BA-01M Manual - NPI Electronic Instruments
Contents
1. 01 always start with the basic settings to avoid oscillations Basic settings Turn all controls to low values less than 1 and OFFSET in range of 5 zero position see chapter 4 2 Turn POWER switch on Now the BA 01M is ready for an initial check with the cell model version 5 1 page 16 01 User Manual 7 Passive Cell Model The 01 can be ordered with a passive cell model as an optional accessory An active cell model is also available on request for ref see Draguhn et al 1997 The passive cell model is designed for use with single electrode amplifiers to check the function of the instrument in the following circumstances 1 just after unpacking to see whether the instrument has been damaged during transport or 2 to train personnel using the instrument or 3 case of trouble see also chapter 10 to check which part of the setup does not work correctly e g to find out whether the amplifier or headstage is damaged or something 1s wrong with the electrodes or holders etc The passive cell model consists only of passive elements i e resistors that simulate the resistance of the cell membrane and the electrodes and capacitances that simulate the capacitance of the cell membrane A switch allows simulation of two different cell types a cell with 50 and 22 pF CELL 1 represents an astrocyte like cell or a small cell with 200 membrane resistance and 100 pF membra2. D Electronic Instruments for the Life Sciences nga e ade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION FOR THE BA 01M INTRACELLULAR BRIDGE MODE AMPLIFIER MODULE FOR EPMS SYSTEMS amp 01 CUR RANGE mV BRIDGE BALANCE BLAS CURRENT CURRENT STIMULUS INPUT OUTPUT 1 10 1 0 1 V nA VERSION 5 1 2014 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 01 User Manual Table of Contents TIS IMPAMUA ERO 3 Ms ALCL YR SSL ATOMS 4 2 JEPNIS 0T Modular 5 2 1 General System Description Operation iore tere eoe 5 2125 eee ire ae eT nr ne ne m a nc re ee 5 2 3 JDNISSE OT DIOUSIDE 5 Te PWR O 5 eani E E aa ee eee 6 Mn ba abso edid 6 6 2 6 Technical 6 EPNTS UT een Ua rer er eee eer ene 6 EPNISSE ere teen ee ee eee eT 6 9 Components noe 7 d gie esa Eo 7 TI 7 4 2 Descripuon or the Front Panel e o ute t ie eiua
3. 60 W depending on the modules plugged in Dimensions 19 rackmount cabinet 3U high 1U 1 3 4 44 45 mm version 5 1 page 34 01 User Manual index abbreviations 3 accessories 7 basic installation 16 basic settings 16 bias current adjustment 20 BIAS current potentiometer 9 bridge balance 23 24 26 BRIDGE BALANCE unit 9 BUZZ EL CLEAR buzz electrode clear unit 11 CAP COMP potentiometer 10 cell model 17 connections and operation 18 description 17 components 7 CURRENT MONITOR connector 13 CURRENT STIMULUS INPUT connector 13 DISPLAY CUR RANGE unit 10 electrical connections 16 electrode 21 artifacts 27 capacity compensation 21 offset compensation 21 version 5 1 selection 23 front panel view 8 gated stimulus unit 14 general description 7 headstage 15 elements 15 HEADSTAGE connector 9 HOLD CUR holding current unit 11 literature 31 model circuit sharp electrode 25 OFFSET potentiometer 9 POTENTIAL OUTPUT connector 14 safety regulations 4 sample experiments 25 sharp electrode 25 suction patch electrode 28 sealing 29 sharp electrode 25 suction electrodes 28 technical data 33 testing 20 trouble shooting 30 tuning 20 page 35
4. b 10 mV gigaseal formed c whole cell configuration established omv 25 ms Figure 17 Approaching the cell forming a gigaseal and establishing the whole cell configuration version 5 1 page 29 01 User Manual 10 Trouble Shooting In the following section some common problems possible reasons and their solutions are described Important Please note that the suggestions for solving the problems are only hints and may not work In a complex setup it is impossible to analyze problems without knowing details In case of trouble always contact an experienced electrophysiologist in your laboratory if possible and connect a cell model to see whether the problem occurring with electrodes and real cells persists Problem 1 After immersing the electrode into the bath there is an unusual high potential offset Possible reasons 1 The Ag AgCl coating of the silver wire in the electrode holder is damaged 2 The Ag AgCl pellet or Ag AgCl coating of the silver wire in the agar bridge are damaged 3 There is an unwanted GND bridge e g caused by a leaky bath 4 The headstage or the amplifier has an error Solutions 1 Chloride the silver wire again 2 Exchange the pellet or chloride the silver wire in the agar bridge 3 Try to find the GND bridge and disconnect it e g by sealing the bath 4 Contact npi Problem 2 Even if no stimulus is given a current flows through the electrode Possible reason 1 The BIAS cu
5. e 100 correspond to 100 selected by toggle switch 12 12 Vg Ret switch display mode selector Toggle switch to select the mode of the DISPLAY 10 VeL the potential at the electrode in mV is displayed the current flowing through the electrode in nA is displayed Rev the resistance of the electrode in MQ 1s displayed The selected mode is indicated by the LEDs mV nA or MQ 8 The resistance is measured accurately regardless of the correct setting of other front panel elements such as OFFSET BRIDGE BALANCE etc Note With high resistance electrodes ReEL gt 20 MQ the displayed value is dependent on the setting of the capacitance compensation Note Resistance is measured correctly also in current range x10 Caution If the mode selector is switched to the 01 automatically applies current pulses of 2 nA to the electrode Therefore it should not be used during recordings from cells since this current may stimulate or damage the cell version 5 1 page 10 01 User Manual 11 x1 x10 current range switch and LED Switch to set the stimulus current range 1 LED off CURRENT STIMULUS INPUT 1 nA V CURRENT OUTPUT 1V nA HOLD CUR 9 99 nA max STEP SIZE 9 99 nA max x10 LED on CURRENT STIMULUS INPUT 10nA V CURRENT OUTPUT 0 1 HOLD CUR 99 9 nA max STEP SIZE 99 9 nA max HOLD CUR holding current unit HOLD CUR nA The HOLD CUR unit consist
