OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF
Contents
1. Electronic Instruments for the Life Sciences pease pade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF THE VA 10X VOLTAMMETRIC AND AMPEROMETRIC AMPLIFIER VERSION 7 0 npi 2014 npi electronic GmbH Bauhofring 16 D 71732 Tamm Germany Phone 49 0 7141 9730230 Fax 49 0 7141 9730240 support npielectronic com http www npielectronic com VA 10X User Manual Table of Contents l Safety SOU IARIONS 3 a SAMMI LOU 4 VA IOX COMPOSING 5 4 VP NOG SY 5 System Descriptio EET 5 4 2 Description of the 5 Se Headsta getea 10 Headstage 10 5 2 3 Electrode Headstage optional 11 EEE EA E A E 12 6 1 Setting up the VA 10X 12 6 2 Testing Basic Functions of the VA 10X 14 Open Circuit aeiia e i Ea a tesa 14 ID ORATE A EEE 14 Dynamic Test Frequency Response 15 6 3 Carbon Fiber Electrode Sinisen 15 6 4 Counter Electrode 2 16 92. MONITORING unit SAN iis MONITORING unit consists of 11 COMMAND MONITOR MONITOR MONITOR MONITOR connector 12 FILTER MONITOR connector and 13 GAIN MONITOR connector BV 47V x 10 mV 11 COMMAND MONITOR x10 mV connector The COMMAND MONITOR voltage provided at this BNC connector is the voltage at the electrode multiplied by a factor of 10 i e it is the sum of the setting at the COMMAND potentiometer 7 multiplied by 10 and the INPUT voltage at the INPUT 10 connector 10 divided by 10 12 FILTER MONITOR connector BNC connector that monitors the setting of the FILTER switch The FILTER is monitored in steps of 1 V as the FILTER knob is dialed clockwise 8 V 7 V 13 GAIN MONITOR connector BNC connector that monitors the setting of the GAIN switch The GAIN is monitored in steps of 1 V as the gain knob is dialed clockwise 1 V 7 V 16 FREQUENCY BOOSTER trim pots optional Trim pots for adjusting the FREQUENCY BOOSTER TIME CONST Trim pot for adjusting the TIME CONSTANT of the FREQUENCY BOOSTER AMPLITUDE Trim pot for adjusting AMPLITUDE of the FREQUENCY BOOSTER Note The BOOSTER is best adjusted by following the procedure described in chapter 6 2 version 7 0 page 9 VA 10X User Manual 5 Headstage The VA 10X comes with the standard headstage range 1000 mV for connecting carbon fiber electrodes via an electrode holder optional A 3 electrode headstage with d
3. 16 6 6 Cyclic 16 CD MRT UMD i 17 8 rechnical Data cens AR Aaaa AER TAARE i 21 version 7 0 page 2 VA 10X User Manual 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 2 3 4 5 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 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 ea
4. Neurophysiol Taylor A R amp Chow H 2001 A microelectrochemical technique to measure trans plasma membrane electron transport in plant tissue and cells in vivo PlantCellEnviron 24 1 6 Urena J Fernandez Chacon R Benot A R Alvarez de Toledo G amp Lopez Barneo J 1994 Hypoxia induces voltage dependent Ca2 entry and quantal dopamine secretion in carotid body glomus cells Proc Natl Acad Sci USA 91 10208 10211 Wightman R M J A Jankowski R T Kennedy K T Kawagoe T J Schroeder D J Leszcezyszyn J A Near E J Dilberto Jr O H Viveros 1991 Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells Proc Natl Acad Sci USA 88 10754 10758 Zhou Z Misler S amp Chow R H 1996 Rapid fluctuations in transmitter release from single vesicles in bovine adrenal chromaffin cells Biophys J 70 1543 1552 Zhou Z amp Misler S 1995 Amperometric detection of stimulus induced quantal release of cate cholamines from cultured superior cervical ganglion neurons Proc Natl Acad Sci USA 92 6938 6942 Zhou Z amp Misler S 1996 Amperometric detection of quantal secretion from patch clamped rat pancreatic beta cells J Biol Chem 271 270 277 version 7 0 page 20 VA 10X User Manual 8 Technical Data Headstage Input voltage range Operating voltage Enclosure Electrode connector Ground connector Co
5. electrochemical monitoring of single secretory events in adrenal chromaffin cells Nature 356 60 63 Chow R H amp von Riiden L 1995 Electrochemical Detection of Secretion from Single Cells in Sakmann B amp Neher E eds Single Channel Recording Plenum Press New York and London Doreian B W Fulop T G amp 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 Huang L Shen H Atkinson M A amp Kennedy R T 1995 Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode Proc Natl Acad Sci 92 9608 9612 Kudernatsch M amp Sutor B 1994 Cholinergic modulation of dopamine overflow in the rat neo striatum a fast cyclic voltammetric study in vitro Neurosci Lett 181 107 112 Paras C D amp Kennedy R T 1995 Electrochemical detection of exocytosis at single rat melanotrophs Anal Chem 67 3633 3637 Smith P A Duchen M R amp Ashcroft F M 1995 Fluorometric and amperometric study of calcium and secretion in isolated mouse pancreatic beta cells PflugersArch 430 808 818 Souvannakitti D Kumar G K Fox A amp Prabhakar N R 2009 Neonatal Intermittent Hypoxia Leads to Long Lasting Facilitation of Acute Hypoxia evoked Catecholamine Secretion from Rat Chromaffin Cells J
