Home

TEC-03X - NPI Electronic Instruments

1. version 4 8 page 54 TURBO TEC 03X User Manual 12 3 Speed of Response and Linearity of the Capacitive Transients For the investigation of voltage activated channels with voltage clamp instruments some special techniques for eliminating the capacitive and leak currents have been introduced such as the P 4 or more general P N protocol see Rudy and Iverson 1992 for overview For these protocols the speed and linearity of response of the clamp system is of great importance As outlined in chapter 12 1 the TEC systems are designed following a control theory procedure called modulus hugging see Froehr 1985 Polder 1984 Polder and Swandulla 1990 Polder and Swandulla 2001 The procedure requires a PI proportional integral controller This procedure is applicable to control systems composed of an element with one large time constant Tm and many small time constants Ti These small time constants can be added to an equivalent time constant Te In case of the TEC control chain the large time constant is formed by the time constant of the cell membrane several hundred of milliseconds and the sum of small time constants resulting from the microelectrodes and the electronics a few ten microseconds Note Here only the proportional part of the PI controller is considered Possible improvement of clamp performance due to series resistance compensation see Ogden 1994 Smith et al 1990 Greeff 2000 Greeff and
2. version 4 8 page 70 TURBO TEC 03X User Manual Speed of response 1 settling time potential output signals after application of square pulses of 1V with 1 MQ electrode resistance Potential electrode lt 10 us Current electrode 50 us VOLTAGE CLAMP Input sensitivity 10 mV Input resistance 2100 KQ HOLD XXX mV ten turn digital control with 0 switch maximum 1000 mV RISE TIME LIMIT 0 0 2 ms GAIN 10 n A V 10000 Lu A V ten turn linear control INTEGRATOR TIME CONSTANT 200 us 2 ms ten turn control OUTPUT CURRENT LIMIT 0 100 linear control NOISE filters set to 10 kHz other settings see below Potential output 100 uV pp Current output 10 nA pp at 10 KHz 2 nA at 500 Hz SPEED of RESPONSE VC Mode 1 settling time 80 us for 10 mV step and 100 us for 100 mV step applied to cell model Rz 1 MQ Rm 100 KQ Cm 0 1 uF standard headstage POWER REQUIREMENTS 115 230 V AC 60 W 1 25 0 63 A fuse SLOW DIMENSIONS 19 rackmount cabinet 19 483 mm wide 14 355 mm deep 5 25 132 5 mm high WEIGHT 8 kg ACCESSORIES PROVIDED 4 Potential headstage standard current headstage other headstages may be substituted on request with order Cable set and connectors for reference current electrode and ground connectors Power cable User manual SSS OPTIONAL ACCESSORIES ordered at additional cost TEC EH Electrode holder and adapter two holders wi
3. The TURBO TEC 03X system can be operated with various software packages It provides special features such as electronic remote selection of modes of operation and monitor telegraph signals for the setting of current gain and filters If the CellWorks software is used some of the functions of the TURBO TEC 03X can be controlled directly by the computer Since the voltage and current clamp techniques are standard techniques of electrophysiology for reviews see Methods in Enzymology Vol 207 Smith et al 1985 Standen et al 1987 Kettenmann and Grantyn 1992 Ogden 1994 only a short procedural description follows This description is based on the diagrams of Figure 1 and Figure 2 Terms and abbreviations in capital letters in the text correspond with the labels at the front panel p s Cel Pel gt ACTIVE POTENTIAL QUTPUT Figure 1 equivalent circuit of TEC amplifiers Potential Measurement For membrane potential measurement all TEC amplifiers use a differential electrode arrangement to record the membrane potential as accurately as possible see Figure 1 i e the membrane potential Vm is measured according to Vm PreL Prer with PreL potential at the potential electrode and Prer potential at the reference electrode A description of the potential headstage is given in chapter 5 1 In order to determine whether both electrodes are inserted into the same cell the potential of the current injecting
4. You can switch the logical level i e high or low of every individual channel by clicking Logic Level In Figure 17 all channels are high You can prove this by connecting a digital multimeter or an oscilloscope to the digital outputs of the breakout box Note Every sub panel shows on the right side of the window a field named Last Error and a dot named Fatal Error If the dot is red an error had occurred during the last test and a number appears in the field Last Error Click the button Error Codes to open a window with explanations about this error code version 4 8 page 44 TURBO TEC 03X User Manual Test without a Breakout Box _ Disconnect the NI board from all peripherals This is important since otherwise the peripherals may interfere with the tests L Start MAX there should be an icon of MAX on the Windows desktop L Click to Devices and Interfaces and select the board you want to test L Launch the Test Panel The Test Panel consists of four sub panels where analog in and outputs counter I O and digital I O ports can be tested see Figure 16 _ The test panel in Analog Input should show some noise _ Go to all of the sub panels and perform as many tests as possible Watch the status dots that prove the function of the respective task Note Every sub panel shows on the right side of the window a field named Last Error and a dot named Fatal Error If the do
5. 25 to 0 Set the MODE OF OPERATION 28 to CC Set the display to POTENTIAL ELECTRODE using switch 7 Turn POWER switch on O O L Now the TEC 03X is ready for an initial check with the cell model 7 Passive Cell Model The TEC 03X can be ordered with a passive TEC Two Electrode Clamp amplifier cell model as an optional accessory An active cell model is also available on request for reference see Draguhn et al 1997 The passive cell model is designed for use with TEC 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 in using the instrument or 3 in case of trouble see also chapter 11 to check which part of the setup does not work correctly e g to find out whether the amplifier or headstages are damaged or something is 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 a capacity simulating the capacity of the cell membrane see Figure 12 and Figure 13 A switch allows simulation of two different membrane resistances 10 kQ and 100 kQ A second switch permits grounding the current electrode see also chapter 8 1 version 4 8 page 30 TURBO TEC 03X User Manual 7 1 Cell Model Description BNC jack SUBCLICK connector Pa
6. D Electronic Instruments for the Life Sciences ease wade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION FOR THE TURBO TEC 03X TWO ELECTRODE CLAMP SYSTEM VERSION 4 8 npi 2013 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 TURBO TEC 03X User Manual Table of Contents INbout this Manual s s eos fere teorie A apelon scutes fto tale eaae Ri wes hn Ses EE 4 ls Safety Beoulatons ooi ti mea e aut ns tex us inte iul tp edisus n eps uidd 6 2 nicsinerv ter cc P D I MR 7 3 TURBO TEC 03X GOompolellts s eoa ISI I PRU es d PR I an teda eni ec 8 4 TURBO TEC 03X System eie nivei rente ease a Ve aee DN aUe Ha ee YR SY S PR Rosa URL AEE ERSE 8 Ale System DescppiloDia ceste te Poesie eve Pret NISL dev etes o labeo eit i Goes leis Doerdes urbis toU 8 Potential Measurement noie ete Peste qe dd tp a PR Ea Ur ERA ede capa ced 9 Current Injection and Measurement ioecc eese eerte tonto ene ah no Sendo a enne Rae ine aae Re eph 10 Current C lamp Mode Ge ree o devictus ui Aq Hie M HE 10 Voltage Clamp Mode 0 mr eevaditedtden rubis d edv at med ead Snow Sgn Mae 10 Control Circuit nV Eoo boot Paces ds ptu aue orsa edente de mt ect ee i MEE 11 Control Modes Of V Castes Pete Senda edad fasti tak amet nacre 11 Improvement of the Control Properties essere enne enne 12 4 2 Description of the
7. Umax Cn REL dUm dt max 150 V 0 1 uF 1 MQ 1500 V s 1 5 mV us It would last 100 us to reach 150 mV provided that the clamp has an ideal characteristic Now the minimum bandwidth of a real clamp system necessary for ideal behavior can be calculated Ts 8 4 Te 100 us gives Te 12 us BW 1 2 n Te 13 kHz with BW bandwidth If we assume that Te is determined by 70 80 by the time constant of the current electrode i e Tea 10 us if Te 12 us it is clear that with electrode resistances in the range of 500 KQ the total capacity related to the current injecting electrode can be maximum 20 pF In this case the maximum cable length is 15 20 cm A cable of 0 5 1 5 m has a capacity in the range of 50 200 pF With such a capacity and an electrode resistance of 1 MQ Te is in the range of 50 200 us and the speed of response would be in a range of 0 5 2 ms Conclusions 1 For adequate VC experiments a clamp gain of 1 5 mA V i e 100 500 internal gain with a current source calibration of 10 uA V is necessary Therefore with pulse amplitudes of 100 200 mV the operational amplifiers in the gain stages will be saturated causing nonlinear components in the capacitive transients 2 The maximum speed of response is determined by the cell capacity the maximum available current and the command amplitude 3 The real speed of response is determined by the time constant associated with the
8. Wischmeyer E Hirsch J R Preisig M ller R Rajan S Engel H Grzeschik K H Daut J and Karschin A 2001 Genetic and functional linkage of Kir5 1 and Kir2 1 channel subunits FEBS Letters 491 305 311 version 4 8 page 60 TURBO TEC 03X User Manual L Ferber M Sporning A Jeserich G DeLaCruz R Watkins M Olivera B M amp Terlau H 2003 A novel conus peptide ligand for K channels J Biol Chem 278 2177 2183 LI Fleischmann B K Duan Y Fan Y Schoneberg T Ehlich A Lenka N Viatchenko Karpinski S Pott L Hescheler J amp Fakler B 2004 Differential subunit composition of the G protein activated inward rectifier potassium channel during cardiac development J Clin Invest 114 994 1001 J Gomez Varela D de la P P Garcia J Giraldez T amp Barros F 2002 Influence of amino terminal structures on kinetic transitions between several closed and open states in human erg K channels J Membr Biol 187 117 133 L Gomez Varela D Barros F Viloria C G Giraldez T Manso D G Dupuy S G Miranda P amp de la P P 2003 Relevance of the proximal domain in the amino terminus of HERG channels for regulation by a phospholipase C coupled hormone receptor FEBS Lett 535 125 130 LI Karschin C Wischmeyer E Preisig M ller R Rajan S Derst C Grzeschik K H Daut J and Karschin A 2001 Expression Pattern in Brain of
9. amp Heinemann S H 2002 Conformational switch between slow and fast gating modes allosteric regulation of voltage sensor mobility in the EAG K channel Neuron 35 935 949 LI Schwake M Athanasiadu D Beimgraben C Blanz J Beck C Jentsch T J Saftig P amp Friedrich T 2006 Structural determinants of M type KCNQ Kv7 K channel assembly J Neurosci 26 3757 3766 L Seebohm G Lerche C Busch A E and A Bachmann 2001 Dependence of 7 Ks biophysical properties on the expression system Pfl gers Arch 2001 L Seebohm G Scherer C R Busch A E and C Lerche 2001 Identification of Specific Pore Residues Mediating KCNQI Inactivation A Novel Mechanism For Long Qt Syndrome J Biol Chem 276 17 13600 13605 LI Seebohm G Lerche C Pusch M Steinmeyer K Br ggemann A and A E Busch 2001 A kinetic study on the stereospecific inhibition of KCNQI and IKs by the chromanol 293B Br J Pharmacol 134 1647 1654 Li Seebohm G Westenskow P Lang F amp Sanguinetti M C 2005 Mutation of colocalized residues of the pore helix and transmembrane segments S5 and S6 disrupt deactivation and modify inactivation of KCNQI K channels J Physiol 563 2 359 368 LI Seebohm G Strutz Seebohm N Ureche O N Baltaev R Lampert A Kornichuk G Kamiya K Wuttke T V Lerche H Sanguinetti M C amp Lang F 2006 Differential Roles of S6 Domain Hinges in the Gating of
10. 2acacpcehiet onatsdassvaederesstacts 28 current headstage 5 cese de tess 26 ODWODS oda GS eae ese eae 28 potential headstage 25 potential headstage elements 25 J junction potential esses 35 L Linear optimiutm eie e eiie tein 32 linearity of response esse 55 O mietllod 5 essct ann Ena 22 M MAX NI test panel epe 42 Maximum speed of response 57 modulus hugging eeeeeeeee 50 N National Instruments NI 42 O Offset Compensation sess 35 operation modes seeseeeeee 10 WOS CUTTS see tie Oe se Uu coc 37 osa 5 see ETE M 50 UNN Sod ue acto usted Eu 38 52 Optional accessories caeso lon ette 8 OPON NL telat eerie 8 OUtput CUIEnt ais es rofa etri e Peres 51 P PI Gonttollers icono ect t PD 50 Potential Registration 9 proportional gain 56 R tear panel 166 sica te eal ate 21 rear panel items TEC 03X CW 23 rear panel View deed o eee iodias 21 Relerenees 2 sco roc iece esa haces 58 S Safety Regulations eese 6 sample experiment seeessee 47 series resistance compensation 12 51 SO miethod sisareni ints 52 SDOEHICaETOTISus e r a nT 69 speed of response ree i ete intenta 55 version 4 8 TURBO TEC 03X User Manual Symmetrical op
11. 8 page 43 TURBO TEC 03X User Manual L Leave AiO at the breakout box open and watch the panel in MAX You should see some noise at the input _ Connect an external waveform to AiO of the breakout box and to an oscilloscope The voltage range of the waveform should not exceed 10 V Select Channel 0 and you should see the external waveform on the screen The waveform on the screen and the oscilloscope should be identical L Perform this test with all other analog inputs L Go to Analog Output and connect an oscilloscope to DACO of the breakout box As Output Mode select Sine Generator and as Channel Selection select 0 Start the sine generator and watch the output at the oscilloscope L Test analog out channel 1 DACI in the same way L Go to Counter I O and keep the default values Start the test with GPCTRO The green button in the field Counter Status indicates that counter 0 works well L Test counter 1 accordingly Test Panel NATIONAL Device Name Device Number INSTRUMENTS PCI 6014 1 Analog Input Analog Output Counter 1 0 Digital 1 0 Line Direction Selection Input C Dutput e e Line Number 7 6 Last Error o Fatal Error Error Codes Logic Level V iv Line State 3 3 9 9 e 9 9 9 Figure 17 testing digital I O ports using MAX L Launch Digital I O and activate all outputs by clicking Output for every channel
12. Electrode 552 d 62 Ge peak Hp eod fuisae ea SEEDS 36 C rrent Electroden ME T TET Nr eae a Oe ate y en at 36 8 4 Capacity Compensation senn N a sido tor i Dude ues ns REC e eniin 36 8 5 Testing Operation Modes iei te eh Seen n NEN SXR CHASE SNR TNR REUS SERT R UAR e YR Eee eR posa 37 tigris S Erde 37 ADICCISC ST eroaisi 37 8 0 Lunimg the VC modens trani gt dete dicia Cines fts eu tcs futs 38 General ConsideratlOis 252 csse oxove s ee nade ee iae DRIN QU Mud E suec ues E E eode dde Aiea 38 Tuning Proce Quire eoe p nen ee ache ce bedienen eae 40 8 7 Installing and Testing a Data Acquisition Board from National Instruments 42 Basic Test of INT 20X and Data Acquisition board sese 42 Test usinga Breakout BOX Scii an a eei ai itii dv ite ato nE arr 43 Test svithout a Breakout BOX ettet n cede tvesseae ue ac sorta eS UR tede Ere redd 45 9 Positioning of BIGCEEOGGS secco ras torio Rete up TRUE qp EA Ke RM acp A iS oen INS E 46 TU Sample Experiment 5 sc sete ENVIAR IY IAS MESS SQNURE STRE E EET AENEAN ABER RRRUE UTERE RR i v osa 47 TT Trouble SHDOUITS io Sic Splice coa do eR dieu eR din E Re testa dies Nd ebur A Ue 49 version 4 8 page 2 TURBO TEC 03X User Manual 12 PR DDOBGDU S anoo cam uela usse ut NS adepti jie stent utente tae uc ot UE UM tame D M a 50 12 1 Theory of Operation e etr tea eie ee er nea A EEE ME TERN HR resa ASi 50 12 2
