and service manuals are provided.

gold-

Axon Instruments, Inc.

1437 Rollins Road

Burlingame, CA 94010

U.S.A.

Phone (415) 340-9988

Telex: 6771237

B-1

Axon Instruments, Inc.

AXOPROBE-1

HEADSTAGES

Types: HS-2 series headstages are standard. Voltage Gain: All headstages record voltage at a unity Headstage Current Gain (H): Select on basis of cell input resistance

(Ri„) and maximum current capacity dmax)H = x

0.0001 H = x 0.01 for Rin greater than approx. 300 Mfl. H = x 0.1 for R,„ approx. 30-300 Mfl. H = X 1 for Ri„ approx. 3-30 Mil. H = x 10 for Ri„ approx. 0.3-3 Mn.

These ranges suggested for optimum performance. Considerable

overlap Is allowable.

Extracellular Electrodes: Operate with any H value (but check Ip,,,

and Input Leakage Current).

lonophoresis: Typically uses H = x 1.

'"Current Setting and Measuring Resistance (RJ: Located inside

headstage. R^, determines H. R^ is nof the input resistance.

'''input Resistance: Inversely proportional to H. '•"'max: Maximum current that can be passed with input grounded via

indicated

Compliance

high-resistance electrodes.

'*'Noise:

Values measured at V^ output with input grounded via ReSingle-pole lowpass filter used to set measurement circuit -3 dB bandwidth. of Vj is non-oyershooting and so -3 dB bandwidth of v^ is equal to measurement circuit bandwidth.

Hum (Power Line Noise): Less than 20 )xV peak-to-peak, grounded

input, input-referred.

for ion-sensitive electrodes with current passing.

sum

of cell resistance

Voltage: ± 30

(Rin) and

Max voltage that can be applied across

Capacitance Neutralization adjusted so step response

A Multipurpose Microelectrode Amplifier

Note:

Numbered Hems are detailed in the TaUe.

electrode resistance

SPECIFICATIONS

gain.

(R^).

November 1986

'"10-90%

'"Capacitance Neutralization Range: Ten-turn potentiometer. L ver-

'"Case/Shield: Case and shield connector connected to Capacitance "'input Leakage Current die,),) vs. Temperature. Temperature depen-

PREAMPS (Two Channels)

Settling Time

(fia_9o):

Two values

shown;

f,o_,o,i,

for voltage step applied to Input via R^; fro_9o.i for current step into same R^. Capacitance Neutralization adjusted for fastest non-overshooting response.

Input Capacitance: Largely eliminated from step response considera-

tions by bootstrapped power supplies and Capacitance Neutralization.

See Settling Time specifications.

sion headstages have smallest range and lowest noise. M version headstages have larger range but more noise.

Capacitance Neutralization Leakage Current: Prevented by removal

of DC potentials from neutralization capacitor

and

shield.

Removal

has 1 s or 10 s time constant. Neutralization or to unity-gain buffered electrode potential (V,.). dance measured near 25°C. At fixed temperature input leakage

current can be adjusted fo zero.

Offset Range: ±500 mV. Ten-turn potentiometers. Electrode. Resistance Compensation Range: 10-^H MO/turn.' Ten-

turn potentiometers. Coupling resistance range, on ME1 control only, is

I-HH2

Mfl/turn.

Test: For. electrode resistance measurement. 100H mV/Mfl or

H mV/Mft. Res. Comp. must be off.

Clear: Forces ±

In Use/Standby: Microelectrode 2 arnplifier

100

nA through electrode.

only.

In Standby piosltion

disables panel meter and Capacitance Neutralization circuit.

PARAMETER

'" R„

'^' Input Resistance

'max (Rin + ^Q)

Nolse(R„, Bandwidth)

V/(Re'

u(Re).

^10-90,

'*' Capacitance Neutralization Range

'•'' Case/Shield Connected To

'«'Ileal,

vs.