6. Neurosci 18 3548 3553 1 Lerner I Trus M Cohen R Yizhar O Nussinovitch I amp 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 L Moore J M Papke J B Cahill A L amp Harkins 2006 Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca2 evoked release of catecholamine Physiol Cell Physiol 291 C270 C281 Ll Petzinger G M Walsh J P Akopian G Hogg E Abernathy A Arevalo P Turnquist P Vuckovic M Fisher B E Togasaki D M amp Jakowec M W 2007 Effects of treadmill exercise on dopaminergic transmission in the 1 methyl 4 pheny l 1 2 3 6 tetrahydropyridine lesioned mouse model of basal ganglia injury J Neurosci 27 5291 5300 Ll Wang P Wang C T Bai J Jackson M B amp 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 amp 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 1 Wang H Chan 5 A Ogier M Hellard D Wang Q Smith C amp Katz D M 2006 Dysregulation of brain derived neurotrophic fa
7. 04 Serotonergic modulation in aplysia I Distributed serotonergic network persistently activated by sensitizing stimuli J Neurophysiol 92 2468 2486 1 Marinesco S Wickremasinghe N Kolkman K E amp Carew T J 2004 Serotonergic modulation in aplysia Cellular and behavioral consequences of increased serotonergic tone J Neurophysiol 92 2487 2496 1 Marinesco S Wickremasinghe N amp 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 LI Dias A F Dernick G Valero V Yong M G James C D Craighead H G amp Lindau M 2002 An electrochemical detector array to study cell biology on the nanoscale Nanotechnology 13 285 289 1 Hafez I Kisler K Berberian K Dernick G Valero V Yong M G Craighead G amp Lindau M 2005 Electrochemical imaging of fusion pore openings by electrochemical detector arrays Proc Natl Acad Sci U S A 102 13879 13884 1 Sp gel C Heiskanen A Acklid J Wolff A Taboryski R Emne us J amp 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 1 Etienne M Schulte A amp Schuhmann W 2004 High resolution cons
8. change the dU dt and so you can evaluate the range of linearity of the amplifier and also the frequency response 1 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 pF capacitor LI The effect of the gain stage and filters can be tested easily if these tests work Gain stage When testing the DC accuracy see chapter 0 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 6 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 10X voltammeter the electrodes must fit to the BNC connector at the input of the headstage 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 metal liquid junction for example using a 3 M KCI 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 adeq
9. ctor expression and neurosecretory function in Mecp2 null mice J Neurosci 26 10911 10915 Xie Z Herring B E amp 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 amp Tse W 2002 Differential Regulation of Exocytosis by a and B SNAPs J Neurosci 22 53 61 Zhang 7 amp Jackson M 2008 Temperature dependence of fusion kinetics and fusion pores in Ca2 triggered exocytosis from PC12 cells J Gen Physiol 131 117 124 LI Zhuge R Decrescenzo V Sorrentino V Lai F A Tuft R A Lifshitz L M Lemos J R Smith C Fogarty K E amp 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 version 7 0 page 18 VA 10X User Manual VA 10 USED FOR RECORDINGS WITH 3 ELECTRODES 1 Marinesco S amp 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 1 Marinesco S amp 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 1 Marinesco S Kolkman K E amp Carew T J 20
10. for typical application Marinesco S and 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 6 2299 2312 version 7 0 page 11 VA 10X User Manual 6 Operation 6 1 Setting up the VA 10X Voltammeter The VA 10X amplifier is shipped as a stand alone 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 the attached electrode so that the electrode tip is near the cell s to be investigated L Install the VA 10X unit in a convenient location near the experimental setup _ Mount the headstage to the manipulator Be careful and avoid static discharge during the handling of the headstage Once the headstage has been mounted it can be connected to the VA 10X via the 8 pole connector Caution Please always adhere to the appropriate safety regulations see chapter 1 Please turn power off when connecting or disconnecting the headstage from the HEADSTAGE connector Remember that the shield of the BNC connector and the enclosure of the headstage are linked to the command potential output driven shield config