13. Front Panel eset aee intendo tok rabie ee desdaaaasavees 13 4 3 Description of the Rear Panel aue oeee Ee a e Po es t n hase USED DO OM gecu e sas eeeaes 21 nnum 22 PECO A WY RR EU 23 Dar JIC AUS ADCS PRETEND ERE 25 Sho Potential TIC ACS a OCF sooo siete daas vbsRastumxetedufgat bui tu E E EEG 25 Headstase Elements cost eibi maa duutd ip dena ista na duse 25 5 2 Current Headstage ai td et ERROR OO va IER Dp dei doe ia voces Pacto pots 26 ODUOGHS xis co E aus ib anto ARX ERA NO xe Sasa cum uci reU S cop b Ful E om nO 28 6 Setting up the TEC O3 X ie erit HERE RNERSA NUS eR IER US Sa ea Pe Eee ERUNT NN ME Ree a aa eigai 29 T Passive Cell Model irentsia nga buda dude dis ion ate cales sanat edes 30 Ris SCell Model Descriptions naa Uia tot a E oH Fre t L peer md a 31 22 COMMeCHONS and Operatloi ymse oree sega URN ea eects E A dat Cem dio 32 Checking the Configuration with the Cell Model eee 32 p Testand TUNNE Procedures itani E tutta an faa E E ADEL LEE 33 8 1 Current Headstage Bias Current Adjustment essere 33 8 2 Offset Compensation esee rry PRAEEST AR US SENTIS AREE NR SR eS o A PR RUSSE e DIES 35 Potential Blectrode s 5 ed oed do aic i stt iso tust ain edi esses du a altes eosi 35 Current Electrode iaeninie ii e pre ecl deli A E a ESAS ive ese eee 35 5 9 Electrode Resistance DeSUe asoseesdcodee tva eiue odetued ee edi eatin A ORA Ca ed me SES 35 Potential
14. IMQ REF Ground REF GND jack TEC CELL MODEL npi CELL membrane switch Switch to ground C SUBCLICK connector Ca Figure 12 TEC passive cell model PeL BNC connector for the potential electrode resistance 1 MQ REF SUBCLICK subclick SMB connector for the reference electrode GND ground connector Rw switch for the cell membrane representing a membrane resistance of either 10 KQ or 100 KQ Cw cell membrane capacity always 100 nF ON GND switch to ground the current electrode ON Cex inside the cell GND CeL connected to ground see also chapter 8 1 CeL SUBCLICK SMC connector for the current electrode resistance 1 MQ version 4 8 page 31 TURBO TEC 03X User Manual Potential Electrode Current Electrode ON GND Rpel 1MQ 1M Reel 1 ko vvv O REF Ue Roath Figure 13 schematic diagram of the TEC passive cell model Connections and Operation Checking the Configuration with the Cell Model O O O O Make the basic settings see chapter 6 _ Connect the Pet BNC jack of the cell model to the BNC connector at the potential Connect the SUBCLICK connector REF to the REF connector at the potential headstage Connect the SUBCLICK connector Cex to the plug at the current headstage Switch the CELL membrane switch see Figure 12 to the desired position Set the GND switch see Figure 12 to ON Turn POWER switch of the amplifier on Now you can adjust the amplifier and app
15. PEL sensitivity x10 mV POTENTIAL OUTPUT 35 GROUND connector Banana jack providing the internal GROUND not connected to PROTECTIVE EARTH version 4 8 page 20 TURBO TEC 03X User Manual 36 AUDIO MONITOR potentiometer E Volume control for the AUDIO MONITOR The potential at the electrode selected by switch 7 is monitored by a sound The pitch of sound is related to the value of the potential 4 3 Description of the Rear Panel cel ABS ere 6 S Figure 5 TURBO TEC 03X rear panel view the numbers are related to those in the text below 1 FUSE holder Holder for the line fuse and line voltage selector For changing the fuse or selecting line voltage open the flap using a screw driver The fuse is located below the voltage selector Pull out the holder indicated by an arrow in order to change the fuse For selecting the line voltage rotate the selector drum until the proper voltage appears in the front 2 Mains connector Plug socket for the mains power plug Important Check line voltage before connecting the TEC amplifier to power Always use a three wire line cord and a mains power plug with a protection contact connected to ground Disconnect mains power plug when replacing the fuse or changing line voltage Replace fuse only by appropriate specified type Before opening the cabinet unplug the instrument 3 PROTECTIVE EARTH connector Banana plug provi
16. RESISTANCE COMPENSATION is done by positive feedback in the control circuit its use can lead very quickly to stability problems Therefore the clamp speed should be improved first through conventional methods The optimal positioning of the electrodes especially of the current electrode is important for best SERIES RESISTANCE COMPENSATION see also chapter 9 By placing the electrode in the center of the oocyte the membrane capacity is charged homogeneously The capacitive current transient is mono exponential and the amplifier can be tuned without ringing around the slow tail of the transient Greeff 2000 L Before the experiment make sure that the electrodes are in optimal position see Figure 19 If you have a micromanipulator that can remember positions you can first position the electrodes without the oocyte Then the position is saved and the electrodes are drawn back the oocyte is placed and the electrodes are brought back into the saved position Set the voltage clamp control mode switch to FAST Apply adequate test pulses without filtering L Tune the amount of SERIES RESISTANCE COMPENSATION 4 Figure 3 while watching the current output The capacitive transient of the current should show a mono exponential decay Overcompensation is indicated by a ringing after the first peak of the capacitive transient see also Figure 18 in chapter 9 left side This is also a sign that electrode position is not optimal
17. SHUTOFF unit with a moderate THRESHOLD DISABLED RESET switch 2 in middle position OSCILLATION SHUTOFF LED green THRESHOLD potentiometer set to a low value but not to the most left position Set the amplifier with the MODE OF OPERATION switch 28 to VC mode The upper display should show the holding potential of 50 mV and the lower display the holding current of 5 uA according to Ohm s law It is very likely that the display shows a holding potential of slightly less than 50 mV because the controller is in NORMAL GAIN ONLY mode see also Appendix chapter 12 and the GAIN is low Increasing GAIN and setting the controller to SLOW Integrator mode will enhance the control loop and therefore increase accuracy LL Hint If the system oscillates as soon as you switch to VC mode switch back to CC mode and check the settings GAIN too high CAPACITY COMPENSATION not 0 THRESHOLD potentiometer of the OSCILLATION SHUTOFF unit at the most left position Control mode switch not to NORMAL GAIN ONLY or SLOW Integrator Ld Apply a test pulse of 20 mV to the cell model by giving a voltage step of 0 2 V to COMMAND INPUT 31 The length of the test pulse should be at least 30 ms L You should see a potential step of 200 mV amplitude at Pex 33 Note If you expect the POTENTIAL display to show the value of the potential step in this case 20 mV amplitude i e 30 mV remember that the display is rather sluggish and may not
18. TEC 03X User Manual POTENTIAL ELECTRODE tests the RESISTANCE by applying current pulses of 10 nA to the respective electrode In off position the ELECTRODE RESISTANCE test is disabled HOLD unit The HOLD unit consists of 12 HOLD current potentiometer and 25 0 switch 12 HOLD current potential potentiometer 10 turn digital control that presets a continuous command signal HOLD potential XXX mV maximum 999 mV for VC or HOLD current X XX uA maximum 9 99 uA for CC Polarity is set by switch 25 25 0 switch Toggle switch to set the polarity of HOLD current potential or to disable HOLD current potential positive HOLD current potential 0 HOLD current potential turned off negative HOLD current potential CURRENT OUTPUT conditioning unit The CURRENT OUTPUT conditioning unit consists of 14 CURRENT FILTER Hz 15 V uA CURRENT OUTPUT SENSITIVITY 21 4 1V 47V CURRENT OUTPUT SENSITIVITY MONITOR connector and 23 1V 47V FREQUENCY MONITOR connector 14 CURRENT FILTER Hz switch 16 position switch to set the corner frequency of the Bessel filter The setting is monitored by 23 15 V uA CURRENT OUTPUT SENSITIVITY switch 7 position switch to set the CURRENT OUTPUT gain The setting is monitored by 21 21 1V 47V CURRENT OUTPUT SENSITIVITY MONITOR connector BNC output connector monitoring the setting of 15 V uA CURRENT OUTPUT SENSITIVITY switch Reso
19. The optimal gain for a VC experiment is in between these two values The overshoot can be reduced by low pass filtering of the command pulse The speed of response of the clamp in case of the modulus optimum can be calculated as T 4 7 Te Ts 8 4 Te with T time until the membrane potential reaches for the first time 100 of the command pulse Ts time to reach steady state within a tolerance of 2 Ts is roughly the duration of the capacitive transient For a system with dampened overshoot T approaches Ts From these formulas it is clear that the performance of the clamp is determined by Te Te is determined by the time constant of the current injecting electrode i e by the electrode resistance stray capacities cable capacities etc Shielded cables have capacities of 60 110 pF m connectors and pipette holders add a few picofarads The potential electrode is equipped with a driven shield and a capacity compensation circuit Therefore this time constant is always much smaller than the time constant associated with the current electrode The time constants of the operational amplifiers are small and can be neglected Example A cable of 10 cm has a capacity of approximately 10 pF with the stray capacities in the headstage and an electrode resistance of 1 MQ cell model This gives a time constant of 10 30 us corner frequencies of 5 15 kHz With Cm 100 nF and Te 20 us 8 kHz bandwidth the gain can be calculated
20. Tuning Procedures for VC Controllers de otesi ote eoe ir e Wed Reina ees d ere hea peveluded 52 Practical Implications cn terio e ER tO ERES Se ARE STAR CY cor Rad es SNR EN E ue gea ER dd 52 12 3 Speed of Response and Linearity of the Capacitive Transients sssse 55 13 I EE E E E E E E 58 14 TURBO TEC 03X Specifications Technical Data eene 69 NOR deh 72 version 4 8 page 3 TURBO TEC 03X User Manual About this Manual This manual should help to setup and use the TURBO TEC 03X system correctly and to perform reliable experiments The manual is divided into 13 chapters Chapter 1 contains important information about safety regulations Chapter 2 gives a brief introduction to the amplifier Chapter 3 lists the components and optional accessories of the TEC 03X In chapter 4 a general description of the TEC 03X system is given and the control elements of front and rear panel are explained Chapter 5 is about the headstages and in chapter 6 the basic connections are described Chapter 7 deals with the passive cell model and its connection to the amplifier Several test and tuning procedures are outlined in chapter 8 including hints for optimizing the clamp Chapter 9 shows how to optimize the clamp properties by correct electrode positioning and chapter 10 describes a basic voltage clamp experiment Chapter 11
21. V range see Figure 15 version 4 8 page 42 TURBO TEC 03X User Manual Configuring Device 1 PCI 6014 System Al 40 Accessory OPC Remote Access Polarity Range Select the default analog input 10 0V 10 0V settings for the device Mode Nonreferenced Single Ended v Cancel Figure 15 standard configuration of E series and B series boards Test using a Breakout Box L Connect the cable from the NI board directly to the breakout box If you use a PCI 1200 connect the NI board without the CellWorks trigger dongle This is important since otherwise the trigger dongle will interfere with the tests L Start MAX there should be an icon of MAX on the Windows desktop L Click to Devices and Interfaces in the left panel and select the board that you want to test L Launch the Test Panel The Test Panel consists of four sub panels where analog in and outputs counter I O and digital I O ports can be tested see Figure 16 First the sub panel Analog Input appears Test Panel NATIONAL Device Name Device Number INSTRUMENTS P6074 1 Analog Input Analog Dutput Counter 1 0 Digital 1 0 Channel D zi 10 004 Input Limits High 10 0000 n Last Error Low 10 0000 0 Fatal Eror Error Codes Data Mode Strip Chart Y Scale Mode Average Reading C One Shot C Auto Scale 0 999969 C Continuous Ful Range Figure 16 Test Panel of MAX version 4
22. a cell model It cannot be performed with an electrode since there are always unknown potentials involved tip potential junction potentials etc Warning High voltage Always turn power off when working directly on the current headstage output see Chapter 1 L Put the holding current switch to position 0 0 switch 25 If you use a cell model only the Ce and GND connectors must be connected L Set the MODE OF OPERATION switch to OFF Important The tuning procedure must not be done in VC mode L Connect the CURR EL connector of the current headstage to ground If parasitic oscillations occur use a 10 kQ resistor for grounding If you use a cell model set the ON GND switch to GND LI Switch the upper digital display 10 to CURRENT ELECTRODE potential output of the current electrode using the electrode selector POTENTIAL 7 Set the reading of the display to 0 using the potentiometer CURRENT ELECTRODE OFFSET 16 Li After tuning the current electrode potential OFFSET connect the cell model see chapter 7 2 If you do not use a cell model simulate an electrode by replacing the 10 kQ resistor with a much larger resistor min 5 10 MQ L1 The digital display and the CURRENT ELECTRODE potential connector CeL x10mV 32 now shows a voltage deflection that is related to the BIAS current of the headstage according to Ohm s Law Cancel this voltage by tuning the headstage C HEADSTAGE BIAS CURRENT potentio
23. as LO Kz125mA V MO K 2 5mA V The standard TEC current source has a calibration of 10 uA V This means that the gain stages related to the GAIN control on the front panel must provide a gain between 125 250 In the TEC system the gain amplifier is composed of two stages x10 fix and 1 100 variable The maximum gain of the variable gain stage can be set with an internal trim potentiometer If a command step of 150 mV is applied the output of the first stage is 1 5V while the second stage goes into saturation if the gain values calculated above are used Therefore the capacitive transients will have large nonlinear components A response with no saturation effects is obtainable only with command signals below 100 mV With larger membrane capacities the saturation effects start even earlier because a higher gain is required In this situation systems with higher output compliance and or headstage with x2 x5 or x10 ranges must be used to improve clamp response In this case the saturation effect of the gain amplifier is avoided Polder and Houamed 1994 Greeff and Polder 1997 Polder et al 1997 version 4 8 page 56 TURBO TEC 03X User Manual The speed of response with x1 headstage and 150 V output from the point of view of control theory is Tr 94 us Ts 168 us Maximum speed of response The speed of an ideal VC system is limited only by the maximum current delivered by the current source dUm dt max
24. current injecting electrode It is strongly dependent on the length of the cable that connects the headstage with the electrode holder Important The speed of response and the linearity of the capacitive transients can be improved considerably if a current headstage with a steeper gain x2 20 uA V x5 50 uA V is used especially in combination with a higher output voltage of 225 V TEC 225 System and an improved series resistance compensation Dietzel et al 1992 Polder and Houamed 1994 Greeff and Polder 1997 Greeff and K hn 2000 version 4 8 page 57 TURBO TEC 03X User Manual 13 References Recording Methods and Voltage Clamp Technique LI Dietzel I D Bruns D Polder H R amp Lux H D 1992 Voltage Clamp Recording In Practical Electrophysiological Methods eds Kettenmann H amp Grantyn R pp 256 262 Wiley Liss New York LI Greeff N G amp Polder H R 1997 An optimized high current oocyte clamp amplifier with ultralinear low noise response In G ttingen Neurobiology Report 1997 eds Elsner N amp Wassle H Thieme Verlag Stuttgart LI Greeff N G Conti F Gaggero E Planck J Polder H R Terlau H amp Weskamp M 2002 TEVC Recording from Xenopus Oocytes Series Resistance and Space clamp Biophys J 82 267a L Polder H R amp Houamed K 1994 A New Ultra High Voltage Oocyte Voltage Current Clamp Amplifier In G ttingen Neurobiology Report 1