6. electrodes or the 10 MQ electrode connector 3 Figure 9 for simulating an experiment with patch electrodes Ld Switch the CELL membrane switch see 4 Figure 9 to the desired position CELL 1 CELL 2 Turn all controls at the amplifier to low values less than 1 and the OFFSET in the range of 5 zero position and the OSCILLATION SHUTOFF in the DISABLED position _J Turn POWER switch of the amplifier on Now you can adjust the amplifier see below and apply test pulses to the cell model The lower position of the CELL membrane switch CELL 1 simulates a cell with a resistance of 50 and a capacitance of 22 pF In the middle position CELL 2 cell membrane with 200 MQ and 100 pF is simulated version 5 1 page 19 01 User Manual 8 Test and Tuning Procedures Important The 01 should be used only in warmed up condition 1 30 minutes after turning power on The following test and tuning procedures are necessary for optimal recordings It is recommended to first connect a cell model to the amplifier to perform some basic adjustments and to get familiar with these procedures It 1s assumed that all connections are built as described in chapter 6 Important Except for Headstage bias current adjustment see 8 1 all adjustments described below should be carried out every time before starting an experiment or after changing the electrode 8 1 Bias Current Adjustment Caution I
7. 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 POWER The 19 cabinet is connected to the CHASSIS connector at the rear panel The CHASSIS 15 linked to protective earth as soon as the PWR 03D 15 connected It can be connected also to the SYSTEM GROUND SIGNAL GROUND on the rear panel of the instrument see Figure 3 Important Always adhere to the appropriate safety measures Figure 3 Rear panel connectors of the EPMS E 07 2 6 Technical Data 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 Power supply 115 230 V AC 60 50 Hz fuse 2 A 1 A slow 45 60 W EPMS E 07 External power supply for EPMS E 115 230 V AC 60 50 Hz fuse 1 6 0 8 A slow Dimensions of External power supply W x D x H 225 mm x 210 mm x 85 mm version 5 1 page 6 01 User Manual 3 BA 01M Components The following items are shipped with BA 01M system Amplifier module for the EPMS 07 system X Headstage GND 2 4 mm connector for headstage User manual Optional accessories Electrode holder Suction electrode holder Remote switch for penetration unit Active cell model Passive cell model see Figure 9 Low noise low bias current headstage with a reduced current range 10 hea
8. od e biete ons 8 RSAC SUMS a assets es rela uth vals athe et 15 15 6 ocne up Me BA tem dtes ih o 16 Rr D 17 DesectrpUoOb 17 Teza Connectons and E 18 o Lest and Tonine 20 Hedadstage Bias Current 20 5 2 Ofset Compensa 21 5 0 Capacitance Compensa 21 Bail IN 23 Be PECOJ Sele Cll OM cai 23 9 Sample 25 9 1 Sample Experiment using a Sharp 25 9 2 Sample Experiment using a Suction Patch 28 10 TrOUDle SNOOUNG 30 r 31 1 rrp ern an ner sere renee err MEME NU 33 lio up 35 version 5 1 page 2 01 User Manual About this Manual This manual should help to setup and use the 01 system correctly and to perform reliable experiments If you are not familiar with the use of instruments for intracellular recording of electrical signals please read the manual completely The experienced user should read at least chapters 1 4 5 8 Important Please read chapter 1 carefully It contains general information about the safety regulations and how to handle highly sensitive electronic in
9. see chapter 8 3 This is very important since a badly compensated input capacitance prevents setting the BRIDGE BALANCE to correct values Apply current pulses to the electrode either using an external stimulator via CURRENT STIMULUS INPUT connector 18 Figure 4 or by using the gated stimulus unit L Watch the POTENTIAL OUTPUT at the oscilloscope and adjust the BRIDGE BALANCE as shown in Figure 12 using the BRIDGE BALANCE potentiometer 5 Figure 4 After adjustment you should see a straight voltage trace without artifacts caused by the potential drop at Rex Figure 12 illustrates the BRIDGE BALANCE procedure using a 100 MQ resistor that represents the electrode The current stimuli were generated using the gated stimulus unit gated by two TTL pulses The amplitude was set to 0 5 nA In the upper diagram the bridge is slightly undercompensated and in the diagram in the middle it is slightly overcompensated The lower diagram shows a well balanced bridge compensated Important BRIDGE BALANCE and CAP COMP must be tuned several times during an experiment since most parameters change during a recording session Figure 15 shows artifacts caused by uncompensated stray capacitance and bridge during recording from a cell It also shows how to cancel these artifacts by tuning with CAP COMP and BRIDGE BALANCE OFFSET deviations can be detected by comparing the readout on the potential display before and after an experiment with the e
10. see Figure 8 that fits into the BNC connector of the headstage or into an electrode holder adapter optional see also Optional accessories in chapter 3 The electrical connection between the electrolyte and the headstage is established using a carefully chlorinated silver wire Chlorinating of the silver wire is very important since contact of silver to the electrolyte leads to electrochemical potentials causing varying offset potentials at the electrode deterioration of the voltage measurement etc for details see Kettenmann and Grantyn 1992 For optimal chlorinating of sliver wires an automated chlorinating apparatus ACL 01 is available contact npi for details Ground provides system ground and is linked to the bath via an agar bridge or a Ag AgCl pellet The headstage is attached to the amplifier with the headstage cable see 4 Figure 8 and a 8 pole connector The headstage is mounted to a holding bar that fits to most micromanipulators or optionally to a mounting plate or a dovetail adapter Note The shield of the BNC connector is linked to the driven shield output and must not be connected to ground The headstage enclosure is grounded 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 version 5 1 page 15 01 User Manual 6 Setting up the BA 01M System The following steps should he
