NPI VA-10M User manual

OPERATING INSTRUCTIONS AND
SYSTEM DESCRIPTION OF THE
VA-10M
VOLTAMMETRIC AND
AMPEROMETRIC AMPLIFIER
FOR EPMS SYSTEMS
VERSION 4.2
npi 2017
npi electronic GmbH, Bauhofring 16, D-71732 Tamm, Germany
Phone [phone]; Fax: [phone]
[email protected]; http://www.npielectronic.com

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Table of Contents
1. Safety Regulations ............................................................................................................ 3
2. EPMS-07 Modular Plug-In System .................................................................................. 4
2.1. General System Description / Operation ..................................................................... 4
2.2. EPMS-07 Housing ....................................................................................................... 4
2.3. EPMS-H-07 Housing ................................................................................................... 4
2.4. EPMS-E-07 Housing ................................................................................................... 4
2.5. EPMS-03 ..................................................................................................................... 5
2.6. PWR-03D .................................................................................................................... 5
2.7. System Grounding ....................................................................................................... 6
EPMS-07/EPMS-03 .................................................................................................... 6
EPMS-E-07 .................................................................................................................. 6
2.8. Technical Data ............................................................................................................. 6
EPMS-07, EPMS-E-07 and EPMS-H-07 .................................................................... 6
EPMS-07 and EPMS-H-07 .......................................................................................... 6
EPMS-E-07 .................................................................................................................. 6
EPMS-03 ..................................................................................................................... 6
3. Introduction ...................................................................................................................... 7
4. VA-10M Components ...................................................................................................... 8
5. VA-10M System ............................................................................................................... 8
5.1. System Description ...................................................................................................... 8
5.2. Description of the Front Panel ..................................................................................... 9
6. Headstage ......................................................................................................................... 12
6.1. Headstage Elements ..................................................................................................... 12
7. Operation .......................................................................................................................... 13
7.1. Setting up the VA-10M ............................................................................................... 13
7.2. Testing Basic Functions of the VA-10M ..................................................................... 14
Open Circuit Test ........................................................................................................ 14
DC Accuracy ............................................................................................................... 14
Dynamic Test / Frequency Response .......................................................................... 15
7.3. Carbon-Fiber Electrodes .............................................................................................. 16
7.4. Reference- / Counterelectrode ..................................................................................... 16
7.5. Amperometric Measurements ..................................................................................... 16
7.6. Cyclic Voltammetry .................................................................................................... 16
8. Literature .......................................................................................................................... 17
9. Technical Data .................................................................................................................. 21
10. VA-10M with 3-Electrode Headstage .............................................................................. 22

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1. Safety Regulations
VERY IMPORTANT: Instruments and components supplied by npi electronic are NOT
intended for clinical use or medical purposes (e.g. for diagnosis or treatment of humans),
or for any other life-supporting system. npi electronic disclaims any warranties for such
purpose. Equipment supplied by npi electronic must be operated only by selected, trained
and adequately instructed personnel. For details please consult the GENERAL TERMS
OF DELIVERY AND CONDITIONS OF BUSINESS of npi electronic, D-71732 Tamm,
Germany.
1) GENERAL: This system is designed for use in scientific laboratories and must be operated
by trained staff only. General safety regulations for operating electrical devices are to be
followed.
2) AC MAINS CONNECTION: While working with the npi systems, always adhere to the
appropriate safety measures for handling electronic devices. Before using any device
please read manuals and instructions carefully.
The device is to be operated only at 115/230 Volt 60/50 Hz AC. Please check for
appropriate line voltage before connecting any system to mains.
Always use a three-wire line cord and a mains power-plug with a protection contact
connected to ground (protective earth).
Before opening the cabinet unplug the instrument.
Unplug the instrument when replacing the fuse or changing line voltage. Replace fuse only
with an appropriate specified type.
3) STATIC ELECTRICITY: Electronic equipment is sensitive to static discharges. Some
devices such as sensor inputs are equipped with very sensitive FET amplifiers, which can
be damaged by electrostatic charge and must therefore be handled with care. Electrostatic
discharge can be avoided by touching a grounded metal surface when changing or
adjusting sensors. Always turn power off when adding or removing modules,
connecting or disconnecting sensors, headstages or other components from the
instrument or 19” cabinet.
4) TEMPERATURE DRIFT / WARM-UP TIME: All analog electronic systems are sensitive
to temperature changes. Therefore, all electronic instruments containing analog circuits
should be used only in a warmed-up condition (i.e. after internal temperature has reached
steady-state values). In most cases a warm-up period of 20-30 minutes is sufficient.
5) HANDLING: Please protect the device from moisture, heat, radiation and corrosive
chemicals.