11. ifferential input see also Optional accessories in chapter 3 and chapter 5 2 is also available For details contact npi Figure 2 Headstage of the VA 10X 5 1 Elements BNC connector for the electrode holder GROUND ground COMMAND command potential output headstage cable to amplifier 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 MQ 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 2 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 version 7 0 page 10 VA 10X User Manual 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 see 4 Figure 2 and an 8 pole connector For maximal fle
12. ing a BNC cable from the data acquisition system to GAIN MONITOR 13 Figure 1 and or the FILTER setting by connecting a BNC cable from the data acquisition system to FILTER MONITOR 12 Figure 1 If you intend to use an external voltage source e g for cyclic voltammetry 1 connect your external voltage source to the INPUT 10 mV connector 10 Figure 1 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 The VA 10X is now ready for measurements version 7 0 page 13 VA 10X User Manual 6 2 Testing Basic Functions of the VA 10X 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 i
13. mmand output Current range Feedback resistor Noise LP FILTER Attenuation corner frequencies GAIN Voltage source OUTPUTS Impedance Linear voltage range FILTERED UNFILTERED INPUTS Resistance COMMAND VOLTAGE MONITORING Impedance GAIN FILTER COMMAND Dimensions Power requirements 1200 mV 12 V Size 40 x 70 x 20 mm driven shield COMMAND potential mounting plate 50 x 70 mm not conducting BNC with shield COMMAND potential 1 mm connector black 1 mm connector red 20 nA max 500 MQ lt pA Bessel Filter 4 pole 24 dB octave 8 pole optional 48 dB octave 20 50 100 200 300 500 700 1 k 1 3k 2k 5k 8k 10k 13k 20k Hz 7 position rotary switch x0 5 xl x2 5 x5 x10 x25 and x50 mV pA COMMAND VOLTAGE set by 10 turn potentiometer range 1000 mV polarity selectable with polarity switch 0 display mV 50 Q 10 V BNC connector sensitivity selectable with GAIN switch FILTERED with LP Bessel FILTER BNC connector sensitivity selectable with GAIN switch 1 MQ INPUT BNC connector sensitivity 10 also displayed 50 Q BNC connector 1 V per step 1 7 V BNC connector V per step 8 V 7 V BNC connector sensitivity x10 19 rackmount cabinet 19 483 mm 10 250mm 1 75 44 mm 115 230 V AC 60 50 Hz fuse 0 4 0 2 A slow version 7 0 page 21
14. nase that regulates exocytosis release kinetics and quantal size J Biol Chem 281 1564 1572 Fischer R J Pevsner J amp Burgoyne R D 2001 Control of Fusion Pore Dynamics During Exocytosis by Munc18 Science 291 875 878 Fulop T Radabaugh S amp Smith C 2005 Activity dependent differential transmitter release in mouse adrenal chromaffin cells J Neurosci 25 7324 7332 Graham M E amp 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 amp Burgoyne R D 2004 Syntaxin Munc 18 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 amp Jackson M B 2004 Transmembrane segments of syntaxin line the fusion pore of Ca triggered exocytosis Science 304 289 292 version 7 0 page 17 VA 10X User Manual Han X amp Jackson M B 2005 Electrostatic Interactions between the Syntaxin Membrane Anchor and Neurotransmitter Passing through the Fusion Pore Biophys J 88 120 122 Jaffe Marty A Schulte A and Chow 1998 Extrasynaptic vesicular transmitter release from the somata of substantia nigra neurons in rat midbrain slices J
15. nce 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 information on the principles of electrochemical measurements at single cells and the fabrication of carbon fiber microelectrodes refer to several recent reviews see also chapter 7 version 7 0 page 4 VA 10X User Manual 3 VA 10X Components The following items are shipped with the VA 10X system VA 10X amplifier Headstage GND connector for headstage 1 mm COMMAND connector for headstage 1 mm REF connector for headstage 1 mm Mains cord Ground bridge User manual lt lt 44444 4 Optional accessories Electrode holder Carbon fiber electrodes 5um 3 electrode headstage with differential input o 4 VA 10X System 4 1 System Description The VA 10X 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 a ten to twenty of nA It can be used for either DC am
16. nin release and motility in guinea pig ileum J Physiol 577 689 704 Bristol A S Sutton M A amp 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 amp 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 amp 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 amp 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 amp 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 amp 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 Evans G J Barclay J W Prescott G R Jo S R Burgoyne R D Birnbaum M J amp Morgan A 2006 Protein kinase B Akt is a novel cysteine string protein ki