11. to electrode PR cell Cs P a P stray By ground C ground Figure 13 Model circuit for intracellular recording using a sharp electrode Cm membrane capacitance Cstray electrode stray capacitance Rex electrode resistance Rm membrane resistance I Connect the electrode cable holder to the BNC connector and Ag AgCl pellet or the agar bridge for grounding the bath with GND at the headstage Make the basic settings see chapter 6 Again It is of major importance that 01 systems are used only in warmed up condition i e 30 minutes after turning power L Adjust BIAS CURRENT to zero if necessary see chapter 8 1 L Reconnect the STIMULUS INPUT and or the STEP GATE INPUT and put an electrode into the electrode holder I Immerse the electrode into bath not in a cell as deep as necessary during experiment Test the capability of the electrode to carry current see chapter 8 5 compensate the potential offset see chapter 8 2 measure the electrode resistance see 9 chapter 4 2 and compensate the input capacitance see chapter 8 3 and Figure 11 Apply current steps to the CURRENT STIMULUS INPUT and adjust the BRIDGE BALANCE to suppress all artifacts on the POTENTIAL OUTPUT see chapter 8 4 version 5 1 page 25 BA 01M User Manual I Now the system is preadjusted for measurements Find cell Approach the desired cell There are several indications
12. Figure 4 BA 01M front panel view the numbers are related to those in the text below version 5 1 page 8 01 User Manual In the following description of the front panel elements each element has a number that is related to that in Figure 4 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 BUZZ unit and are described as units regardless of the order of numbers 1 HEADSTAGE connector The HEADSTAGE 1 connected via a flexible cable and a 8 pole connector to the mainframe see also chapter 5 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 2 BIAS bias current potentiometer With this trim potentiometer the output current of headstage headstage BIAS p current can be tuned to zero range 200 pA see chapter 8 1 3 0 STEP SIZE switch 0 toggle switch to disable the gated stimulus set by STEP SIZE 21 and gated by STEP GATE INPUT TTL 20 or to select the polarity of the gated stimulus gated stimulus positive 0 gated stimulus disabled gated stimulus negative 4 OFFSET potentiometer Control to set the output of the electrode preamplifier to zero te
13. IZE nA digital potentiometer Control to set the amplitude of the gated stimulus current range x1 digital potentiometer nA range 9 99 nA resolution 10 pA current range x10 digital potentiometer XX X nA range 99 9 nA resolution 100 pA 20 POTENTIAL OUTPUT x1l0mV connector BNC connector monitoring the recorded membrane potential with a gain of ten resistance 250 Q range 1000 mV membrane potential POTENTIAL Important Maximum potential that can be monitored is 1000 mV This applies also xTO mwv to operation in current range x10 version 5 1 page 14 01 User Manual 5 Headstage The 01 comes with the standard headstage range 12 nA 120 nA for connecting glass electrodes with high resistances or suction electrodes for whole cell patch clamp recordings with lower resistances via an electrode holder see Figure 8 A low noise low bias current headstage range 1 2 nA 12 nA see also Optional accessories in chapter 3 for measurement of very small currents 15 also available For details contact headstage Figure 8 electrode holder optional and headstage of the BA 01M 5 1 Headstage Elements BNC connector for the electrode holder linked to the driven shield output REF not connected GND Ground connector headstage cable holding bar A U N The electrode filled with electrolyte is inserted into an electrode holder optional
14. Q obtained by application of square current pulses 2 nA display XX XX MQ Bandwidth and speed response Full power bandwidth 10 gt 10 kHz rise time 1096 90 Square pulse 1 V pp 40 us voltage 30 us current 100 MQ 25 us voltage and current 5 MQ version 5 1 page 33 01 User Manual Outputs Resistance 50 0 Current x1 BNC connector sensitivity 1 nA V display XX XX nA resolution 10 pA Current x10 BNC connector sensitivity 0 1 nA V display XXX X nA resolution 100 pA Potential BNC connector sensitivity x10 mV display XXXX mV Inputs Current x1 Current stimulus input via connector sensitivity 1 nA V Step gate input via BNC connector gated stimulus with digital control of current step size nA Max step size 9 99 nA resolution 10 pA polarity selectable with toggle switch holding current range 10 nA adjustable with ten turn control Current x10 Current stimulus input via BNC connector sensitivity 10 nA V Step gate input via BNC connector gated stimulus with digital control of current step size XX X nA Max step size 99 9 nA resolution 100 pA polarity selectable with toggle switch holding current range 100 nA adjustable with ten turn control Dimensions Front panel 24 HP 121 5 mm x 128 5 mm Housing T 175 mm deep EPMS 07 system Power requirements 115 230 V AC 60 50 Hz fuse 2 A slow 45
15. acitance In the diagram in the middle the capacitance is slightly overcompensated and in the lower diagram it is well compensated version 5 1 page 21 01 User Manual undercompensated potential mV 250 200 150 100 50 0 10 20 30 40 50 time ms overcompensated potential mV 600 400 200 o 200 400 600 10 20 30 40 50 time ms compensated potential mV 200 200 150 100 50 0 j im Lp m 0 10 20 30 40 50 time ms potential Figure 11 Tuning of the capacitance compensation using a 100 MQ resistor version 5 1 page 22 01 User Manual 8 4 Bridge Balance If current is passed through an electrode the occurring voltage deflection potential drop at affects the recording of membrane potential Therefore this deflection must be compensated carefully by means of the BRIDGE BALANCE control With the cell model connected or the electrode in the bath the BRIDGE BALANCE control is turned on clockwise until there is no artifact on the POTENTIAL OUTPUT see Figure 12 The procedure can be done in current range x1 or in current range x10 I Make the basic settings at the amplifier see chapter 6 I Connect a cell model or immerse the electrode into bath as deep as necessary during the experiment L Tune the OFFSET to zero see chapter 8 2 and compensate the input capacitance