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2. EPMS-07 Modular Plug-In System
2.1.General System Description / Operation
The npi EPMS-07 is a modular system for processing of bioelectrical signals in
electrophysiology. The system is housed in a 19” rackmount cabinet (3U) has room for up to 7
plug-in units. The plug-in units are connected to power by a bus at the rear panel.
The plug-in units must be kept in position by four screws (M 2,5 x 10). The screws are important
not only for mechanical stability but also for proper electrical connection to the system housing.
Free area must be protected with covers.
2.2.EPMS-07 Housing
The following items are shipped with the EPMS-07 housing:
 EPMS-07 cabinet with built-in power supply
 Mains cord
 Fuse 2 A / 1 A, slow (inserted)
 Front covers
Figure 1: Left: front view of empty EPMS-07 housing.
In order to avoid induction of electromagnetic noise the power supply unit, the power switch
and the fuse are located at the rear of the housing (see Figure 2, right).
2.3.EPMS-H-07 Housing
In addition to the standard power supply of the EPMS-07, the EPMS-H-07 has a built-in high
voltage power supply. This is necessary for all MVCS / MVCC modules, the HVA-100, HV-
TR150 and HVC-03M modules. The output voltage depends on the modules in use.
2.4.EPMS-E-07 Housing
The following items are shipped with the EPMS-E-07 housing:
 EPMS-E-07 cabinet
 External Power supply PWR-03D
 Power cord (PWR-03D to EPMS-E-07)
 Mains chord
 Fuse 1.6 A / 0.8 A, slow (inserted)
 Front covers
The EPMS-E-07 housing is designed for low-noise operation, especially for extracellular and
multi channel amplifiers with plugged in filters. It operates with an external power supply to
minimize distortions of the signals caused by the power supply.

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2.5.EPMS-03
The following items are shipped with the EPMS-07 housing:
 EPMS-07 cabinet with built-in power supply
 Mains cord
 Fuse 0,4 A / 0,2 A, slow (inserted)
 Front covers
Figure 2: Left: front view of EPMS-03 housing. Right: rear panel detail of EPMS-03 and
EPMS-07 housing.
In order to avoid induction of electromagnetic noise the power supply unit, the power switch
and the fuse are located at the rear of the housing (see Figure 2, right).
2.6.PWR-03D
The external power supply PWR-03D is capable of driving up to 3 EPMS-E housings. Each
housing is connected by a 6-pole cable from one of three connectors on the front panel of the
PWR-03D to the rear panel of the respective EPMS-E housing. (see Figure 3, Figure 4). A
POWER LED indicates that the PWR-03D is powered on (see Figure 3, left). Power switch,
voltage selector and fuse are located at the rear panel (see Figure 3, right).
Note: The chassis of the PWR-03D is connected to protective earth, and it provides protective
earth to the EPMS-E housing if connected.
Figure 3: Left: PWR-03D front panel view Right: PWR-03D rear panel view.
Note: This power supply is intended to be used with npi EPMS-E systems only.

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2.7.System Grounding
EPMS-07/EPMS-03
The 19" cabinet is grounded by the power cable through the ground pin of the mains connector
(= protective earth). In order to avoid ground loops the internal ground is isolated from the
protective earth. The internal ground is used on the BNC connectors or GROUND plugs of the
modules that are inserted into the EPMS-07 housing. The internal ground and mains ground (=
protective earth) can be connected by a wire using the ground plugs on the rear panel of the
instrument. It is not possible to predict whether measurements will be less or more noisy with
the internal ground and mains ground connected. We recommend that you try both
arrangements to determine the best configuration.
EPMS-E-07
The 19" cabinet is connected to the CHASSIS connector at the rear panel. It
can be connected to the SYSTEM GROUND (SIGNAL GROUND) on the
rear panel of the instrument (see Figure 4).
The chassis can be linked to PROTECTIVE EARTH by connecting it to the
PWR-03D with the supplied 6-pole cable and by interconnecting the
GROUND and PROTECTIVE EARTH connectors on the rear panel of the
PWR-03D (see Figure 3). Best performance is generally achieved without
connection of the chassis to protective earth.
Important: Always adhere to the appropriate safety measures.
Figure 4: Rear panel connectors of the EPMS-E-07
2.8.Technical Data
EPMS-07, EPMS-E-07 and EPMS-H-07
19” rackmount cabinet, for up to 7 plug-in units
Dimensions: 3U high (1U=1 3/4” = 44.45 mm), 254 mm deep
EPMS-07 and EPMS-H-07
Power supply: 115/230 V AC, 60/50 Hz, fuse 2 A / 1 A slow, 45-60 W
EPMS-E-07
External power supply (PWR-03D) 115/230 V AC, 60/50 Hz, fuse 1.6/0.8 A, slow
Dimensions of external power supply: (W x D x H) 247 mm x 180 mm x 90 mm
EPMS-03
Power supply: 115/230 V AC, 60/50 Hz, fuse 0.4 A / 0.2 A slow
Maximum current supply: 500 mA
Dimensions: 3U high (1U=1 3/4” = 44.45 mm), 245 mm deep, 265 mm wide