25. display the right value depending on the length of the step The same is true for the CURRENT display 8 6 Tuning the VC mode In VC mode there is the problem that the voltage step is often not strictly angular shaped But for instance increasing the clamp speed by tuning the CAPACITY COMPENSATION of the potential electrode or increasing GAIN also increases noise Therefore the settings of the different parameters result always in a compromise between the stability accuracy noise and control speed In this chapter we will give some practical hints how to optimize the accuracy and speed of the clamp The theoretical background of adjustment criteria is discussed in chapter 12 see also Polder and Swandulla 2001 The main considerations are Do I expect rapid or slow responses to voltage changes How much noise can I accept Is it possible to use electrodes with low resistance General The speed and accuracy of the voltage clamp control circuit is mainly determined by the question how much current can be injected and how fast can this happen Thus the more current the system can inject within a short time the better the quality of the clamp see chapter 12 General Considerations The key to accurate and fast recording is a properly built setup e Make sure that the internal system ground is connected to only one point on the measuring ground and originates from the potential headstage Multiple grounding version 4 8 page 38
26. gives hints in case of trouble and chapter 12 deals with theoretical aspects of closed loop circuits voltage clamp control and optimization methods Chapter 13 contains references and technical data are listed in chapter 14 If you are not familiar with the use of instruments for two electrode voltage clamping of cells please read the manual completely The experienced user should read at least chapters 4 5 8 and 12 Important Please read chapter 1 carefully It contains general information about safety regulations and how to handle highly sensitive electronic instruments 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 gt In some chapters the user is guided step by step through a certain procedure These steps are marked with LI Important information hints and special precautions are highlighted in gray Abbreviations cm time constant of the cell membrane Al analog input BW bandwidth CeL current electrode Cm cell membrane capacity DAC analog output Do digital output GND ground Imax maximal current K proportional gain Pm potential electrode Prek potential at the potential electrode Prer potential at the reference electrode Rce current electrode resistance Rm cell membrane resistance version 4 8 p
27. integrator induces a 0 in the transfer function the clamp system will tend to overshoot if a step command is used Therefore the tuning of the controller is performed following optimization rules which yield a well defined system performance AVO and SO see below The various components of the clamp feedback electronics can be described as first or second order delay elements with time constants in the range of microseconds The cell capacity can version 4 8 page 50 TURBO TEC 03X User Manual be treated as an integrating element with a time constant Tm which is always in the range of hundreds of milliseconds Compared to this physiological time constant the electronic time constants of the feedback loop can be considered as small and added to an equivalent time constant Te The ratio of the small and the large time constant determines the maximum gain which can be achieved without oscillations and thus the accuracy of the clamp With the gain adjusted to this level the integrator time constant and small time constant determine the speed of response of the system The clamp performance can be increased considerably if the influence of the current injecting electrode is excluded as far as possible from the clamp loop since the electrode resistance is nonlinear This is achieved if the output of the clamp system is a current source rather than a voltage source In this case the clamp transfer function has the magnitude of a con
28. is switched off and a SERIES RESISTANCE COMPENSATION circuit is activated SERIES RESISTANCE COMPENSATION improves the performance of the clamp system especially if fast voltage activated currents are recorded The amount of compensation is set by 4 DISPLAY unit tutCtTROOE nESSIANCE The DISPLAY unit consists of 7 electrode selector for POTENTIAL 8 mV LED 9 CURRENT display 10 POTENTIAL RESISTANCE display 11 MO LED and 13 electrode selector for RESISTANCE 7 Electrode selector POTENTIAL This switch selects whether the potential of CURRENT ELECTRODE or POTENTIAL ELECTRODE is displayed see 10 below 8 mV LED When lit indicates that POTENTIAL mV is revealed in DISPLAY 10 9 CURRENT display LC Display for the CURRENT passed through the CURRENT electrode in uA X XX uA 10 POTENTIAL RESISTANCE display LC Display for the POTENTIAL at the electrode tip in mV XXX mV or the electrode RESISTANCE in MQ XX X MQ i e 01 0 corresponds to 1 0 MQ The electrode is selected by electrode selector 7 for POTENTIAL measurement and by electrode selector 13 for ELECTRODE RESISTANCE 11 MQ LED When lit indicates that RESISTANCE MQ is revealed in DISPLAY 10 13 Electrode selector ELECTRODE RESISTANCE Switch to select the electrode to measure the RESISTANCE from Three position switch CURRENT ELECTRODE off POTENTIAL ELECTRODE Switching to CURRENT or version 4 8 page 16 TURBO
29. the circuit A RESET is carried out if one wants to reset the circuit after a previous SHUTOFF condition After resetting the OSCILLATION SHUT OFF unit is active again Voltage clamp control unit The voltage clamp control unit consists of 5 GAIN potentiometer 4 PI CONTROLLER potentiometer and 6 control mode switch see also chapter 8 6 5 GAIN potentiometer 10 turn potentiometer to set amplification factor GAIN of the VC error signal see also chapter 4 1 To keep the VC error as small as possible it is necessary to use high GAIN settings but the system becomes unstable and begins to oscillate if the GAIN is set too high Thus the OSCILLATION SHUT OFF circuit see above should be activated when setting this control version 4 8 page 15 TURBO TEC 03X User Manual 4 PI CONTROLLER potentiometer 10 turn potentiometer to set either the time constant of the integrator in SLOW mode or the amount of series resistance compensation in FAST mode see also 6 6 control mode switch Switch to select the control mode Three modes are possible NORMAL In this mode only the proportional gain see above is active The gain is set by 5 SLOW In the SLOW mode the controller is converted into a PI proportional integral system The integrator improves control performance for slow signals e g during recording of ligand gated currents The time constant is set by 4 FAST In the FAST mode the integrator
30. the overshoot of the selected optimization method 4 with the AVO method and 43 with the SO method With the AVO method the response to a command step is very fast with 496 overshoot potential output The response to a disturbance e g an activating channel is slow and has a slight deviation With the SO method the response to an unsmoothed command step is fast with 4396 overshoot potential output The response to a disturbance e g an activating channel is very fast and has a slight deviation Now the steady state error must disappear Note If the SO is used an external command input filter can be used to smooth the command signal and consequently reduce the overshoot according to the requirements of the experiment see also Figure 14 Tuning the series resistance compensation FAST mode The optimization methods mentioned above cannot be applied if series resistance is present and has to be considered version 4 8 page 53 TURBO TEC 03X User Manual Generally the upper speed limit for all optimization methods is determined by the maximum amount of current which the clamp system can force through a given electrode In experimental situations where very high clamp speed is desirable e g recording of gating currents the clamp speed can be improved additionally by optimizing the position of the electrodes and using SERIES RESISTANCE COMPENSATION see also Greeff and Polder 1998 Greeff and Kiihn 2000 Since SERIES
31. the user 1 NORMAL fits to many users In this mode a good compromise between speed accuracy noise and stability is achieved The normal mode can be optimized by the LO method see above 2 SLOW for relative slow recordings e g ligand activated currents In this mode accuracy and stability are increased while speed is decreased Optimization is done according to the AVO or SO method see above 3 FAST for very fast recordings e g fast voltage activated currents In this mode speed and accuracy are increased but the system is very sensitive with a higher noise level and tuning requires more experience Optimization is done by adjusting the amount of current proportional gain of the SERIES RESISTANCE COMPENSATION and optimal positioning of the electrodes see chapter 9 Important First use a cell model for the tuning procedure You will get familiar with the different settings and the consequences for the system without any damage to cells or electrodes version 4 8 page 39 TURBO TEC 03X User Manual Tuning Procedure LI Before you switch to VC mode tune all parameters related to the recording electrodes offset capacity compensation etc in CC mode set GAIN to a low save level and the control mode switch to NORMAL GAIN ONLY see chapter 8 5 Activate the OSCILLATION SHUTOFF unit If you a working on slow currents _ Switch to VC mode and apply identical test pulses to the cell model _ The controller is
32. 1 214 221 LJ Theis S Knutter I Hartrodt B Brandsch M Kottra G Neubert K amp Daniel H 2002 Synthesis and characterization of high affinity inhibitors of the H peptide transporter PEPT2 J Biol Chem 277 7287 7292 L Verri T Kottra G Romano A Tiso N Peric M Maffia M Boll M Argenton F Daniel H amp Storelli C 2003 Molecular and functional characterisation of the zebrafish Danio rerio PEPTI type peptide transporter FEBS Lett 549 115 122 Fast Perfusion Technique ScreeningTool L Baburin I Beyl S amp Hering S 2006 Automated fast perfusion of Xenopus oocytes for drug screening Pflugers Arch 453 117 123 L Khom S Baburin I Timin E N Hohaus A Sieghart W amp Hering S 2006 Pharmacological properties of GABAA receptors containing gammal subunits Molecular Pharmacology 69 640 649 L Khom S Baburin I Timin E Hohaus A Trauner G Kopp B amp Hering S 2007 Valerenic acid potentiates and inhibits GABA A receptors Molecular mechanism and subunit specificity Neuropharmacology 53 178 187 L Stork D Timin E N Berjukow S Huber C Hohaus A Auer M amp Hering S 2007 State dependent dissociation of HERG channel inhibitors Br J Pharmacol 151 1368 1376 Automated Recordings Using the CellWorks EggWorks Software LJ Anson L C et al 1998 Identification of Amino Acid Residues of the NR
33. 2A Subunit That Control Glutamate Potency in Recombinant NRI NR2A NMDA Receptors J Neurosci 18 2 581 598 L Bo X et al 1995 A P2X purino receptor cDNA conferring a novel pharmacological profile FEBS Letters 375 129 133 L Burnashev N et al 1992 Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor Science 257 1415 1419 L Chen P E Johnston A R Mok M H Schoepfer R amp Wyllie D J 2004 Influence of a threonine residue in the S2 ligand binding domain in determining agonist potency and deactivation rate of recombinant NRla NR2D NMDA receptors J Physiol 558 45 58 LI Hausmann R Rettinger J Gerevich Z Meis S Kassack M U Illes P Lambrecht G amp Schmalzing G 2006 The suramin analog 4 4 4 4 carbonylbis imino 5 1 3 benzenetriylbis carbonylimino tetra kis benzenesulfonic acid NF110 potently blocks P2X3 receptors subtype selectivity is determined by location of sulfonic acid groups Molecular Pharmacology 69 2058 2067 Li Kuner Th and R Schoepfer 1996 Multiple structural elements determine subunit specificity of Mg block in NMDA receptor channels Journal of Neuroscience 16 3549 3558 version 4 8 page 66 TURBO TEC 03X User Manual L Kuner Th et al 1996 Structure of the NMDA Receptor Channel M2 Segment Inferred from the Accessibility of Substituted Cysteins Neuron Vol 17 343 352 L Monyer H e
34. 4364 4373 LI Schmitt B M and H Koepsell 2002 An Improved Method For Real Time Monitoring of Membrane Capacitance in Xenopus laevis Oocytes Biophys J 82 1345 1357 Proton Channels Li Nagel G Ollig D Fuhrmann M Kateriya S Musti A M Bamberg E amp Hegemann P 2002 Channelrhodopsin 1 a light gated proton channel in green algae Science 296 2395 2398 LI Nagel G Szellas T Huhn W Kateriya S Adeishvili N Berthold P Ollig D Hegemann P amp Bamberg E 2003 Channelrhodopsin 2 a directly light gated cation selective membrane channel Proc Natl Acad Sci U S A 100 13940 13945 version 4 8 page 68 TURBO TEC 03X User Manual 14 TURBO TEC 03X Specifications Technical Data All following current signal related parameters are for the TEC 03X amplifier with standard 150 V current headstage Parameters for the other systems or systems with selectable current ranges can be calculated from these parameters MODES OF OPERATION CC Current Clamp mode VC Voltage Clamp mode OFF Current and Voltage Clamp disabled EXTERN External control mode the mode of operation can be set by a TTL pulse applied to the MODE SELECT BNC If CellWorks is used some functions can be controlled from CellWorks TEC 03X CW MODE selection toggle switch LED indicators remote selection by TTL pulse HEADSTAGES Potential headstage Differential input for suppression of bath pot
35. 8 page 63 TURBO TEC 03X User Manual L Hausmann R Rettinger J Gerevich Z Meis S Kassack M U Illes P Lambrecht G amp Schmalzing G 2006 The suramin analog 4 4 4 4 carbonylbis imino 5 1 3 benzenetriylbis carbonylimino tetra kis benzenesulfonic acid NF110 potently blocks P2X3 receptors subtype selectivity is determined by location of sulfonic acid groups Molecular Pharmacology 69 2058 2067 L1 Inglis F M Crockett R Korada S Abraham W C Hollmann M amp Kalb R G 2002 The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons J Neurosci 22 8042 8051 LI Jenke M Sanchez A Monje F Stuhmer W Weseloh R M amp Pardo L A 2003 C terminal domains implicated in the functional surface expression of potassium channels EMBO J 22 395 403 L Krause S amp Schwarz W 2005 Identification and Selective Inhibition of the Channel Mode of the Neuronal GABA Transporter 1 Molecular Pharmacology 68 1728 1735 L1 Lu W Zheng B J Xu K Schwarz W Du L Wong C K L Chen J Duan S Deubel V amp Sun B 2006 Severe acute respiratory syndrome associated coronavirus 3a protein forms an ion channel and modulates virus release Proceedings of the National Academy of Sciences 103 12540 12545 L Marquez Klaka B Rettinger J Bhargava Y Eisele T amp Nicke A 2007 Identification of an intersubunit cross link bet
36. 993 Polder and Houamed 1994 Polder and Swandulla 2001 are closely related Modern control theory provides adequate solutions for the design and the optimal tuning of feedback systems Froehr 1985 Most voltage clamp systems are composed only of delay elements i e elements which react with a retardation to a change This type of closed loop systems can be optimized easily by adequate shaping of the frequency characteristic magnitude F jw of the associated transfer function F s output to input ratio in the frequency domain LAPLACE transform of the differential equation of the system Polder and Swandulla 2001 Using controllers with a proportional integral characteristic PI controllers it is possible to force the magnitude of the frequency characteristic to be as close as possible to one over a wide frequency range modulus hugging see Froehr 1985 Polder 1984 Polder and Swandulla 1990 Ploder 1993 Polder and Houamed 1994 Polder and Swandulla 2001 For voltage clamps this means that the controlled membrane potential rapidly reaches the desired command value The PlI controller yields an instantaneous fast response to changes proportional gain while the integral part increases the accuracy by raising the gain below the corner frequency of the integrator i e for slow signals to very high values theoretically to infinite for DC signals i e an error of 096 without affecting the noise level and stability Since the