16. amplifiers with plugged in filters It operates with an external power supply to minimize distortions of the signals caused by the power supply 2 4 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 the one of the three connectors on the front panel of the PWR 03D to the rear panel of the respective EPMS E housing see Figure Figure 3 A POWER LED indicates that PWR 03D is powered on see Figure 1 Power switch voltage selector and fuse are located at the rear panel see Figure 2 Note The chassis of the PWR 03D is connected to protective earth and it provides protective earth to the EPMS E housing if connected version 5 1 page 5 01 User Manual PWRE D3D CUT curn ora Figure 1 PWR 03D front panel view Figure 2 PWR 03D rear panel view Note This power supply is intended to be used with npi EPMS E systems only 2 5 System Grounding EPMS 07 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
17. antyn eds 1992 Practical Electrophysiological Methods Wiley Liss New York Lalley P M Moschovakis U Windhorst 1999 Electrical Activity of Individual Neurons in Situ Extra and Intracellular Recording in U Windhorst and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin New York L Maier N Nimmrich V amp Draguhn 2003 Cellular and network mechanisms underlying spontaneous sharp wave ripple complexes in mouse hippocampal slices J Physiol 550 873 887 L1 Mayer Waarden 1975 Einf hrung in die biologische und medizinische Me technik Schattauer Verlag Stuttgart version 5 1 page 31 01 User Manual Mock M Butovas S amp Schwarz 2006 Functional unity of the ponto cerebellum evidence that intrapontine communication is mediated by a reciprocal loop with the cerebellar nuclei J Neurophysiol 95 3414 3425 I Neher 1974 Elektrische Me technik in der Physiologie Springer Verlag Berlin Ld Nimmrich V Maier N Schmitz D amp Draguhn A 2005 Induced sharp wave ripple complexes in the absence of synaptic inhibition in mouse hippocampal slices Journal of Physiology 563 663 670 1 Ogden DC 1994 Microelectrode Techniques The Plymouth Workshop Handbook Second Edition The Company of Biologists Limited Cambridge 1 Prinz A and P Fromherz 2000 Electrical synapses by guided growth of cultured neuron
18. apacitance compensation yields to very powerful high frequency oscillations see Figure 5 OFF Switching to OFF disables the BUZZ circuit REMOTE In the REMOTE position either a hand held switch or a foot switch can be used to operate the BUZZ circuit remotely The remote device is connected via a grounded BNC cable at connector 16 potential mV 12000 s n M d 8000 4000 0 4000 8000 nN 12000 start Stop time ms Figure 5 BUZZ function of the BA 01M 16 REMOTE connector BNC connector to attach a remote switch to the BUZZ unit see also 12 17 EL CLEAR electrode clear switch The EL CLEAR switch is used to activate the electrode clear circuit that can be used to clean the tip of the electrode by passing large amounts of positive or negative currents The circuit 1 operated by pushing this switch to Imax maximal positive current or Imax maximal ne
19. ch clamp recording using a suction electrode Cm membrane capacitance Cstray electrode stray capacitance Rex electrode resistance Rm membrane resistance Prepare the setup and proceed as described in the previous subchapter 9 1 until you have selected a cell Before immersing the pipette into the bath apply slight positive pressure to the pipette to prevent settling of particles at the tip I Apply test pulses to the pipette about 10 pA The resulting voltage signals at pipette are very small 50 u V with a 5 MQ electrode Approach the cell until the voltage signal changes a Figure 17 Often you can observe slight dent in the cell membrane L Release pressure from the pipette Now forming of the seal is indicated by the voltage deflections getting much larger I If the seal does not form apply gentle suction to the pipette until a gigaseal is established b Figure 17 Apply stronger suction to the pipette or use the BUZZ unit to brake the cell membrane under the pipette and establish the whole cell configuration The whole cell configuration is established if you see the voltage signal getting smaller again c Figure 17 and you read the expected membrane potential Read the membrane potential and if necessary readjust the BRIDGE BALANCE as shown in 8 4 and Figure 14 and Figure 15 Start the experiment version 5 1 page 28 01 User Manual close the ud MEN uM cell membrane
20. dstage 1 maximal current 1 1 2 nA 12 nA Headstage with differential input db Who ub cbe cup L 4 BA 01M System This manual is related to the standard configuration of the BA 01M system with a standard headstage and standard calibrations of bridge balance electrode resistance display range etc as they appear on the front panel Other configurations are available e g if BA 01M system is used only for whole cell patch clamp recordings with suction electrodes the BA 01M system can be delivered with adapted calibrations and a low noise low bias current headstage see Optional accessories in chapter 3 For details contact npi 4 1 System Description The npi BA 01M intracellular recording systems are precise current clamp instruments with a bridge circuit to compensate for the resistance of the recording intracellular electrode For current injection and potential recording a very high impedance voltage to current converter with a special input capacitance compensation circuit is used For methodical reviews see Lalley et al 1999 Richter et al 1996 Ogden 1994 and Boulton et al 1990 The system consists of a module that is plugged in an EPMS 07 housing and a headstage which should be placed close to the recording site The recording electrode is connected to the headstage via an electrode holder see also Figure 8 In some setups there 1 no space for placing the headstage very close to the recording s
21. eady 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 5 1 page 4 01 User Manual 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 V Fuse2A 1 A slow Front covers 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 2 3 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 Front covers SN NN NN The EPMS E 07 housing is designed for low noise operation especially for extracellular and multi channel