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3. Introduction
Recently, electrochemical methods using carbon-fiber microelectrodes have been applied to
measure the release of oxidizable transmitter from single cells, and, even more impressively,
from single exocytotic vesicles. Transmitters that are oxidizable and which, therefore, can be
measured with this approach, include serotonin, dopamine, adrenaline, and noradrenaline. In
addition, some peptides or proteins such as insulin may be oxidizable owing to the presence of
oxidizable amino acids such as cysteine or tyrosine.
Cells that have been studied successfully with this technique include adrenal chromaffin cells,
sympathetic neurons, mast cells, pancreatic beta cells, carotid glomus cells and melanotrophs,
but the list is growing. In addition, in brain slices simultaneous intracellular and voltammetric
studies have been made to correlate intracellular electric signals with transmitter release.
Two useful electrochemical approaches are amperometry and cyclic voltammetry. In
amperometry, a DC potential is applied to a carbon-fiber microelectrode. The applied potential
appears at the interface between the carbon and the mammalian ringer solution. If the potential
is much greater than the redox potential for a given transmitter, then molecules of transmitter
diffusing to the carbon surface are oxidized rapidly yielding a current that can be measured.
The sensitivity of the amperometric approach, in particular, has provided an unprecedented
look at the time course of transmitter release revealing distinct phases of release. On the other
hand, the amperometric approach provides little information about the substance being oxidized
or reduced.
Cyclic voltammetry provides a limited amount of information about the substance being
studied, at some expense to the time resolution. In this approach a cyclically repeating voltage
waveform, typically consisting of voltage ramps, is applied to the carbon-fiber electrode and
the resulting current is plotted as a function of the applied voltage (after subtraction of a
"background" record obtained in the absence of the redox species). Since different substances
have different potentials for oxidation and for reduction one can distinguish transmitters from
each other.
For more detailed informations about the principles of electrochemical measurements at single
cells and the fabrication of carbon-fiber microelectrodes refer to several recent reviews.

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4. VA-10M Components
The following items are shipped with the VA-10 system:
 VA-10M amplifier
 Headstage
 GND connector for headstage (1 mm)
 User manual
Optional accessories:
 Carbon-fiber electrode holder
 Carbon-fiber electrodes (CFE),  5µm
 Headstage with differential input
 Four VA-10 amplifier system:
Modified EPMS housing, 4 VA-10 amplifier modules, and a four-in-one headstage
5. VA-10M System
5.1.System Description
The VA-10M is a sensitive (picoampere range) current amplifier that is intended for
voltammetric measurements with carbon-fiber microelectrodes in biological systems, where the
total currents do not exceed 20 nA. It can be used for either DC amperometry using the built-
in voltage source or it can be operated with user-supplied external voltage waveforms (e.g. for
cyclic voltammetry).
The VA-10M is ideally suited for measurements with carbon-fiber disk microelectrodes having
diameters of 10 µM or less from single cells plated onto glass cover slips. However, it can also
be used for measurements made on superficially located cells in tissue slices. The VA-10M is
not recommended for use in in vivo recordings with carbon-fiber electrodes having long
cylindrical measuring surfaces, because in this case currents approach the µA range and a third
electrode is required to compensate for the IR drop as currents flow through the extracellular
fluid.

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5.2. Description of the Front Panel
Figure 5: VA-10M front panel view
In the following description of the front panel elements each element has a number that is
related to that in Figure 5. The number is followed by the name (in uppercase letters) written
on the front panel and the type of the element (in lowercase letters). Then, a short description
of the element is given.

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(1) UNFILTERED OUTPUT connector
BNC connector providing an unfiltered voltage proportional to the current passed
through the electrode. The signal is amplified by the GAIN factor selected by switch
(3).
(2) FILT. OUTPUT connector
BNC connector providing a filtered voltage proportional to the current passed through
the electrode. The signal is filtered by a low pass filter and amplified by the GAIN
factor selected by switch (3). The corner frequency of the filter is selected by switch
(5).
(3) GAIN mV/pA switch
6-position rotary switch to set the amplification factor (x0.5, x1, x2.5, x5, x10, x25
mV / pA) of the current proportional voltage signal at (1) and (2).
(4) BOOSTER
Trim pots for adjusting the FREQUENCY BOOSTER.
TIME: Trim pot for adjusting the TIME CONSTANT of the FREQUENCY
BOOSTER.
AMP.: Trim pot for adjusting AMPLITUDE of the FREQUENCY BOOSTER.
Note: The BOOSTER is best adjusted by following the procedure described in chapter 7.2
(5) LOW PASS Hz switch
6-position rotary switch to set the corner frequency of the low pass Bessel filter.
The filtered OUTPUT can be obtained at connector (2).
(6) COMMAND voltage display
LCD that indicates the COMMAND voltage applied to the electrode
(range: 1000 mV, display: XXXX mV). The COMMAND voltage is set
by potentiometer (7).