37. 994 eds Elsner N amp H Breer Thieme Verlag Stuttgart L Polder H R Schliephacke R St hmer W amp Terlau H 1997 A new switched mode double electrode clamp amplifier avoiding series resistance errors in G ttingen Neurobiology Report 1997 eds Elsner N amp Wassle H Thieme Verlag Stuttgart LI Polder H R amp Swandulla D 2001 The use of control theory for the design of voltage clamp systems A simple and standardized procedure for evaluating system parameters J Neurosci Meth 109 97 109 LI Polder H R M Weskamp K Linz amp R 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 Oocyte Techniques Book Chapters L St hmer W 1992 Electrophysiological Recording from Xenopus Oocytes in Rudy B amp L E Iverson eds Ion Channels Methods in Enzymology Vol 207 Academic Press San Diego L St hmer W Terlau H and Heinemann S H 1992 Xenopus Oocytes for Two Electrode and Patch Clamp Recording in Kettenmann H amp Grantyn R eds Practical Electrophysiological Methods Wiley Liss New York L St hmer W and A B Parekh 1995 Recording from Xenopus Oocytes in Sakmann B and E Neher eds Single Channel Recording Second Edition Plenum Press New York and London LI St hmer
38. A PI Controller is used The response to an unsmoothed command step is fast with 4396 overshoot potential output The response to a disturbance e g an activating channel is very fast and has a slight deviation Figure 14 tuning VC according LO AVO or SO The potential output is shown version 4 8 page 40 TURBO TEC 03X User Manual If you a working on very fast currents _ Lower the GAIN by approximately 10 L Switch the control mode switch to FAST SERIES RESISTANCE COMPENSATION to activate the series resistance compensation Rise the amount of SERIES RESISTANCE COMPENSATION and watch the current output The capacitive transient seen on the current trace should be mono exponential The critical compensation is achieved when the slow tail of the transient disappears If you see ringing around the slow tail this a sign that the electrodes are not optimally positioned see also chapter 9 Hint SERIES RESISTANCE COMPENSATION is done by positive feedback in the control circuit which can lead very quickly to stability problems Repositioning the electrodes is recommended whenever possible instead of extensive use of SERIES RESISTANCE COMPENSATION see also chapter 9 Note With a standard cell model described in chapter 7 you cannot verify the advantages of the FAST mode because no series resistance is simulated Ask npi for a modified cell model with series resistance simulation Details of how to tune PI controll
39. EC 03X User Manual 12 Appendix 12 1 Theory of Operation The standard configuration for voltage clamping oocytes is the two electrode voltage clamp arrangement St hmer 1992 St hmer et al 1992 Dietzel et al 1992 St hmer 1998 In contrast to previously described clamp systems for review see Smith et al 1985 the amplifiers for oocyte clamping must meet special requirements since oocytes are very large cells with a high membrane capacity up to 100 500 nF and large membrane currents up to 100 uA and more Voltage clamp instruments are closed loop control systems with two inputs external to the control loop An electronic feedback network is used to force the membrane potential of a cell to follow a voltage command setpoint input as fast and as accurately as possible in the presence of incoming disturbances disturbance input correlated with the activities of the cell e g activation of ion channels This is achieved by injecting an adequate amount of charge into the cell The current injected by the clamp instrument is a direct measure of the ionic fluxes across the membrane Ferreira et al 1985 Jack et al 1975 Ogden 1994 Smith et al 1985 The performance evaluation and optimal tuning of these systems can be done by considering only the command input since the mathematical models set point transfer function and the disturbance transfer function see Froehr 1985 Polder 1984 Polder and Swandulla 1990 Polder 1
40. Gu G G amp Singh S 1999 Modulation of dihydropyridine sensitive calcium channels in Drosophila by a cAMP mediated pathway J Neurobiol 39 491 500 L Bhattacharya A Lakhman S S amp Singh S 2004 Modulation of L type calcium channels in Drosophila via a pituitary adenylyl cyclase activating polypeptide PACAP mediated pathway J Biol Chem 279 37291 37297 L Singh A amp Singh S 1999 Unmasking of a novel potassium current in Drosophila by a mutation and drugs J Neurosci 19 6838 6843 L Walther Ch et al 1998 Resting Membrane Properties of Locust Muscle and Their Modulation I Action of Neuropeptides YGGFMRFamide and Proctolin J Neurophysiol 80 771 784 L Walther Ch and K E Zittlau 1998 Resting Membrane Properties of Locust Muscle and Their Modulation II Action of the Biogenic Amine Octopamine J Neurophysiol 80 785 797 L Zordan M A Massironi M Ducato M G Te K G Costa R Reggiani C Chagneau C Martin J R amp Megighian A 2005 Drosophila CAKI CMG protein a homolog of human CASK is essential for regulation of neurotransmitter vesicle release Journal of Neurophysiology 94 1074 1083 version 4 8 page 67 TURBO TEC 03X User Manual Capacitance measurements LJ Cohen R Schmitt B M amp Atlas D 2005 Molecular identification and reconstitution of depolarization induced exocytosis monitored by membrane capacitance Biophys J 89
41. K hn 2000 for details is not considered General Considerations For the TEC systems the small time constants are at least two orders of magnitude below the large time constant The large time constant is the time constant of the membrane and the equivalent time constant is composed of the time constants of the electrodes amplifiers etc Tm Rn Cn Te XTi with Tm large time constant Rm membrane resistance Cm membrane capacity Te equivalent time constant Ti small time constant The performance of a clamp system can be improved if a voltage controlled current source is used for the current injecting electrode In this case the very large time constant hundreds of milliseconds formed by the electrode resistance and the cell capacity can be ignored because the output of the clamp circuit is a current that flows regardless of the resistance of the injecting microelectrode Smith et al 1990 Thus the performance of the clamp is no longer dependent on the electrode resistance as long as the current source is not saturated In this case the clamp gain has the magnitude of a conductance A V version 4 8 page 55 TURBO TEC 03X User Manual The proportional gain of the clamp system can be calculated as follows Froehr 1985 Polder 1984 K Cm 4 Te Linear optimum LO aperiodic response no overshoot K Cm 2 Te Modulus optimum MO or AVO respectively 4 overshoot fastest rise time
42. KCNQ Potassium Channels Biophys J 90 2235 2244 L Strutz Seebohm N Seebohm G Fedorenko O Baltaev R Engel J Knirsch M amp Lang F 2006 Functional Coassembly of KCNQ4 with KCNE Beta Subunits in Xenopus Oocytes Cell Physiol Biochem 18 57 66 L Ullrich S Berchtold S Ranta F Seebohm G Henke G Lupescu A Mack A F Chao C M Su J Nitschke R Alexander D Friedrich B Wulff P Kuhl D amp Lang F 2005 Serum and Glucocorticoid Inducible Kinase 1 SGK1 Mediates Glucocorticoid Induced Inhibition of Insulin Secretion Diabetes 54 1090 1099 L Wuttke T V Seebohm G Bail S Maljevic S amp Lerche H 2005 The New Anticonvulsant Retigabine Favors Voltage Dependent Opening of the Kv7 2 KCNQ2 Channel by Binding to Its Activation Gate Molecular Pharmacology 67 1009 1017 Chloride and epithelial Na Channels LJ Est vez R Schroeder B C Accardi A Jentsch T J amp Pusch M 2003 Conservation of Chloride Channel Structure Revealed by an Inhibitor Binding Site in CIC 1 Neuron 38 47 59 version 4 8 page 62 TURBO TEC 03X User Manual LJ Estevez R Pusch M Ferrer Costa C Orozco M amp Jentsch T J 2004 Functional and structural conservation of CBS domains from CLC chloride channels J Physiol 557 363 378 LI Fong P Rehfeldt A and Jentsch T J 1998 Determinants of slow gating in CIC 0 the voltage gated chlo
43. OTENTIAL OUTPUT PEt Important Capacity compensation is based on positive feedback Therefore over compensation causes oscillations which can damage the preparation or the recording electrodes Therefore the control must be handled with care and before impaling a new cell it must be set to 0 version 4 8 page 36 TURBO TEC 03X User Manual 8 5 Testing Operation Modes Current Clamp The cell s response to current injections is measured in the current clamp CC mode Current injection is performed by means of a current source connected to the current injecting microelectrode Set the amplifier to CC mode using the MODE OF OPERATION switch 28 If not already done tune the BIAS current to 0 see chapter 8 1 Set the CURRENT OUTPUT SENSITIVITY 15 to 1 Compensate the offsets of the current and voltage electrode see chapter 8 2 Set Rm at the cell model to 10k see chapter 7 Set the holding current to 1 uA using the HOLD potentiometer 12 setting 100 reading 1 00 uA and the HOLD current polarity switch 25 set to Make sure that the potential switch 7 is set to POTENTIAL electrode and the ELECTRODE RESISTANCE test is not active The POTENTIAL display should read 10 mV according to Ohm s law The voltage at Per 33 should be 100 mV L OUUU Remember The voltage at Pex is the membrane potential multiplied by 10 LI Disable the holding current and apply a test pulse of 2 uA to the c
44. TASK 1 TASK 3 and a Tandem Pore Domain K1 Channel Subunit TASK 5 Associated with the Central Auditory Nervous System Mol Cell Neurosci 18 632 648 L Kerschensteiner D Monje F amp Stocker M 2003 Structural determinants of the regulation of the voltage gated potassium channel Kv2 1 by the modulatory alpha subunit Kv9 3 J Biol Chem 278 18154 18161 LI Lange A Giller K Hornig S Martin Eauclaire M F Pongs O Becker S amp Baldus M 2006 Toxin induced conformational changes in a potassium channel revealed by solid state NMR Nature 440 959 962 LJ Lerche C Scherer C R Seebohm G Derst C Weii A D Busch A E and K Steinmeyer 2000 Molecular Cloning and Functional Expression of KCNQS a Potassium Channel Subunit That May Contribute to Neuronal M current Diversity J Biol Chem 275 29 22395 22400 LI Lerche C Seebohm G Wagner C I Scherer C R Dehmelt L Abitbol I Gerlach U Brendel J Attali B and A E Busch 2000 Molecular impact of MinK on the enantiospecific block of I Ks by chromanols Br J Pharmacol 131 8 1503 6 L Lerche C Bruhova I Lerche H Steinmeyer K Wei A D Strutz Seebohm N Lang F Busch A E Zhorov B S amp Seebohm G 2007 Chromanol 293B binding in KCNQI Kv7 1 channels involves electrostatic interactions with a potassium ion in the selectivity filter Mol Pharmacol 71 1503 1511 LI Maljevic S Lerch
45. TURBO TEC 03X User Manual should be avoided all ground points should originate from a central point The electrode used for grounding the bath should have a low resistance so that it can pass large currents e Use electrodes with resistances as low as possible e Keep cables short e Use differential potential recording see also chapter 5 1 and Figure 10 e Check regularly whether cables and or connections are broken e Make sure that chloriding of silver wires for the electrodes is proper and that there are no unwanted earth bridges e g salt bridges originating from experimental solutions Only if no intracellular series resistance is considered TEC system can be tuned according to one of three optimization methods see also chapter 12 1 the linear optimum LO that provides only slow response to a command step and a maximal accuracy of 90 97 2 the absolute value optimum AVO that provides the fastest response to a command step with very little overshoot maximum 4 or 3 the symmetrical optimum SO has the best performance compensating intrinsic disturbance signals but shows a considerable overshoot maximum 43 to a step command Under consideration of an existing intracellular series resistance these methods cannot be applied Instead a series resistance compensation can be introduced to optimize clamp performance see also chapter 12 2 Three control modes are implemented to adapt the TEC 03X to the needs of
46. W 1998 Electrophysiologic Recordings from Xenopus Oocytes in P Michael Conn ed Ion Channels Part B Meth in Enzymology Vol 293 Academic Press San Diego L Wagner C A Friedrich B Setiawan I Lang F amp Broer S 2000 The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins Cell Physiol Biochem 10 1 12 version 4 8 page 58 TURBO TEC 03X User Manual Na Channel Gating Currents L Frank J P K hn and Nikolaus G Greeff 1999 Movement of Voltage Sensor S4 in Domain 4 Is Tightly Coupled to Sodium Channel Fast Inactivation and Gating Charge Immobilization Journal of General Physiology Volume 114 1 18 August 1999 LI Greeff N G and F J P K hn 2000 Variable Ratio of Permeability to Gating Charge of rBIIA Sodium Channels and Sodium Influx in Xenopus Oocytes Biophys Journal Vol 79 2434 59 LI Greeff N G and H R Polder 1998 Optimization of a Two Electrode Voltage Clamp for Recording of Sodium Gating Currents from Xenopus Oocytes Biophysical Meeting Kansas City CL K hn EJ P and N G Greeff 2002 Mutation D384N Alters Recovery of the Immobilized Gating Charge in Rat Brain IIA Sodium Channels J Membr Biol 185 145 155 Na Channels LI Hilber K Sandtner W Kudlacek O Glaaser I W Weisz E Kyle J W French R J Fozzard H A Dudley S C amp Todt H 2001 The Selectivity Filter of the Voltage
47. age 4 TURBO TEC 03X User Manual Reet potential electrode resistance Rrer reference electrode resistance Rs series resistance Te equivalent time constant Tea time constant of the current electrode Ti small time constant Tm large time constant T time until the membrane potential reaches 10096 of the command pulse for the first time Ts time to reach a steady state within a tolerance of 296 version 4 8 page 5 TURBO TEC 03X 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 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 6 GENERAL This system is designed for use in scientific laboratories and should be operated by trained staff only General safety regulations for operating electrical 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
48. al connections L Turn POWER off _ Plug the instrument into a grounded outlet L Connect the potential headstage to the POTENTIAL HEADSTAGE connector 20 at the TEC 03X _ Connect the current headstage to the CURRENT HEADSTAGE connector 22 at the TEC 03X _ Connect a cell model see chapter 7 2 Connect a digital analog timing unit or a stimulation device to COMMAND INPUT 31 L Connect a storage oscilloscope or a data recording device i e a computer with data acquisition card to the POTENTIAL OUTPUT Pex and to the CURRENT OUTPUT triggered from the stimulation device We recommend always using an oscilloscope in addition to the computer system Set the desired gain at the CURRENT OUTPUT SENSITIVITY switch 15 and set the CURRENT FILTER 14 to 20k TURBO TEC 03X npi o o Current Potential ADC Oscilloscope Headstage Headstage DAC Stimulation device Figure 11 basic electrical connections version 4 8 page 29 TURBO TEC 03X User Manual Before using the TEC 03X always make the basic settings to avoid oscillations Basic settings L Turn all controls to low values less than 1 and each symmetrical offset adjustment ie C HEADSTAGE BIAS CURRENT CURRENT ELECTRODE OFFSET and POTENTIAL OFFSET potentiometers in the range of 5 zero position see chapter 4 2 Set the CAPACITY COMPENSATION 19 to 0 Disable the HOLD unit by setting the 0 switch
49. anual refers to the standard configuration of the TURBO TEC 03X system consisting of a standard headstage and standard calibrations as written on the front panel Other configurations are available see Optional accessories and Options in chapter 3 For details contact npi 4 1 System Description The TURBO TEC 03X instrument is a voltage current clamp system that operates according to the classic dual microelectrode method This method uses one microelectrode for the registration of membrane potential and one for current injection The equivalent circuit of a TURBO TEC 03X system and the associated block diagram in VC mode are shown in Figure 1 and Figure 2 The TURBO TEC 03X system is based on modern state of the art electronic circuits Its advanced design makes it superior to other amplifiers Some of the special features of TURBO TEC 03X system are differential potential registration and high voltage current version 4 8 page 8 TURBO TEC 03X User Manual source output both to eliminate artifacts induced by the use of microelectrodes full compensation of the current injecting electrode and no need of virtual ground for recording membrane currents The TURBO TEC 03X has an automated electrode resistance test mode which can be used even with the electrodes impaled in a cell see Stiihmer 1992 in Methods in Enzymology Vol 207 In addition a unique oscillation shutoff circuit prevents the cell from damage if oscillations occur