Temp

'" I Output Sensitivity

Maximum Meter Reading

'"" Cx Enhancement/Neutralization Range

UNITS

(iVrms

—

pA/°C

mV/nA

TABLE

HS-2 X 10M

10'"

10,000 (2Mn)

(IMO)

(10kHz)

4 4

(IMn)

Oto22

CapNeut

1.999 |xA

HS-2X1M

10'

10"

1,000

(20Mn)

70 (lOMn) (10kHz)

13 (lOMO)

Oto22

Cap Neut

199.9 nA

HS-2 X 11

10-

10"

1,000

(20Mn)

(lOMO)

(10kHz)

11 12

(lOMfl)

0to8

CapNeut

199.9 nA

HS-2 X 0.1 L

10"

100

(200Mn)

(looMn)

(1kHz)

34 36

(lOOMfi)

0to8

CapNeut

0.3

100

19.99 nA

HS-2 X 0.01 M

10'

10"

(2Gn)

(IGft)

(100Hz)

300

(IGft)

0to22

0.1

1,000

1.999 nA

HS-2M X 0.0001

10"

0.1

(200Gft)

30 (lOCft) (10Hz)

2,000 3,000

(lOOGft)

Ofo22

0.005

0.1V

19.99 pA

B-2

BUZZ

Frequency: Approx 50 Hz-10 kHz. Logarithmic potentiometer. Duration:

Amplitude: 0 to ±30 V. Attenuated by. input capacitance. Linear

Activation:

OUTPUTS "'I, '"Current Meter: Recognizes H of headstage in use and sets decimal

Voltage Meters: Range ±1999 mV. Separate meters for V, and Vj.

V,: Raw headstage voltage. 0.1 % 10V: xio output with Resistance Compensation and Offset. 0.1 % 10(V,-Vj): Difference of 10V, and 100V: xlOO output. AC coupled (1 Hz). 2%. VgATH= xl bath potential. 1% Output Impedances: SOOft.

(10) Q^ Compensation: Used to compensate for the coupling capaci-

LOWPASS FILETERS

Two independent filters: Fl, F2. Second-order.

-3 dB frequencies: Twelve. 2, 5, 10, 20, 50, 100, 200, 500, IK,

Fl Inputs: Switch selected. 10V,, F2 Inputs: Switch selected. 10V,, Bypass Switch: In ACTIVE position signals are filtered. In BYPASS

INTERNAL COMMANDS

Note: DC Current Command: One for each preamp. ±100H nA max.

Pulse Current Command: One for each preamp. ± 1000H nA max.

Step Command: Shared by preamps. Destination switch determines

EXTERNAL COMMANDS

Sensitivities: 20H nAA'

Input Impedance: 100 kft Max. Input Vbltage: ±30 V

CALIBRATION SIGNAL

Intemal:

Extemal:

Accuracy: 1% typical. Audio Monitor: Pitch proportional to V, or Vj. Internal speaker by-

BATH POTENTIAL SUBTRACTION

Signal recorded by bath headstage or by external amplifier Is sub-

Approx. 5-500 ms. Logarithmic potentiometer.

potentiometer.

Front-panel debounced switch, footswitches, or logic

HIGH level on rear-panel connector.

and 1^: /Acfoa/electrode currents. 10-^H mV/nA.

point accordingly. Maximum meter reading is less than headstage

current limit

be measured on I, and Ij outputs. Display selections are I, andlj.

V, meter also displays

tance between electrodes. ENHANCE position adds ME2 voltage via a capacitor into ME2 headstage input, thus matching electrode

responses to common-mode signals. Used for Ion-sensitive electrode recording. NEUTRALIZE position subtracts ME2 voltage from ME2 headstage input. Used for double-barrel current passing.

2K, 5K, 10K Hertz. Continuous rotation.

position signals are wideband.

Commands from all sources sum linearly.

Ten-turn potentiometers.

Ten-turn potentiometers. Activated by HIGH control signal on PULSE GATE input or by front-panel switch.

which preamp command goes to. ± 199.9H nA max. Set on thumbwheel switch. Activated by HIGH control signal on STEP GATE input or by front-panel switch.

by front-panel switch. Input-referred values: 10 mV on x 10 outputs, 1 mV on x 100 outputs, 10H nA on I outputs.

2 mVA' on xlO outputs, 0.2 mVA/ on xlOO outputs, 2H nAA'on I outputs. 100 kft Input impedance.

passed when earphone plugged in.

tracted from xio outputs. Subtraction band-limited to 10 kHz. If bath potential not measured system automatically reverts fo using 0 V as reference potential. Standard headstages work as bath headstages if plugged into bath headstage connector.

{l,^J-

Currents exceeding current meter range can

Vi-Vj.

lOVj.