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COMMAND unit
The COMMAND units consists of (7) COMMAND potentiometer and (8) +/0/-
switch
(7) COMMAND potentiometer
10-turn potentiometer to set the command voltage in DC amperometric
experiments using the internal voltage source (range: 1000 mV).
(8) +/0/- switch
Switch to set the polarity of the COMMAND voltage. In position 0 the
COMMAND voltage generated by the VA-10M is disabled.
(9) INPUT /10 connector
BNC connector to connect an external waveform for fast cyclic voltammetry. The
INPUT voltage is divided by 10 internally and applied to the electrode.
Important: The voltage at the electrode is always the sum of the voltage at INPUT /10 connector
and the setting at the COMMAND voltage potentiometer.
(10) COMMAND MONITOR connector
BNC connector providing the COMMAND voltage at the electrode multiplied by a
factor of 10, i.e. the voltage is the sum of the setting at (7) COMMAND potentiometer
times 10 and the voltage at (9) INPUT /10 connector.
(11) GAIN MONITOR connector
BNC connector that monitors the setting of (3) GAIN switch. Range: +1 to +6 V,
resolution 1 V / STEP (i.e. 3V indicate a GAIN of 2).
(12) HEADSTAGE connector
8-pin connector for the HEADSTAGE cable.
Important: Always turn power off when connecting or disconnecting the headstage.
(13) OVER LEDs
LEDs indicating if the amplifier 10% below it’s positive or negative limit (±10 V). The
linear range of the amplifier is 12 V.

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6. Headstage
The VA-10M comes with the standard headstage (range: 1000 mV) for connecting carbon-
fiber electrodes via an electrode holder (optional).
A 3-electrode headstage with differential input (see also Optional accessories in chapter 4) is
also available. For details contact npi.
Figure 6: Headstage of the VA-10M
6.1.Headstage Elements
1 BNC connector for the electrode holder
2 GROUND: ground
3 COMMAND: command potential output
4 headstage cable to amplifier
5 REFERENCE: not installed
In the 2-electrode headstage the REFERENCE is not installed. The table indicates whether the
headstage is equipped with the standard feedback resistor (500 M) or with a different one. It
is also marked whether the headstage is in 2-electrode or in 3-electrode configuration.
The electrical connections are made like in a conventional patch-clamp headstage (e.g. the
headstage of the EPC-7 (Heka elektronik, Lambrecht, Germany).
The carbon-fiber electrode fits into the BNC connector of the headstage (#1, Figure 6). An
electrode holder (optional) gives additional mechanical stability. Ask npi for details. GROUND
provides the ground and is linked to the bath, e.g. via an Ag-AgCl pellet. COMMAND provides
the command potential at the electrode and remains usually open, but it can be used to optimize
the measurements by connecting it to an electrode shield (see Ogden (1994) for setting up a
driven shield configuration). The headstage is attached to the amplifier with the headstage cable

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(see #4, Figure 6) and a 4-pole connector. For maximal flexibility the headstage is mounted on
a plastic plate by customized screws. Thus, the user can modify the mounting plate according
to his needs, e.g. to mount the headstage to a micromanipulator.
Note: The shield of the BNC connector and the enclosure of the headstage are linked to the
command potential output and must not be connected to ground.
Caution: Please always adhere to the appropriate safety precautions (see chapter 1). Please turn
power off when connecting or disconnecting the headstage from the HEADSTAGE connector!
7. Operation
7.1.Setting up the VA-10M
The VA-10M EPMS amplifier is shipped as a plug-in unit for the EPMS-07 modular system
and equipped with a small headstage with a BNC connector. When the system arrives the
headstage will not be connected to the cabinet.
For biological voltammetric measurements the experimental setup typically consists of a
microscope located within a Faraday cage to minimize noise pickup. A manipulator is used for
positioning the voltammeter headstage with an attached electrode, so that the electrode tip is
near the cell(s) to be studied.
After installing the VA-10M plug-in unit in the EPMS-07 housing (see also chapter 2) mount
the headstage to the manipulator.
Important: Always turn power off when connecting or disconnecting the headstage.
To facilitate noise reduction of the setup, the faraday cage and the microscope may be connected
to the INTERNAL GROUND located on the back of the EPMS cabinet. Needless to say,
grounding for low noise is an art. If you are not familiar with the principles of low noise
connections, you should consult the local electrophysiology expert or electrical engineer (see
also chapter 1).
 Before turning on power, set the switches at the front panel to the following positions:
Gain (#3, Figure 5): 1 mV/pA
LP Filter (#5, Figure 5): 5k
+/0/- (#8, Figure 5): 0
 Turn power on. The reading of the displays in the modules are an indicator for a working
power supply.
As mentioned above the VA-10M can be used for DC amperometry, taking advantage of the
internal voltage source, or it can be used with user-supplied external waveforms, e.g. for cyclic
voltammetry.
 The LCD display should read 0. Use the COMMAND potentiometer (#7, Figure 5) to apply
a constant voltage for DC amperometry. The 3-position polarity switch can be set to “+’ or
“-”, depending on the polarity of the desired command potential. The command voltage is