17. perometry using the built in voltage source or it can be operated with user supplied external voltage waveforms e g for cyclic voltammetry The VA 10X is ideally suited for measurements from single cells plated onto glass cover slips and with carbon fiber disk electrodes having diameters of 10 uM or less However it can also be used for measurements made on superficially located cells in tissue slices The VA 10X 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 HA range and a third electrode is required to compensate for the IR drop as currents flow through the extracellular fluid 4 2 Description of the Front Panel In the following description of the front panel elements each element has a number that is related to that in Figure 1 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 Some elements are grouped in functional units e g OUTPUT unit and are described as units regardless of the order of numbers version 7 0 page 5 VA 10X User Manual 035008 42 3003 3 Figure 1 VA 10X Front panel view page 6 version 7 0 VA 10X User Manual 1 HEADSTAGE connector The HEADSTAGE is connected via a flexible cable and an 8 pole connector to the mainframe Impo
18. r 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 version 7 0 page 14 VA 10X User Manual 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 1 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 1 Start with AMPLITUDE and TIME CONSTANT turned into the left most position counter clock wise and increase first AMPLITUDE and then TIME CONSTANT by turning the trim pots clockwise By changing the amplitude and or frequency you
19. refore 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 10 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 substa
20. rtant Always turn power off when connecting or disconnecting the headstage 2 GAIN switch The GAIN of the output signals is controlled by a 7 position rotary switch that selects gains of 0 5 1 2 5 5 10 25 and 50 mV pA OUTPUT unit The OUTPUT unit consists of 3 OVER OVER LED 14 OUTPUT FILTERED connector and 15 OUTPUT UNFILTERED connector UNFILTERED FILTERED 3 OVER OVER LED The VA 10X has an over voltage indicator with two LEDs which light up if the amplifier is near it s positive or negative limit 10 V 14 OUTPUT FILTERED connector The OUTPUT FILTERED BNC connector provides a voltage that is proportional to the current passed through the electrode mV pA with GAIN 1 This voltage is FILTERED by a Bessel filter see 4 and scaled by the GAIN factor see 2 15 OUTPUT UNFILTERED connector The OUTPUT UNFILTERED BNC connector provides a voltage that is proportional to the current passed through the electrode mV pA with GAIN 1 see also 3 This voltage is scaled by the GAIN factor see 3 and not filtered 4 LP FILTER switch 16 position switch to select corner frequencies of the Bessel low pass filter Corner frequencies 20 50 100 200 300 500 700 1k 1 3k 2k 3k 5k 8k 10k 13k 20k Hz version 7 0 page 7 VA 10X User Manual INPUT unit The INPUT unit consists of 5 INPUT coupling switch and 10 INPUT 10 mV connector INPUT 10 5 INPUT co
21. rth 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 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 with 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 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 HANDLING Please protect the device from moisture heat radiation and corrosive chemicals version 7 0 page 3 VA 10X User Manual 2 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 the
22. s 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 1 Connect a 100 MQ 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 0 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 mV BNC connector 10 Figure 1 Important If an external voltage source is used the 3 position toggle switch controlling the internal voltage source 6 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 connecto
23. sufficient for most measurements 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 try to touch the cell membrane with the electrode 6 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 connect an external voltage source to the INPUT 10 mV connector 10 Figure 1 at the front panel of the VA 10X 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 version 7 0 page 16 VA 10X User Manual 7 Literature VA 10 Typical recordings m Bai J Wang C T Richards D A Jackson M B amp 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 amp 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 seroto