Matched to 0.01%

lOVj,

10(V,-Vj), V^,, 100V,, I,.

lOVj,

VBATH-

'^e2' lOOVj, Ij.

Activated by HIGH control signal on CAL. GATE injjut or

Proportional to applied voltage. Input-referred values:

GROUNDING

Signal ground is isolated fnjm chassis and power ground.

CONTROL INPUTS

Above 3 V accepted as logic HIGH. Below 2 V accepted as logic

LOW. Inputs protected to ±15 V.

PAIRING BRACKET (BR-1)

BR-1 bracket (optional extra) for mounting two headstages

HEADSTAGE DIMENSIONS

Case is 2.25 x 1.14 x 0.87" (57.2 x 29.0 x 22.1 mm). Mounting rod

is 4 (102) or VB (6.3, 7.9 or 9.5). Specify required mounting rod diameter with order. Cable length is 10 feet (3 m).

HEADSTAGE CONNECTORS

Sockets for microelectrode input, shield drive and ground output

are 0.08" (2 mm) diameter. Input socket is Teflon insulated.

CABINET DIMENSIONS

7 (177)

19"

SUPPLY REQUIREMENTS

Line VolUge: 100-125 VAC or 200-250 VAC. User selectable by an

internal switch. Line Frequency: 50-60 Hz. Power: 20 W. Fuse: Line Filter: RFI filter is included. Line

ACCESSORIES PROVIDED

Operator's Manual Service Manual

2 mm plugs for use with headstage Low-capacitance test resistor for each headstage

Spare fuse

Footswitches to operate Buzz of both electrodes.

ORDERING INFORMATION

When ordering please specify:

3. Diameter (D) of headstage mouriting rods.

Unless you specify otherwise, the AXOPROBE-1 A will be supplied

with one HS-2 H = x0.1L and one HS-2 H = xlL headstage, each with D = Vif. (7.9 mm). Domestic and international sales are direct from the factory.

10%

AXOPROBE-1 As by a single group. For non-simultaneous

purchases, 10% discount applies to second and subsequent

AXOPROBE-1 As purchased by a single group within 12 months.

Discount must be requested when placing order.

WARRANTY

12 months parts and labor from date of receipt.

SERVICE

Service is available at the factory. A detailed service manual is

supplied with each AXOPROBE-1 A.

For further information call us. A factory expert will be pleased to

answer your technical and ordering inquiries.

long.

Available mounting rod diameters (D) are Vi, Vio

high,

19 (483) wide, 12.5 (317) deep. Mounts in standard

rack. Handles included.

0.5 A slow. 5 x 20 mm.

cord:

Shielded line cord is provided.

Current gain (H) and type of two headstages provided. Current gain (H) and type of any extra headstages.

discount applies to simultaneous purchase of two or more

a pair.

AXON INSTRUMENTS, INC.

1429 Rollins Road Burlingame, CA 94010

U.S.A.

Phone: (415) 340-9988 / Telex: 677-1237

c-1

CAL.

CONT.

Cx COMP

EXT.

F1/F2

'l/'2

ME1/2

GLOSSARY

Calibration.

Continuous.

Cross capacitance compensation.

External.

Lowpass

Headstage current gain.

Current in microelectrode 1/2.

Microelectrode 1/2.

filter.

FRONT PANEL ABBREVIATIONS

VBATH

Vel/Ve2

V1/V2

10V1/10V2

10(Vi-V2)

IOOV1/IOOV2

Bath electrode potential. Unity gain.

Raw electrode potential. Unity gain.

Unity-gain electrode potential. Derived by dividing 10Vi/10V2byten.

xio electrode potential. Includes Resistance Compensation, Offset, bath potential subtraction, Calibration.

Difference between

XlOO

electrode potential. Includes Calibration. AC coupled.

IOV1

and IOV2.

D-1

QUICK GUIDE TO OPERATIONS

The controls and operation of the AXOPROBE-1 A are very briefly described in this section. Detailed

explanations are given in the alphabetically organized section E of this manual.

D1.