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displayed at the LCD in mV. This voltage is applied to the electrode mounted on the
headstage.
 If you intend to read the signal from the VA-10M into a data acquisition system, connect a
BNC cable from the acquisition system to FILT. OUTPUT (#2, Figure 5) or UNFILTERED
OUTPUT (#1, Figure 5). Additionally, you can monitor the GAIN setting by connecting a
BNC cable from the acquisition system to GAIN MONITOR (#11, Figure 5).
 If you intend to use an external voltage source you need to connect your external voltage
source to (9) INPUT /10 connector. Remember that the input voltage will be scaled down
by a factor of 10 at the headstage. Note that, when an external voltage source is used, the 3-
position toggle switch controlling the internal voltage source should be set to “0”, unless
you want to sum the external voltage with the internal voltage source.
Important: The voltage at the electrode is always the sum of the voltage at INPUT /10 connector
and the setting at the COMMAND voltage potentiometer.
The VA-10M is now ready for measurements.
7.2.Testing Basic Functions of the VA-10M
All tests should be made in a noise free environment (e.g. Faraday cage or metal box connected
to GROUND). Please be careful, the headstage is sensitive to electrostatic discharges (see also
chapter 1). Please note that the headstage enclosure is NOT connected to GROUND, it is
connected to the COMMAND signal applied to the microelectrode.
Special notice for 3-electrode headstage: The 3-electrode headstage differs from the standard
headstage in having an additional 1 mm electrode connector (REFERENCE) between the
GROUND and COMMAND connectors for measuring the bath potential. This signal is
processed electronically, so that the command potential is floating with respect to the bath
potential. The REFERENCE input must not be open. It has to be connected to GND for these
tests.
Before starting the tests, check that if everything is set to zero that there is no offset at the output
BNC connectors or digital meter. Also please check that the headstage enclosure (driven shield)
is also at zero, e.g. with a digital meter. Then do the following tests:
Open Circuit Test
 Do not connect anything to the electrode BNC. With no command signals, the current
should be zero.
 Connect a pulse to the command input BNC connector. You should observe only capacitive
transients and NO current during the pulse.
DC Accuracy
 Connect a 100 M or another high value resistor from the electrode BNC to ground.
Caution: Do not use the BNC shield or the headstage enclosure for grounding since they are
connected to COMMAND!

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 Apply a command signal of 100 mV DC to the headstage from the COMMAND setting of
the voltammeter. Alternatively, connect a DC signal of 1 V to the INPUT /10 BNC
connector.
Important: If an external voltage source is used, the 3-position toggle switch controlling the
internal voltage source (#8) should be set to “0”. If the switch is set to “-“ or “+”, the voltage at
the electrode is the sum of the external voltage and the internal voltage source.
 Check with a digital meter that the headstage enclosure and the shield of the headstage BNC
connector are at the COMMAND potential of 100 mV.
 The COMMAND MONITOR output BNC should provide the correct signal of 1 V.
 At the current output BNC should be a signal corresponding to Ohm´s Law and multiplied
by the selected gain factor.
 Changing the polarity or magnitude of the command signal must lead to corresponding
output signals, especially at the CURRENT OUT BNC connectors (according Ohm´s Law).
Dynamic Test / Frequency Response
For this test a good signal generator with a ramp (triangle / sawtooth) output and an oscilloscope
is required.
 Connect a 1 pF capacitor to the electrode BNC at the headstage. To this capacitor connect
a triangle wave generator, with approx. 0.5 V pp and 20-100 Hz.
 This ramp is transferred into a small current following the formula:
Ic=C*dU/dt.
where dU/dt is the slope of the triangle signal (V/sec).
 Observe the current at the UNFILTERED output using an oscilloscope.
Note: The observed current is always double since you change from a positive (+) slope to
a negative (-) slope [x- (-x) = 2x)].
Note The amplitude of the current is also influenced by the accuracy of the capacitor and
the connecting wires.
 Start with AMP. and TIME turned into the left most position (counter-clock wise) and
increase first AMP. and then TIME by turning the trim pots clockwise. By changing the
amplitude and/or frequency you change the dU/dt, and so you can evaluate the range of
linearity of the amplifier and also the frequency response.
 The BOOSTER is set correctly, if the current output is as square as possible. This also
depends on the quality of the triangle wave at the 1 pF capacitor.
 The effect of the gain stage and filters can be tested easily, if these tests work.
Gain stage: When testing the DC accuracy change the setting of the GAIN and observe the
correct signal magnitude at the output BNC.
Filter: If the booster is set correctly connect the oscilloscope to the FILTERED output and
change the filter corner frequency. You should see the changes on the shape of the pulses.