22. electrodes and stray capacitances Cstray form a low pass filter which deteriorates the shape of recorded intracellular signals see also Figure 13 The frequency response of the amplifier 15 improved considerably by using the capacitance compensation function This function is based on positive feedback negative capacitance circuit The tuning of the capacitance compensation control is performed using pulses applied to the CURRENT STIMULUS INPUT or pulses provided by the electrode resistance test circuit With the cell model connected or the electrode in the bath the CAP COMP control is turned clockwise until there is no artifact on the POTENTIAL OUTPUT see Figure 11 Make the basic settings at the amplifier see chapter 6 I Connect a cell model or immerse the electrode into the bath as deep as necessary during the experiment L Switch the display mode selector 12 Figure 4 to VEL L Tune the OFFSET to zero see chapter 8 2 I Switch the display mode selector 12 Figure 4 to Ret or apply pulses to the CURRENT STIMULUS INPUT and watch the POTENTIAL OUTPUT L Compensate the input capacitance as shown in Figure 11 using the CAP COMP potentiometer 6 Figure 4 Figure 11 illustrates the capacitance compensation procedure using a 100 MQ resistor that represents the electrode The pulses were generated using the automated electrode resistance test circuit of the 01 The upper diagram shows an undercompensated cap
23. gative current version 5 1 page 12 01 User Manual 18 CURRENT STIMULUS INPUT 1 10 nA V connector Analog input BNC connector for applying signals from an external stimulus source The voltage signal that is connected here 1 transformed Smet to a proportional current at the electrode with a sensitivity of nA V or SMS NEU 10 nA V The scaling is dependent on the current range setting please see also 11 For instance an input voltage of 5 V 1s transformed to an output current of 5 nA in current range x1 and to an output current of 50 nA in current range x10 The signal form remains unchanged The amplitude of the output current signal current stimulus is determined by the amplitude of the CURRENT STIMULUS INPUT Two examples for operation in current range x1 are given in Figure 6 In A the amplitude of the CURRENT STIMULUS INPUT is 1 V that gives a current stimulus of nA in B the CURRENT STIMULUS INPUT amplitude 1 2 V that is transformed into a current stimulus of 2 nA CURRENT STIMULUS INPUT current stimulus gt PULL UL Figure 6 Input output relation using CURRENT STIMULUS INPUT Important The current injected through the electrode is always the sum of the input signal at CURRENT STIMULUS INPUT 18 the holding current set by HOLD CUR 9 and switch 14 and the gated stimulus set by STEP SIZE 21 and switch 3 19 CURRENT OUTPUT 1 0 1 nA V connector BNC con
24. ical devices should 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 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 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 TEMPERATURE DRIFT WARM UP TIME analog electronic systems are sensitive to temperature changes Therefore all electronic instruments containing analog circuits should be used only in a warmed up condition 1 after internal temperature has reached st
25. ing the configuration _J Turn POWER switch of the amplifier off a For simulation of an experiment using a suction electrode L Connect the BNC jack labeled 10 of the cell model to the BNC connector Pet of the headstage b For simulation of an experiment using a sharp electrode L Connect the BNC jack labeled 50MQ of the cell model to the BNC connector Pet at the headstage For headstages with SMB connector use the supplied SMB to BNC adapter For a and b L Connect GND of the cell model to GND of the headstage Important When using the differential headstage optional the REF connector must not be left open It must be connected to ground version 5 1 page 18 01 User Manual Simulation of electrode the bath L Set switch 4 Figure 9 to the upper position L Set switch 5 Figure 9 to GROUND position The 1 resistor simulates the resistance of the bath solution This can be used to train cancellation of offsets using the bridge balance and using the capacity compensation Simulation of SEAL formation L Set switch 4 Figure 9 to the upper position L Set switch 5 Figure 9 to SEAL position The 1 GQ resistor simulates the SEAL resistance when forming a GIGASEAL in patch clamp experiments Simulation of intracellular recording Intracellular recordings can be mimicked with one of two cells with different properties Use the 50 MQ electrode connector 1 Figure 9 for an experiment with sharp
26. ite In that case the electrode holder can be connected to the headstage via an electrode adapter see Optional accessories in chapter 3 electrode connectors use a driven shield approach for details of this approach see Ogden 1994 to minimize the effect of the connecting cables In addition all headstages are equipped with a ground connector GND The standard system 1s equipped with a headstage capable of injecting a maximal current of approximately 12 nA into a resistance of 100 MQ version 5 1 page 7 01 User Manual With this headstage the system can be used either with high resistance sharp electrodes for intracellular recordings or with low resistance suction electrodes for whole cell patch clamp recordings To cover all the needs of electrophysiological research all systems have a large variety of operation and control elements such as BUZZ cell penetration mode EL CLEAR electrode clear facility ten turn controls for BRIDGE BALANCE CAP COM capacitance compensation OFFSET and HOLD CUR holding current an automated electrode resistance test a digital DISPLAY for potential membrane current and electrode resistance a STEP GATE INPUT and linear CURRENT STIMULUS INPUT 4 2 Description of the Front Panel npi BRIDGE BALANCE 100 MQ 7 CURRENT CURRENT STEP GATE POTENTIAL HEADSTAGE STIMULUS INPUT OUTPUT INPUT OUTPUT 1 10 1 0 1 V nA TIL x10 mV