24. tant distance mode alternating current scanning electrochemical microscopy AC SECM Electrochem Commun 6 288 293 1 Hengstenberg A Dietzel I D amp Schuhmann W 1999 Visualization of biological activities using the scanning electrochemical microscope In Monitoring Molecules in Neuroscience ed Rollema H Abercombie E Sulzer D amp 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 1 Hengstenberg A Dietzel I D Bl chl A amp Schuhmann W 1999 Zell Zell Kommunikationsprozesse mittels elektrochemischer Rastermikroskopie BioForum 10 595 599 GIT Verlag Darmstadt Germany 1 F Schulte A Hartwich G amp 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 version 7 0 page 19 VA 10X User Manual REFERENCES METHODS Alvarez de Toledo G Fernandez Chacon R amp Fernandez J M 1993 Release of secretory products during transient vesicle fusion Nature 363 554 557 Britt J P amp McGehee D S 2008 Presynaptic opioid and nicotinic receptor modulation of dopamine overflow in the nucleus accumbens Journal of Neuroscience 28 1672 1681 Chow R H von Rtiden L amp Neher E 1992 Delay in vesicle fusion revealed by
25. uate version 7 0 page 15 VA 10X User Manual Carbon fiber disk microelectrodes with small diameter 5 10 um range can be obtained from npi or ALA Scientific Instruments The electrodes are manufactured using an anodic electrophoretic insulation method see Schulte A and R Chow 1996 Anal Chem 68 3054 3058 6 4 Counter Electrode The counter electrode used for biological measurements is typically an 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 6 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 so far To generate a command potential for DC amperometry there should be no control voltage at the INPUT 10 mV BNC and the INPUT 10 mV coupling switch should be set to OFF The 3 position command toggle switch 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
26. upling switch The switch must be set to ON if an external waveform is used If no external waveform is used the switch should be set to OFF to avoid coupling of noise 10 INPUT 10 mV connector If an external waveform is used the external source is connected to the INPUT 10 mV BNC connector The voltage supplied is divided by ten internally before reaching the headstage Important If an external voltage source is used the 3 position toggle switch controlling the internal voltage source 6 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 COMMAND unit The COMMAND unit consists of 6 COMMAND polarity switch 7 COMMAND potentiometer and 8 COMMAND voltage display 6 COMMAND polarity switch This switch selects the polarity of the COMMAND voltage corresponds to a range of 0 1000 mV to a range of 0 1000 mV and 0 disables the internal voltage source 7 COMMAND potentiometer 10 turn potentiometer for dialing the COMMAND voltage for DC amperometry if the internal voltage source is used The polarity is set by switch 6 and the amplitude is displayed at 8 8 COMMAND voltage display LC display that monitors the amplitude of the COMMAND voltage XXXX mV 9 POWER switch POWER POWER switch to turn on off the power version 7 0 page 8 VA 10X User Manual
27. uration and must not be connected to ground To improve noise reduction of the setup the faraday cage and the microscope may be connected to the INTERNAL GROUND located on the back of the VA 10X 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 1 Set the switches at the front panel to the following positions Gain 1 mV LP Filter 20 kHz 3 position command polarity switch 0 Li Turn on the power The reading of the display in the module is an indicator for a present power supply The LCD should read 0 As mentioned above the VA 10X 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 1 For use of the internal voltage source the 3 position command toggle has to be switched to or depending on the polarity of the desired command potential The command voltage is displayed at the LCD in mV This voltage is applied to the electrode mounted on the headstage version 7 0 page 12 VA 10X User Manual If you intend to read the signal from the VA 10X into a data acquisition system LI connect a BNC cable from the data acquisition system to the FILTERED UNFILTERED connector 14 or 15 Figure 1 Additionally you can monitor the GAIN setting by connect
28. xibility 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 driven shield configuration 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 5 2 3 Electrode Headstage optional 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 Therefore the command potential is independent from any bath potential that may occur Usually an Ag AgCl silver electrode or pellet is used for measuring the bath potential Important If REF is not used REF must be connected to GROUND electronic GmbH D 71732 Tamm lt ELECTRODE www npielectronic com ze OF 2 Ay Ww w OA 2 5 2 2 mm 2 1 sts 0 Figure 3 VA 10X 3 electrode headstage with CFE electrode holder optional Reference
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