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7.3.Carbon-Fiber Electrodes
Most voltammetric measurements in today’s biological investigations involve the use of
carbon-fiber electrodes. These electrodes can be purchased or you can make your own. For use
with the VA-10M voltammeter the electrodes must fit to the BNC connector at the input of the
amplifier. Two types of connection are commonly used:
1) direct connection via a BNC pin that is soldered onto the end of the electrode or
2) connection via a metal/liquid junction, for example using a 3 M KCl solution to interface
the end of a carbon fiber to a Ag/AgCl wire.
For the first type of connection no special holder is required. For the metal/liquid junction type
a special electrode holder must be used. For some electrodes a patch-pipette holder is adequate.
Carbon fiber disk microelectrodes with small diameter (5-10 µm range) can be obtained from
npi or ALA Scientific Instruments. The electrodes are manufactured using an anodic
electrophoretic insulation method (Schulte, A. and R. Chow, 1996, Anal. Chem. 68, 3054-
3058).
7.4.Reference- / Counterelectrode
The counter electrode used for biological measurements is typically a Ag/AgCl pellet (a
sodium-saturated calomel electrode is sometimes used). The pellet should be immersed into the
recording chamber and connected via a thin wire to the ground input of the headstage (#2,
Figure 6).
7.5.Amperometric Measurements
For high time resolution measurements of transmitter release from single vesicles DC
amperometry is the appropriate approach. In this approach, the carbon-fiber electrode is
energized with a command potential that exceeds the redox potential of the transmitter being
studied. In practice, a command potential of equal to or greater than +650 mV is sufficient for
measurements of all major oxidizable transmitters that have been studied to date.
When generating a command potential for DC amperometry, there should be no control voltage
at the INPUT /10 BNC. The 3-position command toggle switch (#8, Figure 5) should be set,
for example to the “+” position. Then, the desired potential can be dialed in with the 10-turn
potentiometer. As indicated above +650 mV is sufficient for most measurements with cells.
The amperometric signal is diffusion based. Thus, the distance between the carbon-fiber
electrode detecting face and the cell surface must be kept to a minimum. For maximum signal
size and most rapid kinetics it is possible to touch the cell membrane with the electrode.
7.6.Cyclic Voltammetry
In order to facilitate the identification of the transmitter being released, it is possible to use
various voltage waveforms. One common approach is to apply fast voltage ramp potentials, i.e.
to perform fast cyclic voltammetry.
For this application, it is necessary to use an external voltage source connected to the
INPUT /10 connector (#9, Figure 5) at the front panel of the VA-10M. Because one has to relate
the measured current to the applied instantaneous voltage, the current and the applied voltage
should be recorded simultaneously with a data acquisition system.