27. lay display mode selector 12 Figure 4 in position VEL is related to the BIAS current of the headstage according to Ohm s Law Cancel this voltage by tuning the headstage BIAS current potentiometer 2 Figure 4 L Accuracy Now in both modes potential current the display should read 000 Due to the limited resolution of the display unbalanced offsets and thermal drifts an offset of 001 to 002 may occur on the display This small deviation can be trimmed internally but this procedure 15 necessary only if very small currents in the pA range are recorded version 5 1 page 20 01 User Manual 8 2 Offset Compensation If an electrode is immersed into the bath solution an offset voltage will appear even if no current is passed This offset potential is the sum of various effects at the tip of the electrode filled with electrolyte tip potential junction potential etc This offset voltage must be compensated 1 set to zero carefully with the OFFSET control 4 Figure 4 before recording from a cell When adjusting the OFFSET make sure that no current flows through the electrode Thus it is recommended to disconnect CURRENT STIMULUS INPUT and to disable STEP GATE INPUT TTL and the HOLD CUR unit see chapter 4 2 If a cell model 15 connected the OFFSET control should read a value around 5 otherwise it 1 likely that the headstage or the amplifier is damaged 8 3 Capacitance Compensation High resistances of
28. lectrode in the tissue but not in a cell 8 5 Electrode Selection Electrodes must be tested before use This is done by applying positive and negative current pulses and by compensating with the BRIDGE BALANCE control Electrodes which show significant changes in resistance rectification cannot be used for intracellular recordings By increasing the current amplitude the capability of the electrode to carry current can be estimated The test current must cover the full range of currents used in the experiment Sometimes the performance of electrodes can be improved by breaking the tip or by using the BUZZ or ELECTRODE CLEAR facilities of the amplifier version 5 1 page 23 01 User Manual undercompensated potential mV 6 50 100 150 200 250 300 350 400 time ms overcom pensated potential mV 6 time ms compensated potential mV 3 a 2 1 ON d 1 zu 3 T T T T T T T 0 50 100 150 200 250 300 350 400 time ms potential Figure 12 Tuning of the BRIDGE BALANCE using 100 resistor version 5 1 page 24 01 User Manual 9 Sample Experiments In the following the basics of a simple experiment are described either using a sharp or a suction electrode It is assumed that all connections are built as described in chapter 6 Before starting remove the cell model 9 1 Sample Experiment using a Sharp Electrode Rep
29. lp you set up BA 01M correctly Always adhere to appropriate safety measures see chapter 1 Usually the BA 01M is shipped mounted in an EPMS 07 housing If a single 01 is delivered the user has to mount the module in the EPMS 07 housing This is done performing the basic installation steps Basic installation n n n After Turn off the EPMS 07 system Remove two front covers from the EPMS 07 housing Plug in the BA 01M and fasten the amplifier module with four screws The screws are important not only for mechanical stability but also for proper electrical connection to the EPMS 07 housing installation the 01 is attached to the setup by assembling the electrical connections It is assumed that a cell model will be attached The connection of the Ag AgCl pellet or the agar bridge for grounding the bath is described in chapter 9 2 Electrical connections n n n Connect the headstage to the HEADSTAGE connector 1 Figure 4 at the BA 01M Connect a cell model see chapter 7 if you want to test the system with a cell model Connect a digital analog timing unit or a stimulation device to CURRENT STIMULUS INPUT or to STEP GATE INPUT TTL if you intend to use the gated stimulus unit Connect a store oscilloscope or a data recording device 1 a computer with data acquisition card to the POTENTIAL OUTPUT and to the CURRENT MONITOR triggered from the stimulation device Before using the
30. me ms potential current current r 3 0 r 2 5 2 0 0 0 160 current nA 7 3 0 2 5 2 0 0 0 160 current nA y 3 0 2 5 1 2 0 Figure 15 Artifacts caused by the recording electrode The measurements were done using a cell model with 100 MQ membrane resistance 100 pF membrane capacitance and version 5 1 100 MQ electrode resistance A Cstray and VreL not compensated bridge not balanced Cstray compensated and Vret not compensated Cstray and compensated bridge balanced Cm membrane capacitance Cstray electrode stray capacitance Rex electrode resistance Rm membrane resistance Tcm time constant of the cell membrane VREL potential drop at Rex see also Figure 13 page 27 01 User Manual 9 2 Sample Experiment using a Suction Patch Electrode If suction electrodes are used for whole cell recordings they are usually called pipettes Thus in this subchapter pipette means suction electrode to amplifier a m i ae d p Po d fy if Fa ks e 1 A 1 1 LI p 11 Fi M i Fa p Fi 11 il l1 A ror LA ii ii m iy if fy 9 yt 4 4 I SS amp Af kt gt UU ta e oe Mee r z e le NM 7 Pa ill lt lt en al ri all R ground Figure 16 Model circuit for whole cell pat
31. n turn potentiometer symmetrical i e 0 mV 5 on the dial range 200 mV see chapter 8 2 The BRIDGE BALANCE unit consists of 7 100 10 MQ range switch and 5 BRIDGE BALANCE potentiometer 7 100 10 MQ range switch Switch to set the range of the BRIDGE BALANCE potentiometer 100 MQ position 0 MQ to 1000 10 MQ position 0 MQ to 100 5 BRIDGE BALANCE MQ potentiometer If current is passed through the recording electrode the potential deflection caused at the electrode resistance is compensated with this control ten turn potentiometer clockwise calibrated in MQ range set by switch 7 see also chapter 8 4 version 5 1 page 9 01 User Manual 6 CAP COMP capacitance compensation potentiometer clockwise range 0 30 pF see chapter 8 3 Control for compensation of the input capacitance ten turn potentiometer Caution This circuit is based on a positive feedback circuit Overcompensation leads to oscillations that may damage the cell DISPLAY CUR RANGE unit CUR RANGE mi MO The DISPLAY unit consists of 8 mV nA MO LEDs 10 display 12 VeL Ig switch and 11 CUR RANGE switch 8 mV nA LEDs LEDs which indicate the unit of the reading of the display 10 10 Display Display for the recorded potential in mV XXX mV the electrode current in XX XX nA or the electrode resistance in MQ XXX i