50. as a high impedance floating output Therefore the zero point the zero of the bias current of the current source must be defined i e without an input signal there should not be an output current Since the high voltage FET amplifiers that are used become warm from the internal heat dissipation and their characteristics are strongly temperature dependent the calibration procedure has to be done periodically by the user The tuning procedure is done using the C HEADSTAGE BIAS CURRENT control and one resistance of a few kQ and one of a few MQ or a cell model It is based on Ohm s Law U R DJ If the headstage generates an output current this current will cause a voltage deflection at a test resistor If this test resistor has a low resistance of only a few kQ this voltage deflection is nearly zero and a possible reading at the digital display originates only from a possible offset of the electrode which can be cancelled using the CURRENT ELECTRODE OFFSET 16 potentiometer Replacing the low resistance resistor by one of a much higher resistance may lead to another voltage reading at the digital display This voltage deflection then originates only from the BIAS output current and is proportional to this output current according to Ohm s law Using the C HEADSTAGE BIAS CURRENT control the monitored voltage can be set to 0 version 4 8 page 33 TURBO TEC 03X User Manual The tuning procedure is performed using high value resistors or
51. ce compensation unit allow recording of very large voltage activated currents and even gating currents from whole oocytes Greeff et al 2000 The TURBO TEC 03X can be operated with a reduced bandwidth of 10 Hz to allow simultaneous single channel recordings with patch clamp amplifiers An excellent introduction into recording techniques preparation of oocytes etc can be found in Methods in Enzymology Vol 207 1992 and also in the chapter by Stuehmer et al in Practical Electrophysiological Methods The basics of microelectrode techniques and voltage clamp principles are described comprehensively in the Plymouth Workshop Handbook Ogden 1994 version 4 8 page 7 TURBO TEC 03X User Manual 3 TURBO TEC 03X Components The following items are shipped with the TURBO TEC 03X system X TURBO TEC 03X amplifier V Potential headstage 4 Current headstage 4 GND connectors for headstage 2 6 mm V Connectors for current and reference electrode V Power cord V User manual Note If an electrode holder set TEC EH SET is ordered the connector for the current electrode is not supplied Optional accessories Electrode adapter Electrode holder Electrode holder set Passive cell model see chapter 7 Active cell model t gF Options Interface for CellWorks Four pole Bessel current filter 15V or 45V or 225V output compliance Current headstage with four ranges 0555 4 TURBO TEC 03X System This m
52. d be no voltage The TURBO TEC 03X CW is designed to work with data acquisition boards from National Instruments NI The installation process of a data acquisition board from NI is carried out in two steps detailed information is given in the CellWorks Short Manual 5 1 and in the User Manual of the respective NI board Briefly for PC 1 Software installation This has to be performed prior to the hardware installation The setup routine from NI installs the driver for the data acquisition board and a program called MAX Measurement amp Automation Explorer Once the software is installed a reboot is required and the computer has to be shut down to install the hardware 2 Hardware installation Find a free PCI slot install the NI board and start the operating system The NI board will be recognized automatically and the driver is configured with the configuration dialog that starts Now the computer is ready to measure data with CellWorks The NI program MAX is a tool for configuring and testing data acquisition boards from NI The following steps can be carried out to test some basic functions of the NI board independent of CellWorks and the TURBO TEC 03X CW Although the tests can be performed also without a breakout box we recommend using a breakout box for the tests because then you can really prove the in and outputs Important The NI board has to be configured in Unreferenced Single Ended mode with bipolar output and 10
53. d fluorene aspartic acid and diaminopropionic acid analogs as potent inhibitors of the high affinity glutamate transporter EAAT2 Molecular Pharmacology 68 974 982 Foltz M Mertl M Dietz V Boll M Kottra G amp Daniel H 2005 Kinetics of bidirectional H and substrate transport by the proton dependent amino acid symporter PATI Biochem J 386 607 616 LI Gorbunov D Gorboulev V Shatskaya N Mueller T Bamberg E Friedrich T amp Koepsell H 2007 High affinity Cation Binding to Transporter OCT1 Induces Movement of Helix 11 and Blocks Transport after Mutations in a Modelled Interaction Domain between Two Helices Mol Pharmacol LI Kottra G and H Daniel 2001 Bidirectional electrogenic transport of peptides by the proton coupled carrier PEPT1 in Xenopus laevis oocytes its asymmetry and symmetry J Physiol 536 2 495 503 L Picollo A amp Pusch M 2005 Chloride proton antiporter activity of mammalian CLC proteins CIC 4 and CIC 5 Nature 436 420 423 version 4 8 page 65 TURBO TEC 03X User Manual L Schmitt B M amp Koepsell H 2005 Alkali Cation Binding and Permeation in the Rat Organic Cation Transporter rOCT2 J Biol Chem 280 24481 24490 L Theis S Hartrodt B Kottra G Neubert K amp Daniel H 2002 Defining minimal structural features in substrates of the H peptide cotransporter PEPT2 using novel amino acid and dipeptide derivatives Mol Pharmacol 6
54. dditional headstage In addition the current source method provides an improved frequency response of the voltage clamp control circuit On all TEC systems for oocyte recordings the bandwidth of the current injection electronics can be limited to approximately 10 Hz by means of a switch BANDW on the current headstage see Figure 9 and Figure 10 This enables simultaneous macrocurrent recording with the TEC system and single channel recording with the patch amplifier without degradation of the patch amplifier signals from the TEC operation Current Clamp Mode CC In the current clamp mode the cell s response to current injections is measured Current injection is performed by means of a current source connected to the current injecting microelectrode regardless of the electrode resistance see Figure 1 Therefore only a current input conditioning unit is necessary for the adequate shaping of the current input signal COMMAND INPUT In addition the TEC 03X is equipped with a HOLD unit for applying a constant holding current The polarity is controlled by a switch Voltage Clamp Mode VC The voltage clamp mode is based on a closed loop control see Figure 2 In voltage clamp mode the membrane potential is forced by a controller to maintain a certain value or to follow an external command That allows measurement of ion fluxes across the cell membrane separate from capacitive currents This is the most complex mode of operation with th
55. ding mains ground see below 4 INTERNAL GROUND connector Banana plug providing internal ground see below 5 CURRENT SENSITIVITY connector BNC connector providing a voltage monitoring the position of the CURRENT OUTPUT SENSITIVITY switch 1 V to 7 V 1V STEP See also 21 Figure 3 version 4 8 page 21 TURBO TEC 03X User Manual 6 FILTER CURRENT connector BNC connector providing a voltage monitoring the position of the CURRENT FILTER switch 7 V to 8 V 1V STEP See also 23 Figure 3 Grounding TEC instruments have two ground systems 1 the internal ground called INTERNAL GROUND represents the zero level for the recording electronics and is connected to the recording chamber and the BNC input output sockets 2 mains ground PROTECTIVE EARTH is connected to the 19 cabinet and through the power cable to the protection contact of the power outlet GROUND outlets are located on both headstages and on the front panel For both grounds there is an outlet on the rear panel GROUND black socket internal system ground PROTECTIVE EARTH green yellow socket mains ground 19 cabinet All TEC systems have a high quality toroid transformer to minimize stray fields In spite of this noise problems could occur if other mains operated instruments are used in the same setup The internal system ground GROUND sockets should be connected to only one point on the measuring ground of the recording chamber
56. ductance A V Other advantages of this arrangement are that the clamp current can be determined by a differential amplifier with no need of virtual ground see Greeff and Polder 1997 Polder and Houamed 1994 and that the bandwidth of the feedback system can be altered easily e g for noise suppression during simultaneous patch clamp recordings see St hmer 1992 St hmer et al 1992 St hmer and Parekh 1995 This output circuit is equipped with large bandwidth high voltage operational amplifiers To avoid deterioration of clamp performance caused by electrode overload the output current has to be limited by an electronic circuit to a safe level With electrodes in the range of one MQ and a voltage of 150 V the maximum current will be 150 uA With this current a cell with a capacity of 100 nF can be depolarized by 100 mV in approximately 100 us which comes close to the theoretically possible speed of response without any detectable deviations from the command level With an output compliance of 225 V and a x2 or x5 range current injecting headstage currents up to 500 uA can be injected see Greeff and Polder 1997 Polder and Houamed 1994 The accuracy of a two electrode clamp system and the speed of response is determined by the cell capacity the resistance of the current injecting microelectrode that limits the maximum amount of injected current and the equivalent time constant and accuracy of the potential recording and feedback
57. e C Seebohm G Alekov A K Busch A E amp Lerche H 2003 C terminal interaction of KCNQ2 and KCNQ3 K channels J Physiol 548 2 353 360 LI Marble D D Hegle A P Snyder E D Dimitratos S Bryant P J amp Wilson G F 2005 Camguk CASK enhances Ether a go go potassium current by a phosphorylation dependent mechanism J Neurosci 25 4898 4907 L1 Hou P Di A Huang P Hansen C B and Nelson D J 2000 Impermeability of the GIRK2 weaver channel to divalent cations Am J Physiol 278 C1038 C1046 L Putzke C Wemhoner K Sachse F B Rinne S Schlichthorl G Li X T Jae L Eckhardt I Wischmeyer E Wulf H Preisig Muller R Daut J amp Decher N 2007 The acid sensitive potassium channel TASK 1 in rat cardiac muscle Cardiovasc Res 75 59 68 version 4 8 page 61 TURBO TEC 03X User Manual L Reuveny E 2002 Trapping the sensor Neuron 35 814 815 L Sarac R Hou P Hurley K M Hriciste D Cohen N A amp Nelson D J 2005 Mutation of critical GIRK subunit residues disrupts N and C termini association and channel function J Neurosci 25 1836 1846 LI Schenzer A Friedrich T Pusch M Saftig P Jentsch T J Grotzinger J amp Schwake M 2005 Molecular determinants of KCNQ Kv7 K channel sensitivity to the anticonvulsant retigabine J Neurosci 25 5051 5060 J Schonherr R Mannuzzu L M Isacoff E Y
58. e TEC 03X Special precautions must be taken while tuning the control circuit in order avoid stability problems see chapters 8 6 and 12 Important Although in VC mode one is primarily interested in recording the current flowing across the membrane the clamp circuit primarily controls the membrane potential The better the potential is controlled i e the smaller the VC error signal command voltage minus membrane potential the more accurate is the recording of membrane currents version 4 8 page 10 TURBO TEC 03X User Manual Figure 2 block diagram of VC mode The COMMAND INPUT is used for the COMMAND signal and as in the CC mode the HOLD unit can apply a constant holding voltage Note If no external command signal is connected to the COMMAND INPUT make sure that an appropriate HOLDING potential in voltage clamp mode is selected preferable close to the resting membrane potential of the cell Without any holding potential the cell will be clamped to 0 mV if the amplifier is switched to VC mode and that may unintentionally stimulate or damage the cell Control Circuit in VC A detailed description of the basic principals of voltage clamp controls can be found in the literature e g Methods in Enzymology Smith et al 1987 Ogden 1994 In the control circuit see Figure 1 and Figure 2 the command signal and recorded potential are subtracted to give the VC error signal This signal is applied to an amplifier with ext
59. e deviation is in the same range as with the AVO method The overshoot can be reduced by adequate shaping of the command pulse by a delay unit Froehr 1985 Polder and Swandulla 1990 Polder and Swandulla 2001 This method is preferred for slowly activating currents such as those evoked by agonist application The upper speed limit for all optimization methods is determined by the maximum amount of current which the clamp system can force through a given electrode see chapter 12 3 Practical Implications In the following some practical implications of the theory discussed earlier in this chapter are outlined It is assumed that you have read at least chapters 6 to 8 12 1 and 12 2 that all connections are set up as described in chapter 6 and that the system is in VC mode with the initial settings described in chapter 8 5 Although most of the parameters of the control chain are not known during an experiment it is possible to tune the clamp controller by optimizing the response to a test pulse applied to the COMMAND INPUT The main criterion of tuning is the overshoot seen at the potential output Since the SO method provides the tightest control it will be most sensitive to parameter settings and requires much experience version 4 8 page 52 TURBO TEC 03X User Manual Note The transitions between the optimization methods are blurred and the tuning procedure is adapted to the experimental requirements Often the adequate tuning
60. e 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 current electrode Possible reason 1 The BIAS current is not adjusted Solution 1 Adjust the BIAS current according the procedure described in chapter 8 1 Problem 3 The system oscillates see also voltage clamp in chapter 8 5 Possible reason 1 The capacitance of the electrode is overcompensated 2 There is too much series resistance compensation Solution 1 Turn the CAPACITY COMPENSATION potentiometer 30 Figure 3 to the most left position and compensate the input capacitance again 2 Turn the SERIES RESISTANCE COMPENSATION potentiometer 4 Figure 3 to a lower value Problem 4 With the cell model connected the Rex display does not show the correct value within a tolerance of 2 Possible reason 1 The headstage has an error Solution 1 Contact npi version 4 8 page 49 TURBO T
61. e is dependent on the calibration of the current headstage see chapter 5 2 and the reading is correct only in position x1 Potential Electrode LI Set the ELECTRODE RESISTANCE 13 switch to POTENTIAL ELECTRODE The display 10 shows the resistance of the potential electrode in XX X MQ Current Electrode LI Set the ELECTRODE RESISTANCE 13 switch to CURRENT ELECTRODE The upper digital display 10 shows the resistance of the current electrode in XX X MQ Important Since the amplitude of the current pulses is relatively small at least for oocytes the electrode resistance can be checked even if the electrode is inside the cell 8 4 Capacity Compensation The frequency response of the potential electrode low pass characteristic due to stray capacities is compensated for by a feedback circuit negative capacity compensation CAPACITY COMPENSATION and a driven shield arrangement for an overview see Ogden 1994 Since in oocyte experiments microelectrodes are usually in the one MQ range or below for most experiments it is not required to use the CAPACITY COMPENSATION The tuning of the capacity compensation control is performed using pulses applied to the COMMAND INPUT or pulses provided by the electrode resistance test circuit The TEC 03X has to be in CC mode see chapter 8 5 With the cell model connected or the electrode in the bath the CAPACITY COMPENSATION control is turned clockwise until there is no artifact on the P