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8. Literature
VA-10 typical recordings
 Bai, J., Wang, C. T., Richards, D. A., Jackson, M. B., & Chapman, E. R. (2004). Fusion pore
dynamics are regulated by synaptotagmin*t-SNARE interactions. Neuron 41, 929-942.
 Barclay, J. W., Craig, T. J., Fisher, R. J., Ciufo, L. F., Evans, G. J., Morgan, A., & Burgoyne,
R. D. (2003). Phosphorylation of Munc18 by protein kinase C regulates the kinetics of
exocytosis. J Biol.Chem. 278, 10538-10545.
 Bertrand, P. P. (2006). Real-time measurement of serotonin release and motility in guinea pig
ileum. J Physiol. 577, 689-704.
 Bristol, A. S., Sutton, M. A., & Carew, T. J. (2004). Neural circuit of tail-elicited siphon
withdrawal in aplysia. I. Differential lateralization of sensitization and dishabituation.
Journal of Neurophysiology 91, 666-677.
 Bristol, A. S., Marinesco, S., & Carew, T. J. (2004). Neural Circuit of Tail-Elicited Siphon
Withdrawal in Aplysia. II. Role of Gated Inhibition in Differential Lateralization of
Sensitization and Dishabituation. Journal of Neurophysiology 91, 678-692.
 Britt, J. P. & McGehee, D. S. (2008). Presynaptic opioid and nicotinic receptor modulation
of dopamine overflow in the nucleus accumbens. Journal of Neuroscience 28, 1672-1681.
 Chan, S. A., Chow, R., & Smith, C. (2003). Calcium dependence of action potential-induced
endocytosis in chromaffin cells. Pflugers Arch. 445, 540-546.
 Chan, S. A., Polo-Parada, L., Landmesser, L. T., & Smith, C. (2005). Adrenal chromaffin
cells exhibit impaired granule trafficking in NCAM knockout mice. J Neurophysiol. 94,
1037-1047.
 Ciufo, L. F., Barclay, J. W., Burgoyne, R. D., & Morgan, A. (2005). Munc18-1 Regulates
Early and Late Stages of Exocytosis via Syntaxin-independent Protein Interactions.
Molecular Biology of the Cell 16, 470-482.
 Doreian, B. W., Fulop, T. G., & Smith, C. B. (2008). Myosin II activation and actin
reorganization regulate the mode of quantal exocytosis in mouse adrenal chromaffin cells.
Journal of Neuroscience 28, 4470-4478.
 Evans, G. J., Barclay, J. W., Prescott, G. R., Jo, S. R., Burgoyne, R. D., Birnbaum, M. J., &
Morgan, A. (2006). Protein kinase B/Akt is a novel cysteine string protein kinase that
regulates exocytosis release kinetics and quantal size. J Biol.Chem. 281, 1564-1572.
 Fischer, R. J., Pevsner, J., & Burgoyne, R. D. (2001). Control of Fusion Pore Dynamics
During Exocytosis by Munc18. Science 291, 875-878.
 Fulop, T., Radabaugh, S., & Smith, C. (2005). Activity-dependent differential transmitter
release in mouse adrenal chromaffin cells. J Neurosci. 25, 7324-7332.
 Graham, M. E., & Burgoyne, R. D. (2000). Comparison of Cysteine String Protein (Csp) and
Mutant a-SNAP Overexpression Reveals a Role for Csp in Late Steps of Membrane Fusion
in Dense-Core Granule Exocytosis in Adrenal Chromaffin Cells. J.Neurosci. 20, 1281-1289.
 Graham, M. E., Barclay, J. W., & Burgoyne, R. D. (2004). Syntaxin/Munc18 interactions in
the late events during vesicle fusion and release in exocytosis. Journal of Biological
Chemistry M400827200.
 Han, X., Wang, C. T., Bai, J., Chapman, E. R., & Jackson, M. B. (2004). Transmembrane
segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis. Science 304, 289-292.
 Han, X. & Jackson, M. B. (2005). Electrostatic Interactions between the Syntaxin Membrane
Anchor and Neurotransmitter Passing through the Fusion Pore. Biophys.J. 88, L20-L22.