32. ne capacitance CELL 1 represents an neuron like cell Electrode immersed into the bath or SEAL formation can be mimicked as well The headstage of the amplifier can be connected to one of two different types of electrodes see below 7 1 Cell Model Description ELC MOD GROUND 1kQ 10MQ CELL 2 200MQ 100pF CELL 1 50MO L 22 pF 50M0 GROUND SND electronic GrnbH D 71732 Tomm Gemnany www npielectronic com Figure 9 passive cell model 1 3 connectors for the headstage 1 electrode resistance 50 MQ 3 electrode resistance 10 MQ 2 GND ground connector to be connected to GND jack of headstage 4 CELL switch for cell membrane representing a membrane of either 50 MQ and 22 pF CELL 1 or 200 MQ and 100 pF CELL 2 In GROUND upper position the electrodes are connected to ground via kQ resistor In SEAL lower position are connected to a 1 GQ resistor simulating the formation of a GIGASEAL with a patch electrode version 5 1 page 17 01 User Manual Whole cell Patch sharp Microelectrode Driven Shield BNC connector oO A cqnwcoenrq mcn_ BNC connector 10 Re 50 MQ Q CELL 2 CELL 1 GROUND 2 SEAL m lt Cr Cm gt Rm 1 1GQ 200 MQ 100 pF 22 pF 50 GND Figure 10 Schematic diagram of the passive cell model 7 2 Connections and Operation It is assumed that all connections are built as described in chapter 6 Check
33. nector monitoring the stimulating current passed through the electrode resistance 250 range approximately 12 nA in current range xl and approximately 120 nA in current range x10 version 5 1 page 13 01 User Manual STEP SIZE mA 000 M The gated stimulus unit consists of 3 0 switch STEP SIZE switch 20 STEP GATE INPUT TTL connector and 21 STEP SIZE nA digital potentiometer STEP GATE INPUT 3 0 STEP SIZE switch 0 toggle switch to disable the gated stimulus set by STEP SIZE 21 and gated by STEP GATE INPUT TTL 20 or to select the polarity of the gated stimulus 4 gated stimulus positive 0 gated stimulus disabled gated stimulus negative 20 STEP GATE INPUT TTL connector With this input a current step gated stimulus can be generated set by the digital potentiometer STEP SIZE 21 and the polarity switch 3 This current step is gated by a positive digital pulse 3 15 V applied to the BNC connector The duration of the current step is set by the duration of gating signal The amplitude of the current step is set by STEP SIZE 21 Two examples for current range x1 are given in Figure 7 In A STEP SIZE 21 is set to 100 i e 1 nA in B STEP SIZE 21 is set to 200 i e 2 nA STEP GATE INPUT gt 3 V current stimulus A d U Loy U L 2 5V B JU U UL PU UI Figure 7 Input output relation using STEP GATE INPUT TTL 21 STEP S
34. rrent is not adjusted Solution 1 Adjust the BIAS current according the procedure described in chapter 8 1 Problem 3 The system oscillates Possible reason 1 The capacitance of the electrode is overcompensated Solution 1 Turn the CAP COMP potentiometer 5 Figure 4 to the most left position and compensate the input capacitance again Problem 4 With the cell model connected display does not show the correct value within a tolerance of 2 Possible reason 1 The capacitance of the electrode is not compensated using the 100 MQ electrode 2 The headstage has an error Solution 1 Turn the CAPACITY COMPENSATION potentiometer 5 Figure 4 to the most left position and compensate the input capacitance again 2 Contact version 5 1 page 30 01 User Manual 11 Literature 1 Ogden DC 1994 Microelectrode Techniques The Plymouth Workshop Handbook Second Edition The Company of Biologists Limited Cambridge I Polder H R M Weskamp Linz amp Meyer 2004 Voltage Clamp and Patch Clamp Techniques Chapter 3 4 272 323 in Dhein Stefan Mohr Friedrich Wilhelm Delmar Mario Eds Practical Methods in Cardiovascular Research Springer Berlin Heidelberg and New York 2004 1 Windhorst U and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin Heidelberg New York L1 Behrend O Branoner F Zhivkov Z amp Ziehm U 2006 Neural responses
35. s from the snail Lymnaea stagnalis Biol Cybern 82 1 15 Prinz A A amp Fromherz P 2003 Effect of neuritic cables on conductance estimates for remote electrical synapses J Neurophysiol 89 2215 2224 J Purves 1981 Microelectrode Methods for Intracellular Recording and Ionophoresis London Academic Press _J Rotte C Krach C Balfanz S Baumann A Walz B amp Blenau W 2009 Molecular characterization and localization of the first tyramine receptor of the American cockroach Periplaneta americana Neuroscience 162 1120 1133 Schaette R Gollisch T amp Herz V M 2005 Spike Train Variability of Auditory Neurons in vivo Dynamic Responses Follow Predictions from Constant Stimuli Journal of Neurophysiology 93 3270 3281 Schnell B Joesch M Forstner F Raghu S V Otsuna H Ito K Borst A amp Reiff D F 2010 Processing of horizontal optic flow in three visual interneurons of the Drosophila brain J Neurophysiol 103 1646 1657 1 Schoen I amp Fromherz P 2007 The Mechanism of Extracellular Stimulation of Nerve Cells on an Electrolyte Oxide Semiconductor Capacitor Biophys J 92 1096 1111 L Vogel A Hennig M amp Ronacher 2005 Increase of neuronal response variability at higher processing levels as revealed by simultaneous recordings Journal of Neurophysiology 93 3548 3559 _I Volgushev M Malyshev A Balaban P Chistiako
36. s of 9 HOLD CUR nA potentiometer and 14 0 switch 9 HOLD CUR nA holding current potentiometer With this control a constant current holding current can be generated ten turn potentiometer clockwise calibrated in nA Imax 9 99 nA current range x1 or 99 9 nA current range x10 The polarity of this holding current is set by toggle switch 14 14 0 holding current switch Switch to disable holding current generation or to set the polarity of the holding current current positive 0 holding current disabled current negative BUZZ EL CLEAR buzz electrode clear unit The BUZZ EL CLEAR unit consists of 13 BUZZ DURATION potentiometer 15 BUZZ OFF REMOTE switch 16 REMOTE connector and 17 EL CLEAR switch BUZZ REMOTE version 5 1 page 11 01 User Manual 13 BUZZ DURATION potentiometer Control to set the duration of the BUZZ potentiometer clockwise linear range 3 ms to 110 ms The duration is dependent on the setting of CAP COMP 5 It 1s effective in both modes REMOTE and BUZZ see also 15 15 BUZZ OFF REMOTE switch Toggle switch to set the mode of BUZZ operation BUZZ Switching to BUZZ activates the BUZZ circuit duration set by 13 To facilitate the penetration of the cell membrane the BUZZ circuit is provided which is based on oscillations caused by overcompensating the capacitance compensation system The overcompensation of c