62. e membrane potential is recorded CC Current Clamp EXT EXTernal control if this position is selected the mode of operation can be set by a TTL pulse applied to the MODE SELECT BNC 29 If CellWorks is used some functions can be controlled from CellWorks TEC 03X CW MODE OF OPERATION version 4 8 page 19 TURBO TEC 03X User Manual 29 MODE SELECT connector BNC connector for remote control of the MODE of operation A TTL signal is connected here to select the mode of operation remotely HI VC LO CC 31 COMMAND INPUT connector or tC BNC connector for an external COMMAND in VC mode sensitivity 10 mV or in CC mode sensitivity 1 uA V EE The voltage signal that is connected here is transformed to a proportional current at the electrode in CC mode or COMMAND voltage in VC mode The signal form remains unchanged Two examples are given in Figure 4 The amplitude of the output current or voltage signal current voltage stimulus is determined by the amplitude of the input voltage signal input voltage signal output current signal Figure 4 input output relation using COMMAND INPUT in CC mode 32 POTENTIAL OUTPUT connector Cer Kion BNC connector monitoring the POTENTIAL at the tip of the CURRENT electrode CEL sensitivity x10 mV POTENTIAL OUTPUT 33 POTENTIAL OUTPUT connector PEL TUM BNC connector monitoring the POTENTIAL at the tip of the POTENTIAL r electrode
63. e moved place it near the cell membrane and the potential electrode to optimize differential potential recording Insert the current electrode into the oocyte There are two indications that the current electrode is inside the cell If you apply a test current pulse to the cell the potential read by the potential electrode changes according to Ohm s law If you switch the POTENTIAL display to CURRENT ELECTRODE see chapter 8 2 you read the same membrane potential as read by the potential electrode After penetration with both electrodes the voltage responses of the cell to the test pulses in CC mode should reflect the cell membrane resistance and time constant Start the experiment If you intend to work in VC mode tune the system in CC mode then switch to VC mode and adjust the clamp as described in chapter 8 6 version 4 8 page 48 TURBO TEC 03X User Manual 11 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 with the cell model Problem 1 After immersing an electrod
64. ectrodes in a sequence of microphotographs A shows the tips coming from 45 from above just before touching the oocyte and B the electrodes in their final position The reconstruction in C gives the position relative to the center of the oocyte Figures are kindly provided by Nikolaus G Greeff Greeff 2000 version 4 8 page 46 TURBO TEC 03X User Manual 10 Sample Experiment In the following the basics of a simple experiment are described It is assumed that all connections are built as described in chapter 6 Before starting remove the cell model Again It is of major importance that the TEC 03X systems are used only in warmed up condition i e 20 to 30 minutes after turning power on to current headstage to potential headstage 4 current potential electrode electrode RREF cell to potential headstage eo AAA gt M reference electrode C m m Figure 20 model circuit for voltage clamp recording from a cell e g an oocyte using low resistance electrodes Cm membrane capacity Rm membrane resistance Rcer current electrode resistance Rrrr potential electrode resistance Rrer reference electrode resistance Rs series resistance Adjust BIAS CURRENT to 0 if necessary see chapter 8 1 Turn off the amplifier Reconnect the COMMAND INPUT Connect the Ag AgCl pellet or the agar bridge for grounding the bath with GND at the potential headstage O O O Connect the refer
65. electronic systems Therefore the design of the potential recording site is very important A differential potential registration with a reference electrode that registers the bath potential minimizes errors due to resistances outside the cell in series with the cell membrane Driven shield and capacity compensation circuits are used to improve the speed of response In some cases a series resistance compensation circuit for series resistance inside the cell which adds a current proportional gain can improve the clamp performance considerably Greeff and Polder 1997 Greeff 2000 Greeff and K hn 2000 The use of such a circuit enhances the speed of response and improves the accuracy of the clamp system But the noise level is also increased because both circuits are positive feedback loops In addition to the elements of the clamp loop itself this oocyte clamp amplifier has some additional units that facilitate experiments such as electrode resistance test units oscillation shut off unit adequate output signal amplification filtering and display units facility for compensating capacitive currents etc version 4 8 page 51 TURBO TEC 03X User Manual 12 2 Tuning Procedures for VC Controllers The initial settings see voltage clamp in chapter 8 5 guarantee only a stable clamp that is not very accurate and insufficiently rapid for certain types of experiments e g investigation of fast voltage activated ion channels or gating c
66. ell model by giving a voltage step of 2 V to COMMAND INPUT 31 The length of the test pulse should be at least 50 ms L You should see a potential step of 200 mV amplitude at Per 33 Due to the membrane capacity the step is smoothed Note If you expect the POTENTIAL display to show the value of the potential step in this case 20 mV amplitude remember that the display is rather sluggish and may not display the right value depending on the length of the step The same is true for the CURRENT display Voltage Clamp In voltage clamp mode the membrane potential is forced by a controller to maintain a certain value or to follow an external command That allows measurement of ion fluxes across the cell membrane This is the most complex mode of operation with the TEC 03X Special precautions must be taken while tuning the control circuit in order avoid stability problems _ Make sure that the amplifier works correctly with the cell model in CC mode see above LJ Set the holding potential to 50 mV using the HOLD potentiometer 12 setting 050 reading 050 mV and the HOLD potential polarity switch 25 set to Remember The HOLDING potentiometer sets the holding current in CC mode and the holding potential in VC mode L Set the control mode switch 6 to NORMAL GAIN ONLY LI Set the CAPACITY COMPENSATION to 0 and the GAIN 5 to 1 version 4 8 page 37 TURBO TEC 03X User Manual L Enable the OSCILLATION
67. ence electrode to REF at the potential headstage L Insert potential and current electrode into the electrode holders Check if the silver wires of both electrodes are well chlorided and in contact with the electrode solution and connect them to the respective headstages Immerse both electrodes into the bath and make the basic settings see chapter 6 _ Turn on the amplifier version 4 8 page 47 TURBO TEC 03X User Manual m L Compensate the potential offset for both electrodes see chapter 8 2 measure the electrode resistance for both electrodes see chapter 8 3 The resistances should be 700 KQ to 1 2 MQ for the potential electrode and 500 kQ to 1 MQ for the current electrode As mentioned above it is usually not necessary to compensate for the electrode capacity for the potential electrode see chapter 8 4 Set the upper display to POTENTIAL ELECTRODE Insert the potential electrode into the oocyte The potential electrode is inside the cell if you read a membrane potential of about 50 mV to 60 mV It s a good idea to activate the audio monitor Then you can look through the microscope while hearing the membrane potential Remember The membrane potential of the oocyte is strongly dependent on the condition of the oocyte leaky or not the experimental solutions and the membrane proteins channels transporters that are expressed d m If your reference electrode can b
68. ent receptor potential domain of vanilloid receptor I in channel gating J Neurosci 27 11641 11650 LI Kottgen M Benzing T Simmen T Tauber R Buchholz B Feliciangeli S Huber T B Schermer B Kramer Zucker A Hopker K Simmen K C Tschucke C C Sandford R Kim E Thomas G amp Walz G 2005 Trafficking of TRPP2 by PACS proteins represents a novel mechanism of ion channel regulation The EMBO Journal 24 705 716 Incorporation of Proteins in the Oocyte Membrane LI Caprini M Fava M Valente P Fernandez Ballester G Rapisarda C Ferroni S amp Ferrer Montiel A 2005 Molecular Compatibility of the Channel Gate and the N Terminus of S5 Segment for Voltage gated Channel Activity Journal of Biological Chemistry 280 18253 18264 L1 Clyne J D Wang L F amp Hume R I 2002 Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor J Neurosci 22 3873 3880 LI Everts I Petroski R Kizelsztein P Teichberg V I Heinemann S F amp Hollmann M 1999 Lectin Induced Inhibition of Desensitization of the Kainate Receptor GluR6 Depends on the Activation State and Can Be Mediated by a Single Native or Ectopic N Linked Carbohydrate Side Chain J Neurosci 19 916 927 L Gu Q B Zhao J X Fei J amp Schwarz W 2004 Modulation of Na K pumping and neurotransmitter uptake by beta amyloid Neuroscience 126 61 67 version 4
69. entials cmr gt 80 dB Input resistance gt 10 Q operating voltage 15 V Electrode connector BNC with driven shield driven shield range 15 V output impedance 250 Q Reference connector bath gold plated SUBCLIC grounded shield Ground connector 2 3 mm connector or headstage enclosure Size 65x25x25 mm headstage enclosure is connected to ground Holding bar diameter 8 mm length 10 cm Current headstage standard voltage Operating voltage range X150 V standard Input resistance gt 10 Q can be internally trimmed Electrode connector gold plated SMC connector grounded shield Size 105x55x35 150V Current range 150 uA 1 MO standard Current range switch optional x0 1 x1 x2 x5 other ranges available Bandwidth and speed of response Full power bandwidth Rg 2 0 gt 100 kHz Rise time 10 90 30 us current pulse of 100 uA applied to Ret 1 MQ Bandwidth switch wide band or 10 Hz for simultaneous patch clamp recordings Current electrode parameter controls Offset compensation ten turn control 500 mV version 4 8 page 69 TURBO TEC 03X User Manual Potential electrode parameter controls Capacity compensation range 0 30 pF ten turn control Offset compensation 300 mV ten turn control POTENTIAL OUTPUTS Potential electrode sensitivity x10 mV output impedance 50 Q output voltage range 15 V Current electrode sensitivity x10 mV output impedance 250 O ou
70. ents concerning current clamp mode of the amplifier are located at the right half of the front panel Elements related to voltage clamp mode are found on the left side of the front panel Each control element has a label and frequently a calibration e g CURRENT OUTPUT SENSITIVITY V u A version 4 8 page 13 TURBO TEC 03X User Manual G9 G9 G2 oo 6909 Q9 AUDIO MONITOR TURBO TEC 03X e npi O 0 0 Figure 3 TURBO TEC 03X front panel view the numbers are related to those in the text below version 4 8 page 14 TURBO TEC 03X User Manual 1 POWER switch POWER Switch to turn POWER on switch pushed or off switch released OSCILLATION SHUTOFF unit OSCILLATION SHUTOFF The OSCILLATION SHUTOFF unit consists of 2 DISABLED RESET switch 3 OSCILLATION SHUTOFF LED and 34 THRESHOLD potentiometer In SHUTOFF condition the amplifier is set into CC mode and all outputs including holding current and CAPACITY COMPENSATION are disabled The inputs and the ELECTRODE RESISTANCE test are activated 3 OSCILLATION SHUTOFF LED Indicates whether the OSCILLATION SHUTOFF circuit is in SHUTOFF condition LED red or not LED green 34 THRESHOLD potentiometer Control to set the activation THRESHOLD of the OSCILLATION SHUTOFF circuit potentiometer linear clockwise range 0 1200 mV 2 DISABLED RESET switch Switch to DISABLE the OSCILLATION SHUTOFF unit or to RESET
71. ernally controlled variable gain proportional gain set with the GAIN control The amplified error signal is fed into the current source which injects the necessary amount of current into the cell to compensate for the ionic fluxes across the cell membrane labeled active in Figure 1 and Cell activity in Figure 2 and as a result to keep the membrane potential as close as possible to the command signal The injected current is recorded by a differential amplifier in the current headstage and is under stable conditions a direct measurement of the ion movements across the cell membrane Note To keep the VC error signal as small as possible it is desirable to use high GAIN settings But if the GAIN is set too high the system becomes unstable and begins to oscillate Therefore the OSCILLATION SHUT OFF unit should be activated when setting the GAIN Control Modes of VC The control circuit of the TEC 03X systems is operated with two 10 turn controls and a mode select switch One of three modes can be selected version 4 8 page 11 TURBO TEC 03X User Manual NORMAL Gain only SLOW Integrator FAST Series resistance compensation In the NORMAL mode only the proportional gain described above is active The gain is set by the GAIN potentiometer In the SLOW mode the controller is converted to a PI proportional integral system The added integrator improves control performance for slow signals The SLOW mode is designed
72. ers and some theoretical aspects are described in chapter 12 2 version 4 8 page 41 TURBO TEC 03X User Manual 8 7 Installing and Testing a Data Acquisition Board from National Instruments The following procedure is related to installation of E series and B sereies data acquisition hardware using NI DAQ 6 9 3 Newer DAQ versions and M series boards have a slightly different MAX user interface Please contact npi electronic if you encounter problems in installation data acquisition hardware with the TEC 03X CW Basic Test of INT 20X and Data Acquisition board 4 Disconnect the INT 20X from all devices including multifunction I O board from NI 4 Test connection between digital ground and analog ground Measure at the shields of the respective BNCs Ai and Do respectively and at the banana plugs AGND and DGND respectively The grounds should not be connected The resistance between AGND and the Ai channels should be around 1 MQ The resistance between DGND and the Do channels should be around 250 Q Q Connect the NI board to the INT 20X but no other devices Prove the ground connections again All BNC shields and ground plugs should now be connected and should have connections to the ground at the computer Check this by connecting AGND to a metal screw of the computer housing Q Check the resistance between Ai channels and AGND The resistance must be 1 MQ If not check if there is voltage at the Ai channel normally there shoul
73. for recording ligand gated currents which in general are slower than voltage activated currents In the FAST mode the integrator is switched off and a series resistance compensation circuit is activated The amount of compensation is set by a ten turn control Series resistance compensation improves the performance of the clamp system especially if fast voltage activated currents are recorded PI Controller SLOW Mode The TEC 03X system is equipped with Proportional Integral PI control loops The VC error signal command minus recorded signal is amplified by the GAIN amplifier and applied in addition to an integrator with variable time constant Consequently amplification becomes very large for signals with frequencies below the corner frequency of the integrator reciprocal to the time constant and this improves the control process Briefly the integrator can be understood as an automatic gain setting for slow signals improving clamp accuracy The adjustment of the PI control loop is described in chapter 12 see also Polder and Swandulla 2001 Series Resistance Compensation FAST Mode The differential recording arrangement see chapter 5 1 Figure 1 and Figure 10 suppresses only series resistances outside the cell In most cells there is also an internal series resistance e g the resistivity of the cytoplasm of cell organelles etc These series resistances could cause a current proportional potential error in the voltage cla