REST POSITIONS OF CONTROLS

Current meter display: l.| Step Command: 000.0

OFF

ME1 Panel meters: V^, IN USE Capacitance Neutralization: Counterclockwise Resistance Compensation: Counterclockwise (zero) Offset: Mid position (approx. 5.0) DC Current Command: OFF

Counterclockwise (zero)

Pulse Current Command: OFF

Counterclockwise (zero) C^ Compensation: Counterclockwise, IN Output Calibration: OFF Buzz frequency: Mid position

Buzz duration: Mid position Buzz amplitude: Mid position

Audio Monitor OFF

Counterclockwise

Filters: lOV^.IOVg

10 kHz Active

D-2

D2.

HEADSTAGES

HS-2 series headstages are standard. Two supplied with

All HS-2 headstages record voltage at unity

Available in several headstage current gains (H). Front-panel controls read directly in indicated units when

H =

x1.

All H values are powers of 10. Small H values for high-resistance cells and electrodes. Large H

values for passing large currents.

H = x10, H = xO.OOOl for ion-sensitive electrodes.

Headstages normally supplied in L version (low-noise, low capacitance-neutralization range). M version can be supplied to compensate large capacitances.

Red connector Microelectrode input Gold connector Driven shield; case Yellow connector Ground output

x1,

xO.1,

xO.OI for general purpose.

gain.

AXOPROBE-1

D3.

MICROELECTRODE 1 (ME1)

Complete intracellular/extracellular electrometer.

Capacitance Neutralization:

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

Resistance Compensation:

Compensates electrode voltage drop during current passing. Resistance (scaled by H) read on ten-

turn

dial.

If cross-capacitance compensation (0,^ COMP) is "on" and in the NEUT (neutralize)

position,

Test Current:

Applies constant current to electrode. Electrode response in mV corresponds to resistance.

Resistance Compensation setting must be zero.

then this control compensates electrode coupling resistance, scaled by H2 (H of ME2).

Offset:

Compensates ±500 mV electrode tip potential. Works an xlO output only. Use to zero electrode

voltage while extracellular.

DC Current Command:

D-3

For injection of constant current. Magnitude set on ten-turn indicates when current injection activtrted.

Pulse Current Command:

For injection of pulsed or constant current. Magnitude set on ten-turn CONT. position for continuous activation. EXT. GATE position for gating by logic HIGH level applied to rear-panel PULSE GATE input LED indicates activation.

Clear

Passes large hyperpolarizing and depolarizing current to clear blocked electrodes or to impale

Display:

Digital panel meter displays either

Buzz:

Use for cell impalement. Connects oscillating voltage to microelectrode. Parameters of oscillation set in Buzz Parameters section. Activate by front-panel pushbutton switch, or by external switch or

V.|

V.| — V2.

dial.

Polarity set on switch. LED

dial.

Polarity set on switch.

cell.

logic device to connect 5 V TO BUZZ input on rear panel.

D4.

MICROELECTRODE 2 (ME2)

Independent intracellular/extracellular electrometer similar to ME1. One difference is Standby switch replacing Display switch. Standby position used when ME2 not required (disables digital panel meter and capacitance neutralization). Second difference is that when cross compensation is used it is applied from ME2 to

ME1,

not vice versa.

D-4

D5.

BUZZ PARAMETERS:

For optimization of headstage oscillation used to assist impalement. Frequency, Duration and Amplitude of oscillation independently set.

D6.

FILTERED OUTPUTS

Two independent two-pole filters. One of six output signals can be selected for each filter. Twelve —3 dB cutoff frequencies can be selected. Bypass/Active switch determines whether signal is filtered (active

position) or widet}and (filter bypassed).

D7.

C^ COMP

IN:

Enhances capacitance coupling of V2 into V.|. Speeds ME1 response to common signals so that

common signals are recorded identically by ME1 and ME2. Useful for ion-sensitive electrode recording.

OUT: Neutralizes resistance and capacitance coupling of V2 into V.|. Useful for current passing through

one ban-el of a double-ban-el electrode.

D8.

OUTPUT CAL

Adds a 100 mV signal to current outputs, xlO and xlOO voltage outputs. Input-referred values are 10H nA,

10 mV and 1 mV respectively. CONT. position for continuous activation. EXT. GATE position for gating by logic HIGH level applied to rear-panel CAL GATE input. Additional calibration signals separately generated by applying a signal to the EXT. CAL SIGNAL input on the rear panel.

D9.