37. struments Signs and conventions In this manual all elements of the front panel are written in capital letters as they appear on the front panel System components that are shipped in the standard configuration are marked with V optional components with In some chapters the user is guided step by step through certain procedure These steps are marked with Important information and special precautions are highlighted in gray Abbreviations Cm cell membrane capacitance Cstray electrode stray capacitance GND ground Imax maximal current Rm cell membrane resistance Ret electrode resistance Tcm time constant of the cell membrane potential drop at REx version 5 1 page 3 01 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 operated only by trained staff General safety regulations for operating electr
38. t is important that this tuning procedure is performed ONLY after a warm up period of at least 30 minutes The BA 01M system is equipped with a voltage to current converter with a very high output impedance which is connected to the recording electrode The zero current of this unit is tuned with the BIAS current control see Figure 4 in chapter 4 2 The tuning procedure must be performed regularly at least once a month since the bias current changes over time If very small currents are used in the 10 pA range the procedure must be repeated in shorter intervals The tuning procedure is performed using high value resistors and or a cell model It cannot be performed with an electrode since there are always unknown potentials involved tip potential junction potentials I Disconnected all input signals except the headstage Put the holding current switch to position 0 0 switch 14 Figure 4 Connect the connector of headstage to ground Note This cannot be done with the cell model Please use a wire to connect the input of the BNC connector on the headstage to GND of the headstage Do not use the shield of the BNC connector since it is connected to driven shield Tune the OFFSET to zero using the OFFSET control 4 Figure 4 see also chapter 8 2 _J Remove wire and attach cell model a resistor with value of about 5 across the same connection The value shown at the disp
39. that the electrode is very close to the cell membrane the electrode resistance increases the bridge balance appears undercompensated extracellular action potentials APs are recorded the acoustic monitor signal changes L Set the DURATION potentiometer to one fourth and apply BUZZ to the electrode If you are lucky the tip of the electrode is now inside cell L If necessary readjust BRIDGE BALANCE and or CAP COMP as shown in Figure 14 and Figure 15 using current stimuli that do not activate 10n channels or transporters L You read the membrane potential and can apply current pulses to the cell After penetration the voltage responses of the cell to the test pulses should reflect the cell membrane resistance and time constant L Start the experiment overcompensated compensated undercompensated mv 20 ms odo ooo LL m 1 20 ms Figure 14 Adjustment of the bridge balance after penetrating a cell version 5 1 page 26 01 User Manual potential mV 100 80 60 40 20 0 20 40 uncompensated t cm a gee REL Tem we MEL C stray 20 40 60 80 100 120 140 time ms compensated potential mV 80 4 70 4 60 50 4 40 30 potential mV 60 4 50 40 4 30 4 20 20 40 60 80 100 120 140 time ms Cstray and compensated 20 40 60 80 100 120 140 ti
40. to water surface waves in the midbrain of the aquatic predator Xenopus laevis laevis Eur J Neurosci 23 729 744 _I Blomeley C P Kehoe A amp Bracci E 2009 Substance P Mediates Excitatory Interactions between Striatal Projection Neurons Journal of Neuroscience 29 4953 4963 LJ Burrell B D amp Sahley C L 2004 Multiple forms of long term potentiation and long term depression converge on a single interneuron in the leech CNS J Neurosci 24 4011 4019 _I Burrell B D amp Sahley C L 2005 Serotonin Mediates Learning Induced Potentiation Of Excitability Journal of Neurophysiology 94 4002 4010 LJ Drion G Bonjean M Waroux O Scuvee Moreau J Liegeois J F Sejnowski T J Sepulchre R amp Seutin V 2010 M type channels selectively control bursting in rat dopaminergic neurons Eur J Neurosci 31 827 835 L Gollisch T Schutze H Benda J amp Herz A V 2002 Energy integration describes sound intensity coding in an insect auditory system J Neurosci 22 10434 10448 1 Grey B amp Burrell B D 2010 Co induction of LTP and LTD and its regulation by protein kinases and phosphatases J Neurophysiol 103 2737 2746 L Hutzler M Lambacher A Eversmann B Jenkner M Thewes R amp Fromherz P 2006 High resolution multi transistor array recording of electrical field potentials in cultured brain slices J Neurophysiol Kettenmann H amp Gr
41. va M Volgushev S amp Wolf 2008 Onset dynamics of action potentials in rat neocortical neurons and identified snail neurons quantification of the difference PLoS ONE e1962 L Zeck G and P Fromherz 2001 Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip PNAS 9 18 10457 10462 version 5 1 page 32 01 User Manual 12 Technical Data 01 Input voltage range 1000 mV Operating voltage 15 V Enclosure Size 23 x 70 x 26 mm grounded Holding bar Size length 150 mm 8 mm Electrode connector BNC with driven shield Ground connector 2 4 mm connector REF connector optional SMB connector Input resistance 10P Q internally adjustable Current range x1 12 nA into 1 GO Current range x10 120 nA into 100 MQ Electrode parameter controls BIAS range 200 pA current adjustable with trim potentiometer OFFSET range 200 mV ten turn control capacitance compensation range 0 30 pF ten turn control Cell penetration overcompensation of capacitance compensation BUZZ duration 3 110 ms dependent on the setting of capacitance compensation timer controlled with linear control accessible with remote switch Electrode clear circuit Application of max DC currents Imax Or Imax Bridge balance Adjustable with ten turn control 0 1000 MQ Electrode resistance test 10 mV M
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