74. g K currents larger then 1004 A with current electrodes up to ca 500 KQ 200 KQ The maximum current is ca 300 uA 500 uA For measuring smaller currents another headstage with four ranges is available x0 1 x0 2 x0 5 and x1 All current input and output signals have to be multiplied by the factor that is set at the 4 position switch at the headstage e g in the x0 1 position all current input and output signals must be divided by 10 and the reading of the current electrode resistance must be multiplied by 10 Note We recommend always measuring the electrode resistance in x1 position version 4 8 page 28 TURBO TEC 03X User Manual 6 Setting up the TEC 03X The following steps should help you to set up the TEC 03X correctly Always adhere to the appropriate safety measures see chapter 1 The headstages are very sensitive and may be damaged by static electricity This can be avoided by touching a grounded metal surface when handling headstages If a headstage is not used the input should always be connected to ground either using an appropriate connector or with aluminum foil wrapped around the headstage After unpacking the TEC 03X is attached to the setup by assembling the electrical connections It is assumed that a passive 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 All numbers are related to those in Figure 3 Electric
75. gated Sodium Channel Is Involved in Channel Activation J Biol Chem 276 27831 27839 LJ Hilber K Sandtner W Kudlacek O Schreiner B Glaaser I W Sch tz W Fozzard H A Dudley S C amp Todt H 2002 Interaction between Fast and Ultra slow Inactivation in the Voltage gated Sodium Channel J Biol Chem 277 37105 37115 LI Szendroedi J Sandtner W Zarrabi T Zebedin E Hilber K Dudley S C Fozzard H A amp Todt H 2007 Speeding the Recovery from Ultra Slow Inactivation of Voltage Gated Na Channels by Metal Ion Binding to the Selectivity Filter A Foot on the Door Biophys J LI Volk T Konstas A A Bassalay P Ehmke H amp Korbmacher C 2004 Extracellular Na removal attenuates rundown of the epithelial Na channel ENaC by reducing the rate of channel retrieval Pflugers Arch 447 884 894 Ca Channels LI Hoda J C Zaghetto F Koschak A amp Striessnig J 2005 Congenital Stationary Night Blindness Type 2 Mutations 229P G369D L1068P and W1440X Alter Channel Gating or Functional Expression of Cavl 4 L type Ca2 Channels Journal of Neuroscience 25 252 259 K Channel Gating Currents LJ McCormack K W J Joiner and St H Heinemann 1994 A Characterization of the Activating Structural Rearrangements in Voltage Dependent Shaker K Channels Neuron Vol 12 301 315 version 4 8 page 59 TURBO TEC 03X User Manual Expression of Plant Channel
76. he CURRENT ELECTRODE OFFSET 16 potentiometer Note If a cell model is connected the OFFSET controls should read values around 5 otherwise it is likely that the headstages or the amplifier are damaged If microelectrodes are used unusual high OFFSETs are a sign of badly chlorinated silver wires or unwanted grounding of the bath 8 3 Electrode Resistance Test The electrode resistance is dependent on the tip diameter of the electrodes and may reveal whether electrodes are broken or clogged Therefore a resistance measurement test for both microelectrodes is included in the TEC 03X The test operates independently of any other adjustments assuming that all microelectrodes are in contact with a grounded bath zero potential The measured resistance is independent of tip potentials and is automatically displayed on the upper digital display in MQ Furthermore the electrode resistance can be tested even if the electrode is inside a cell The measurement is performed by applying square current pulses of 10 nA to the respective microelectrode The voltage deflection caused by this injection is recorded and processed to give a direct reading in MQ on the digital display version 4 8 page 35 TURBO TEC 03X User Manual Important The electrode resistance test is also a test of the correct function of the respective headstage The resistance test gives only a rough estimate of the electrode resistance The value for the current electrod
77. he appropriate safety regulations see chapter 1 Please turn power off when connecting or disconnecting the potential headstage from the POTENTIAL HEADSTAGE connector version 4 8 page 18 TURBO TEC 03X User Manual 22 CURRENT HEADSTAGE connector The CURRENT HEADSTAGE is connected via a flexible cable and a 15 pole connector to the mainframe see also chapter 5 2 CAUTION CURRENT HEADSTAGE HGH VOUAGE Caution Please always adhere to the appropriate safety regulations see chapter 1 Please turn power off when connecting or disconnecting the current headstage from the CURRENT HEADSTAGE connector CURRENT OUTPUT unit The CURRENT OUTPUT unit consists of 24 CURRENT OUTPUT FROM HEADSTAGE connector and 26 CURRENT OUTPUT connector 24 CURRENT OUTPUT FROM HEADSTAGE connector BNC connector providing the CURRENT OUTPUT signal directly from the CURRENT HEADSTAGE sensitivity 0 1 V uA 26 CURRENT OUTPUT connector BNC connector providing the CURRENT OUTPUT signal after passing the CURRENT OUTPUT conditioning unit see also CURRENT OUTPUT conditioning unit earlier in this chapter 27 CC LED 30 VC LED cc LEDs indicating the selection of Current Clamp mode 27 or Voltage Clamp mode 30 28 MODE OF OPERATION switch Switch to select the MODE OF OPERATION VC Voltage Clamp OFF In this position the amplifier is in current clamp mode but does not apply any current to the cell i e only th
78. is rather large It is not intended that the current headstage is attached directly to a micromanipulator An electrode holder adapter is used instead The headstage is connected to the 19 cabinet by a flexible shielded cable through the CURRENT HEADSTAGE connector curent headstage Figure 9 current headstage with optional electrode holder and optional electrode holder adapter of the TEC 03X system version 4 8 page 26 TURBO TEC 03X User Manual The current headstage has the following elements 1 CeL Connector for the current electrode grounded shield 2 BANDWIDTH 10 Hz switch for low bandwidth 10 Hz operation WIDEBAND full bandwidth 10 Hz low noise for simultaneous patch clamp recording 3 headstage cable to amplifier Usually the current electrode is connected to the headstage via an electrode holder that fits into the electrode adapter that is linked to the current electrode connector The electrical connection between the electrolyte and the headstage is established using a carefully chlorinated silver wire in the electrode holder 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 and other problems for details see Kettenmann and Grantyn 1992 For optimal chlorinating of silver wires an automated apparatus is available contact npi for detai
79. is to be operated only at 115 230 V 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 discharges 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 CURRENT INJECTION HIGH VOLTAGE HEADSTAGE The current injection headstage has a 150 V output compliance In addition some TEC headstages are equipped with a driven shield electrode c
80. l The optimal configuration for the headstage connections is shown in Figure 10 This arrangement differential measurement arrangement ensures the most accurate measurement of the membrane potential The reference electrode REF is placed near the membrane of the oocyte and measures the bath potential extracellular potential The bath potential is subtracted from the intracellular potential recorded by the intracellular electrode Per Electrodes used for intracellular potential measurement in oocytes typically have resistances of 700 kQ up to 1 2 MQ Important The shield of the BNC connector is linked to the driven shield output and must not be connected to ground If REF is not used it must be connected to ground Warning This headstage contains amplifiers which may be damaged by static electricity This can be avoided by touching a grounded metal surface when changing or adjusting the electrodes If a headstage is not used the input should always be connected to ground either using an appropriate connector or with aluminum foil wrapped around the headstage In addition it is extremely important that the instrument is turned off when changing the headstage 5 2 Current Headstage The current headstage see Figure 9 contains the circuits for current injection and recording as well as the preamplifier for potential measurement of the current electrode see also chapter 8 2 and chapter 9 Since high voltage amplifiers are used it
81. ls The electrode adapter can be mounted into a commercial micromanipulator near the bath while the headstage is placed somewhere nearby Electrodes used for current injection into oocytes typically have resistances of 500 kQ to 1 MQ The unique advantage of the instruments in the Turbo TEC series is the voltage controlled current source output V C or V I converter for electrical compensation of the disturbances from the microelectrode during current injection i e high resistance and stray capacity see Polder 1984 Polder and Swandulla 1990 This current source is built into the current headstage The use of the current source output allows that the current is measured en route to the electrode This is an improvement in ease of use compared to the virtual ground method that requires an additional headstage Furthermore the current source method also provides an improved frequency response of the voltage clamp control circuit On all TEC systems for oocyte recordings the bandwidth of the current injection electronics can be limited to approximately 10 Hz by means of a switch BANDW on the current headstage see Figure 9 and Figure 10 This allows the use of a patch clamp amplifier for single channel recording simultaneously to recording macro currents with the TEC system without excessive noise from the two electrode clamp loop In this mode the clamp circuit is capable of following only slow changes i e to keep the steady state I
82. lution 1 V STEP i e 3V indicate a GAIN of 0 5 version 4 8 page 17 TURBO TEC 03X User Manual 23 8V 4 7V CURRENT FILTER FREQUENCY MONITOR connector BNC output connector monitoring the setting of 14 CURRENT FILTER Hz switch Resolution 1 V STEP i e 5V indicate a filter frequency of 10 kHz 16 CURRENT ELECTRODE OFFSET potentiometer Control to compensate the current electrode potential ten turn potentiometer symmetrical i e 0 mV 5 on the dial range 500 mV see chapter 8 2 17 CURRENT HEADSTAGE BIAS potentiometer With this 10 turn potentiometer the output current of the CURRENT HEADSTAGE headstage BIAS current can be tuned to 0 see chapter 8 1 18 POTENTIAL ELECTRODE OFFSET potentiometer Control to compensate the potential electrode potential ten turn potentiometer symmetrical i e 0 mV 5 on the dial range 300 mV see chapter 8 2 19 CAPACITY COMPENSATION potentiometer Control for the capacity compensation of the POTENTIAL electrode ten turn potentiometer clockwise range 0 30 pF see chapter 8 4 Usually not used in oocyte experiments Caution This circuit is based on a positive feedback circuit Overcompensation leads to oscillations that may damage the cell 20 POTENTIAL HEADSTAGE connector The POTENTIAL HEADSTAGE is connected via a flexible cable and a 12 pole connector to the mainframe see also chapter 5 1 Caution Please always adhere to t
83. ly test pulses to the cell model Connection to the BNC and SUBCLICK connector respectively gives access to the cell via a potential and current electrode with 1 MQ resistance In the upper position the Rm switch simulates a cell version 4 8 page 32 TURBO TEC 03X User Manual membrane with a resistance of 10 kQ In the lower position a cell membrane with 100 kQ is simulated The membrane capacity is always 100 nF see also chapter 8 5 8 Test and Tuning Procedures Important The TEC 03X should be used only in warmed up condition i e 20 to 30 minutes after turning power on The following test and tuning procedures are necessary for optimal recordings It is recommended first to connect a cell model to the amplifier to perform some basic adjustments and to get familiar with these procedures It is assumed that all connections are as described in chapter 6 All numbers are related to those in Figure 3 8 1 Current Headstage Bias Current Adjustment Caution It is important that this tuning procedure is performed ONLY after a warm up period of at least 30 minutes The tuning procedure must be performed regularly at least once a month with great care since the bias current changes over time and it determines the accuracy of the TEC system The TEC 03X is equipped with a high voltage current source that is connected to the current injecting electrode and performs the current injection see chapter 4 1 This current source h
84. meter 17 The current is 0 if the voltage deflection is 0 Now all current outputs CURRENT OUTPUT FROM HEADSTAGE 24 CURRENT OUTPUT 26 and the CURRENT DISPLAY 9 should also read 0 version 4 8 page 34 TURBO TEC 03X 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 i e set to 0 carefully with the OFFSET controls 16 and 18 before recording from a cell The OFFSET compensation is done in CC mode of the amplifier When adjusting the OFFSETs make sure that no current flows through the electrodes Thus it is recommended to disconnect COMMAND INPUT 31 and to disable the HOLD unit see chapter 4 2 Potential Electrode LI Switch the reading of the upper digital display 10 to POTENTIAL ELECTRODE using the electrode selector POTENTIAL 7 The display 10 shows the potential of the potential electrode in XXX mV LI Compensate the OFFSET with the POTENTIAL OFFSET 18 potentiometer Current Electrode LI Switch the reading of the upper digital display 10 to CURRENT ELECTRODE using the electrode selector POTENTIAL 7 The display 10 shows the potential of the current electrode in XXX mV L Compensate the OFFSET with t
85. microelectrode is recorded by a buffer amplifier in the current headstage Both potential recording units can be compensated for offsets version 4 8 page 9 TURBO TEC 03X User Manual The frequency response of the potential electrode low pass characteristic due to stray capacities is compensated for by a feedback circuit negative capacity compensation CAPACITY COMPENSATION and a driven shield arrangement for an overview see Ogden 1994 Since in most oocyte experiments microelectrode resistances are usually in the one MQ range or below it is not necessary to use CAPACITY COMPENSATION Current Injection and Measurement The current injection is performed by means of a glass microelectrode that is connected to the current headstage CeL A description of the current headstage is given in chapter 5 2 The unique advantage of the instruments in the Turbo TEC series is the voltage controlled current source output V C or V I converter for electrical compensation of the disturbances from the microelectrode during current injection i e high resistance and stray capacity see Polder 1984 Polder and Swandulla 1990 Polder and Swandulla 2001 This current source is built into the current headstage The use of a current source output allows measurement of the current en route to the electrode This is an improvement in accuracy over the virtual ground method and also an improvement in ease of use since it does not require an a