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 Jaffe, E. H, Marty, A., Schulte, A. and Chow, R.H. (1998). Extrasynaptic vesicular transmitter
release from the somata of substantia nigra neurons in rat midbrain slices. J.Neurosci. 18,
3548-3553.
 Lerner, I., Trus, M., Cohen, R., Yizhar, O., Nussinovitch, I., & Atlas, D. (2006). Ion
interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-
induced exocytosis. J Neurochem. 97, 116-127.
 Moore, J. M., Papke, J. B., Cahill, A. L., & Harkins, A. B. (2006). Stable gene silencing of
synaptotagmin I in rat PC12 cells inhibits Ca2+-evoked release of catecholamine. Am.J
Physiol Cell Physiol. 291, C270-C281.
 Petzinger, G. M., Walsh, J. P., Akopian, G., Hogg, E., Abernathy, A., Arevalo, P., Turnquist,
P., Vuckovic, M., Fisher, B. E., Togasaki, D. M., & Jakowec, M. W. (2007). Effects of
treadmill exercise on dopaminergic transmission in the 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine-lesioned mouse model of basal ganglia injury. J Neurosci. 27, 5291-5300.
 Souvannakitti, D., Kumar, G. K., Fox, A., & Prabhakar, N. R. (2009). Neonatal Intermittent
Hypoxia Leads to Long-Lasting Facilitation of Acute Hypoxia-evoked Catecholamine
Secretion from Rat Chromaffin Cells. J.Neurophysiol.
 Wang, P., Wang, C. T., Bai, J., Jackson, M. B., & Chapman, E. R. (2003). Mutations in the
effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in
a nonadditive manner. J Biol.Chem. 278, 47030-47037.
 Wang, C. T., Bai, J., Chang, P. Y., Chapman, E. R., & Jackson, M. B. (2006). Synaptotagmin-
Ca2+ triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore
opening and fusion pore dilation. J Physiol. 570, 295-307.
 Wang, H., Chan, S. A., Ogier, M., Hellard, D., Wang, Q., Smith, C., & Katz, D. M. (2006).
Dysregulation of brain-derived neurotrophic factor expression and neurosecretory function
in Mecp2 null mice. J Neurosci. 26, 10911-10915.
 Xie, Z., Herring, B. E., & Fox, A. P. (2006). Excitatory and Inhibitory Actions of Isoflurane
in Bovine Chromaffin Cells. Journal of Neurophysiology
 Xu, J., Xu, Y., Ellis-Davies, G. C. R., Augustine, G. J. & Tse, F. W. (2002). Differential
Regulation of Exocytosis by α- and β-SNAPs. J.Neurosci. 22, 53–61.
 Zhuge, R., Decrescenzo, V., Sorrentino, V., Lai, F. A., Tuft, R. A., Lifshitz, L. M., Lemos, J.
R., Smith, C., Fogarty, K. E., & Walsh, J. V., Jr. (2006). Syntillas release Ca2+ at a site
different from the microdomain where exocytosis occurs in mouse chromaffin cells.
Biophys.J. 90, 2027-2037.
VA-10 used for recordings with 3 electrodes
 Marinesco, S., & Carew, T. J. (2002). Serotonin Release Evoked by Tail Nerve Stimulation
in the CNS of Aplysia: Characterization and Relationship to Heterosynaptic Plasticity.
J.Neurosci. 22, 2299–2312.
 Marinesco, S., & Carew, T. J. (2002). Improved electrochemical detection of biogenic amines
in Aplysia using base-hydrolyzed cellulose-coated carbon fiber microelectrodes.
J.Neurosci.Meth. 117, 87-97.
 Marinesco, S., Kolkman, K. E., & Carew, T. J. (2004). Serotonergic modulation in aplysia.
I. Distributed serotonergic network persistently activated by sensitizing stimuli. J
Neurophysiol. 92, 2468-2486.
 Marinesco, S., Wickremasinghe, N., Kolkman, K. E., & Carew, T. J. (2004). Serotonergic
modulation in aplysia. II. Cellular and behavioral consequences of increased serotonergic
tone. J Neurophysiol. 92, 2487-2496.

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 Marinesco, S., Wickremasinghe, N., & Carew, T. J. (2006). Regulation of behavioral and
synaptic plasticity by serotonin release within local modulatory fields in the CNS of Aplysia.
J Neurosci. 26, 12682-12693.
VA-10 used for recordings with electrode arrays
 Dias, A. F., Dernick, G., Valero, V., Yong, M. G., James, C. D., Craighead, H. G., & Lindau,
M. (2002). An electrochemical detector array to study cell biology on the nanoscale.
Nanotechnology 13, 285-289.
 Hafez, I., Kisler, K., Berberian, K., Dernick, G., Valero, V., Yong, M. G., Craighead, H. G.,
& Lindau, M. (2005). Electrochemical imaging of fusion pore openings by electrochemical
detector arrays. Proc.Natl.Acad.Sci.U.S.A 102, 13879-13884.
 Spégel, C., Heiskanen, A., Acklid, J., Wolff, A., Taboryski, R., Emnéus, J., & Ruzgas, T.
(2007). On-Chip Determination of Dopamine Exocytosis Using Mercaptopropionic Acid
Modified Microelectrodes. Electroanalysis 19, 263-271.
VA-10 used for scanning electrochemical microscopy
 Etienne, M., Schulte, A., & Schuhmann, W. (2004). High resolution constant-distance mode
alternating current scanning electrochemical microscopy (AC-SECM).
Electrochem.Commun. 6, 288–293.
 Hengstenberg, A., Dietzel, I. D., & Schuhmann, W. (1999). Visualization of biological
activities using the scanning electrochemical microscope. In: Monitoring Molecules in
Neuroscience. ed.: Rollema, H., Abercombie, E., Sulzer, D., & Zackheim, J., Proceedings
of the 8th international conference on in vivo methods, 19-23 June 1999, S.U.N.Y. at Stony
Brook, New York, USA, 47-48.
 Hengstenberg, A., Dietzel, I. D., Blöchl, A., & Schuhmann, W. (1999). Zell-Zell-
Kommunikationsprozesse mittels elektrochemischer Rastermikroskopie. BioForum 10, 595-
599, GIT Verlag, Darmstadt, Germany.
 Turcu, F., Schulte, A., Hartwich, G., & Schuhmann, W. (2004). Label-Free Electrochemical
Recognition of DNA Hybridization by Means of Modulation of the Feedback Current in
SECM. Angew.Chem.Int.Ed Engl. 43, 3482-3485.
VA-10 used for lipid bilayers
 Horner, A., Antonenko, Y. N., & Pohl, P. (2009). Coupled diffusion of peripherally bound
peptides along the outer and inner membrane leaflets. Biophys.J. 96, 2689-2695.

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