CURRENT DISPLAY

Meter displays average current. Decimal point automatically placed to suit H. Display is in nA for H = xl,

xO.l.xO.OI;

pAforH =

x0.0001;

^forH = x10. Display

or I2.

D-5

DIG.

STEP COMMAND

D/A converter generates precision command voltages. Destination switch selects either MEI or ME2 to be target for command. Thumbwheel switch sets magnKude with 0.05% resolution. Magnitudes are scaled by H. CONT position for continuous activation. EXT. GATE position for activation by logic HIGH level applied to rear-panel STEP GATE input.

D11.

AUDIO MONITOR

Pitch of audible tone depends on potential of selected Input (V.| or V2). Potentiometer sets volume. Earphone can be plugged into phone jack. If

D12.

BATH POTENTIAL

Use a low-resistance microelectrode with an HS-2 headstage to record potential of bath solution (Vgy^ji^). This potential is sutjtracted from 10V.| and lOVj outputs. If not required, do not plug headstage into rearpanel connector. Vg^^-p^ recorded t)y outside equipment can be subtracted by connecting to EXT. connector

D13.

INPUTS AND OUTPUTS

Located on rear-panel, but Fl OUT and F2 OUT repeated on front-panel.

F1,F2 outputs:

Filtered outputs.

so,

speaker is disabled.

Vp^^ji.

output

Raw electrode potential.

10V output:

Modified electrode potential. xlO potential.

gain.

Includes Offset Resistance Compensation, CAL and bath

D-6

10(V^-V2) output:

Difference signal.

100V output:

AC-coupled (1 Hz) electrode potential x100

VBATH

°"*P"t=

Potential recorded by bath electrode.

I output

Electrode current.

Logic Levels:

Over 3 V accepted as logic HIGH. Below 1 V accepted as logic LOW. ±15 V safe operating range.

Step Gate input:

Logic HIGH activates Step Command.

Pulse Gate input

Logic HIGH activates Pulse Current.

gain.

Includes CAL

100 mV CAL Gate input:

Logic HIGH activates 100 mV OUTPUT CAL

EXT. CAL Signal input:

Voltage on this input converted into proportional calibration signal.

EXT. ME Command input

Voltage on this input converted into proportional current

EXT. Vgy^ji^ input:

Bath potential recorded by outside equipment subtracted from 10V outputs.

V.| Blank Gate Input:

Logic HIGH causes

Vg.|

to be sampled and

held.

Used for stimulus artifact rejection.

Buzz ME input:

Logic HIGH on this input activates Buzz.

+5V output:

Used with footswitches to generate logic HIGH level for Buzz. Protected by 150 ohm series resistor.

D-7

E-1

DETAILED GUIDE TO OPERATION

The controls and operation of the AXOPROBE-1 A are described In this section. The topics are arranged in alphabetical order.

E-2

AUDIO MONITOR

The audio monitor is a voltage controlled oscillator (VCO) that drives a small speaker A switch is used to select

V.|

or V2 as the control voltage (V^). As V^ varies, a so too does the pitch of the audio tone.

When

= 0 the frequency is about 2.25 kHz. This frequency drops by approximately 3 octaves as V^

decreases to-100 mV.

The Audio Monitor enables changes in the electrode potential to be recognized without having to look at the

oscilloscope or panel meters. Thus one can detect a successful cell impalement while still looking through the microscope. There is also an abrupt change in tone when a new electrode first touches the solution. Thus the electrode can be lowered towards the preparation very rapidly and stopped as soon as the tone change indicates contact with the solution.

The volume control on minimum makes the tone inaudible. The volume control can be left in its usual position and the Audio Monitor switched off by using the center position of the input-selector switch.

An earphone can be plugged into the phone jack provided. This disables the speaker

E-3

BATH PROBE

In certain experimental circumstances it is desirable to make all voltage measurements relative to a reference point in the bathing solution rather 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 a unity-gain headstage is plugged into the rear-panel Bath Headstage connector, measurements by both MEI and ME2 are made relative to the potential recorded by this headstage. The bandwidth of the bath potential is limited to 10 kHz t}efore it is subtracted from the potentials recorded by ME1 and ME2. The bath microelectrode cannot be used for current passing.

If there is no unity-gain headstage plugged into the Bath Headstage connector, a reference potential from an extemal amplifier can be subtracted by connecting It to the EXT. Vg^-p^ connector

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