86. mp mode i e an unwanted change in the membrane potential during a current flow This change can be partially compensated by current proportional amplification in the control circuit The series resistance compensation circuit compensates resistances in series with the cell membrane by feeding a fraction of the recorded current signal back into the control loop The output signal is enhanced in a current proportional manner and the effect of the series resistance is minimized The integrator is switched off Important Series resistance compensation is done by a positive feedback in the control circuit which has a tendency to oscillate Thus whenever possible repositioning of the electrodes is recommended to minimize series resistance effects This compensation circuit should be used only as a last resort and with OSCILLATION SHUTOFF unit activated Improvement of the Control Properties Control circuits with negative feedback tend to be unstable as a result of delays inherent to the system e g low pass characteristics of the microelectrodes or positive feedback caused by capacitive couplings between the electrodes version 4 8 page 12 TURBO TEC 03X User Manual In voltage clamp systems the control properties can be substantially improved by shielding the electrodes from each other Often shielding of the current electrode suffices to reduce the coupling capacity between the electrodes This shield can be connected to GND of the cur
87. mportant The controller must be used in P mode INTEGRATOR OFF since parasitic oscillations may occur due to the limited bandwidth of the current source two integral components in a closed loop form an oscillator see Froehr 1985 for details Warning The current injection headstage has a 150 V output compliance After turning on the instrument it must be ensured that the interior contact and the shield of the electrode plug and of the cable that is connected to this plug cannot be touched In addition it is extremely important that the instrument is turned off when changing or adjusting the electrodes version 4 8 page 27 TURBO TEC 03X User Manual CURRENT HEADSTAGE POTENTIAL HEADSTAGE CAUTION A HIGH VOLTAGE PEL REF GND POTENTIAL ELECTRODE CURRENT ELECTRODE REFERENCE ELECTRODE Figure 10 connections of the headstages Options The TEC 03X can be ordered with a current headstage providing four ranges x0 1 x1 x2 and x5 The x1 position corresponds to the calibrations at the front panel In the x0 1 position all current input and output signals must be divided by 10 and the reading of the current electrode resistance must be multiplied by 10 In the x2 x5 position the current signals have to be multiplied by 2 by 5 while the reading of the current electrode resistance must be divided by 2 by 5 These modes are suitable for recording very large currents e
88. nd negative modulation of GABA A receptor function by niflumic acid a nonsteroidal anti inflammatory drug Mol Pharmacol 64 753 763 L1 Wetzel C H Oles M Wellerdieck C Kuczkowiak M Gisselmann G and H Hatt 1999 Specificity and Sensitivity of a Human Olfactory Receptor Functionally Expressed in Human Embryonic Kidney 293 Cells and Xenopus Laevis Oocytes J Neurosci 19 17 7426 7433 Transporter expressed in the Oocyte Membrane CL Boll M Foltz M Rubio Aliaga I Kottra G amp Daniel H 2002 Functional characterization of two novel mammalian electrogenic proton dependent amino acid cotransporters J Biol Chem 277 22966 22973 L Carpaneto A Geiger D Bamberg E Sauer N Fromm J amp Hedrich R 2005 Phloem localized proton coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force Journal of Biological Chemistry 280 21437 21443 L Cui W W Low S E Hirata H Saint Amant L Geisler R Hume R L amp Kuwada J Y 2005 The zebrafish shocked gene encodes a glycine transporter and is essential for the function of early neural circuits in the CNS J Neurosci 25 6610 6620 LI Dunlop J MclIlvain H B Carrick T A Jow B Lu Q Kowal D Lin S Greenfield A Grosanu C Fan K Petroski R Williams J Foster A amp Butera J 2005 Characterization of novel aryl ether biaryl an
89. nnector for connecting a multi function I O board of the B E or M Series of National Instruments 8 Ao0 and Aol connectors BNC output connectors providing DACO and DACI of the National Instruments board Important These signals are generated from the NI board and Ao0 DACO is connected additionally to the command input port of the amplifier If the user unintentionally connects a signal to COMMAND INPUT BNC at the front panel this signal will be added to the value of the command input signal for the TEC 03X CW generated by CellWorks A procedure to test the data acquisition board from National Instruments is described in chapter 8 7 version 4 8 page 24 TURBO TEC 03X User Manual 5 Headstages The TEC 03X comes with two standard headstages the POTENTIAL HEADSTAGE for recording the membrane potential and the CURRENT HEADSTAGE for injecting current into the cell 5 1 Potential Headstage The potential headstage is housed in a small box containing the buffer amplifiers to record the membrane potential see Figure 8 The enclosure of the headstage is linked to ground A metal bar is mounted to this box allowing direct attachment to a micromanipulator The headstage is connected to the 19 cabinet by a flexible shielded cable via the POTENTIAL ELECTRODE connector The recording microelectrode can be connected by using an electrode holder with a BNC socket The electrical connection between the electrolyte and the headstage i
90. now in P mode proportional only Watch the potential output and increase the GAIN so that no overshoot appears LI Switch the control mode switch to SLOW INTEGRATION to activate the integrator The controller is now in PI mode proportional integral Tune the GAIN again see above LI Watch the potential output and tune the time constant until the overshoot of the desired tuning method appears see also Figure 14 Response to a command variable Response to a disturbance variable step Linear optimum LO aperiodic response P Controller un e no slow response no overshoot slow response large deviation Absolute value optimum AVO PI Controller fastest response 4 overshoot del c slow response slight deviation Symmetrical optimum SO Unsmoothed com mand variable PI Controller Smoothed com mand variable 1 fast response 43 overshoot slow response 8 overshoot dires very fast response slight deviation LO Only a P Controller is used The response to a command step is slow and has no overshoot potential output The response to a disturbance e g an activating channel is slow and has a large deviation AVO A PI Controller is used The response to a command step is very fast with 4 overshoot potential output The response to a disturbance e g an activating channel is slow and has a slight deviation SO
91. of a clamp system can be tested by specific test signals e g stimulus evoked signals etc Very important All parameters that influence clamp performance microelectrode offsets capacity compensation etc must be optimally tuned before starting the PI controller tuning procedure see chapter 8 Always activate the OSCILLATION SHUTOFF unit The tuning procedure involves the following steps Again The main criterion of tuning is the amount of overshoot seen at the potential output Tuning of the proportional gain L Set the voltage clamp control mode switch to NORMAL GAIN only _ Use the command input without smoothing and apply adequate identical pulses to the cell e g small hyperpolarizing pulses _ The controller is in P mode proportional only Watch the potential output and rise the GAIN so that no overshoot appears LO method The response to a command step is slow and has no overshoot potential output The response to a disturbance e g synaptic input or an activating channel is slow and has a large deviation Since the integral part of the controller is disconnected a steady state error in the range of a few percents will be present Tuning the integrator SLOW mode _ Reconnect the integrator to form the complete PI controller by setting the voltage clamp control mode switch to SLOW _ Apply adequate test pulses without filtering _ Adjust the integrator time constant 4 Figure 3 to achieve
92. on Journal of Biological Chemistry C500064200 L Collins A Wang H amp Larson M K 2005 Differential Sensitivity of Kir2 Inward Rectifier Potassium Channels to a Mitochondrial Uncoupler Identification of a Regulatory Site Molecular Pharmacology 67 1214 1220 L Decher N Pirard B Bundis F Peukert S Baringhaus K H Busch A E Steinmeyer K amp Sanguinetti M C 2004 Molecular Basis for Kv1 5 Channel Block CONSERVATION OF DRUG BINDING SITES AMONG VOLTAGE GATED K CHANNELS Journal of Biological Chemistry 279 394 400 LI Decher N Renigunta V Zuzarte M Soom M Heinemann S H Timothy K W Keating M T Daut J Sanguinetti M C amp Splawski I 2007 Impaired interaction between the slide helix and the C terminus of Kir2 1 a novel mechanism of Andersen syndrome Cardiovasc Res 75 748 757 L Delling M Wischmeyer E Dityatev A Sytnyk V Veh R W Karschin A amp Schachner M 2002 The neural cell adhesion molecule regulates cell surface delivery of G protein activated inwardly rectifying potassium channels via lipid rafts J Neurosci 22 7154 7164 LI Derst C Hirsch J R Preisig M ller R Wischmeyer E Karschin A D ring F Thomzig A Veh R W Schlatter E Kummer W and Daut J 2001 Cellular localization of the potassium channel Kir7 1 in guinea pig and human kidney Kidney International 59 2197 2205 L Derst C Karschin C
93. onnector After turning on the instrument do not touch the interior contact and the shield of the electrode plug and of the cable that is connected to this plug In addition it is extremely important that the instrument is turned off when changing or adjusting the electrodes HANDLING Please protect the device from moisture heat radiation and corrosive chemicals version 4 8 page 6 TURBO TEC 03X User Manual 2 Introduction This instruction manual describes the most important functions and operation modes of the TURBO TEC 03X Voltage Current Clamp amplifier A short introduction to the theory and practice of the voltage clamp and current clamp technique is also included as far as it is necessary for understanding the operation of this instrument Many books and articles are available on these techniques A selection of this literature is given at the end of this manual see chapter 13 The TURBO TEC 03X is an accurate and extremely fast voltage and current clamp V C amplifier for studying large membrane currents It is based on the standard two electrode approach and is an ideal system for recording from oocytes A significant improvement over other two electrode clamp amplifiers is that the TURBO TEC 03X fully compensates the current injecting microelectrode and needs no virtual ground for recording membrane currents In addition a powerful PI proportional integral voltage clamp controller and an improved series resistan
94. rent headstage The potential electrode can be shielded using a driven shield arrangement see Ogden 1994 Note In experiments with oocytes shielding of the electrodes is usually not necessary If shielding is required in special experimental situations we recommend learning about this technique from articles such as Ogden 1994 and Smith 1985 The correct setting of the C compensation increases the speed of response of the control loop but also increases the noise The settings of the different parameters result in a compromise between the stability accuracy noise and control speed Adjustment criteria are discussed in chapter 12 also see Polder and Swandulla 2001 Some practical hints are given in sections 8 6 and 12 2 4 2 Description of the Front Panel The TURBO TEC 03X system is made up of a 19 basic system with a built in power supply and two headstages a smaller one for potential recording and a bigger one for current injection and recording In the following description numbering of the items is based on the diagram in Figure 3 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 Current output unit and are described as units regardless of the order of numbers In general the front panel of the TURBO TEC 03X is arranged so that elem
95. ride channel of Torpedo marmorata Am J Physiol 274 C966 C973 L Liantonio A Picollo A Babini E Carbonara G Fracchiolla G Loiodice F Tortorella V Pusch M amp Conte C D 2005 Activation and inhibition of kidney CLC K chloride channels by fenamates Molecular Pharmacology 96 165 173 LI Nagel G Szellas T Riordan J R Friedrich T and K Hartung 2001 Non specific activation of the epithelial sodium channel by the CFTR chloride channel EMBO reports Vol 21 249 254 LI Nagel G Barbry P Chabot H Brochiero E Hartung K amp Grygorczyk R 2005 CFTR fails to inhibit the epithelial sodium channel ENaC when expressed in Xenopus laevis oocytes J Physiol 564 671 682 LI Sacchi O Rossi M L Canella R amp Fesce R 2003 Voltage and activity dependent chloride conductance controls the resting status of the intact rat sympathetic neuron J Neurophysiol 90 712 722 LI Schnizler K Saeger B Pfeffer C Gerbaulet A Ebbinghaus Kintscher U Methfessel C Franken E M Raming K Wetzel C H Saras A Pusch H Hatt H amp Gisselmann G 2005 A Novel Chloride Channel in Drosophila melanogaster Is Inhibited by Protons Journal of Biological Chemistry 280 16254 16262 TRP Channels L Garcia Sanz N Valente P Gomis A Fernandez Carvajal A Fernandez Ballester G Viana F Belmonte C amp Ferrer Montiel A 2007 A role of the transi
96. s LI Becker D I Dreyer St Hoth J D Reid H Busch M Lehnen K Palme and R Hedrich 1996 Changes in voltage activation Cs sensitivity and ion permeability in H5 mutants of the plant K channel KAT1 Proc Natl Acad Sci USA Vol 93 pp 8123 8128 LJ Michard E Lacombe B Poree F Mueller Roeber B Sentenac H Thibaud J B amp Dreyer I 2005 A unique voltage sensor sensitizes the potassium channel AKT2 to phosphoregulation J Gen Physiol 126 605 617 L Philippar K Buchsenschutz K Abshagen M Fuchs I Geiger D Lacombe B amp Hedrich R 2003 The K channel KZMI mediates potassium uptake into the phloem and guard cells of the C4 grass Zea mays J Biol Chem 278 16973 16981 LI Tsunoda S P Ewers D Gazzarrini S Moroni A Gradmann D amp Hegemann P 2006 H4 pumping rhodopsin from the marine alga Acetabularia Biophys J K Channels LI Baltaev R Strutz Seebohm N Korniychuk G Myssina S Lang F amp Seebohm G 2005 Regulation of cardiac shal related potassium channel Kv 4 3 by serum and glucocorticoid inducible kinase isoforms in Xenopus oocytes Pflugers Arch 450 26 33 L Bayrhuber M Vijayan V Ferber M Graf R Korukottu J Imperial J Garrett J E Olivera B M Terlau H Zweckstetter M amp Becker S 2005 Conkunitzin S1 is the first member of a new Kunitz type neurotoxin family structural and functional characterizati
97. s established using a carefully chlorinated silver wire in the electrode holder 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 silver wires an automated chlorinating apparatus is available ACI 01 contact npi for details GND and REF are connected by flexible cables with appropriate connectors potential headstage 2 E EUNT 4 Py AMPLIFIER HEADSTAGE ws m 1s dd LONE electrode 7 ee EX erro holder Figure 8 potential headstage and electrode holder optional Headstage Elements 1 BNC connector for the electrode holder optional 2 REF reference electrode connector 3 GND ground connector 4 Headstage cable to amplifier Two electrodes an intracellular microelectrode Pm potential electrode and an extracellular electrode REF reference electrode are required for potential measurement Both are connected to high impedance buffers input resistance higher than 10 Q in the potential headstage In addition the bath surrounding the cell must have a solid ground connection Ag AgCl pellet or Agar bridge see Figure 10 that can carry large membrane currents flowing during voltage clamp experiments with oocytes version 4 8 page 25 TURBO TEC 03X User Manua
98. see Figure 10 and should originate from the potential headstage The enclosures of both headstages are grounded Multiple grounding should be avoided and all ground points should originate from a central point to avoid ground loops 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 This connection can be disrupted to avoid ground loops see Ogden 1994 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 version 4 8 page 22 TURBO TEC 03X User Manual TEC 03X CW In order to connect the amplifier directly to a computer with a National Instruments NI data acquisition board of the E Series the TEC 03X CW has additional connectors at the rear panel see Figure 6 2 TVNOUO3MIQIS O8 NOIISIKSOv viva O 1000F 299999 f j Figure 6 breakout box connectors of the TEC 03X CW 1 BIDIRECTIONAL COMMUNICATION connector option Optional 50 pole SCSI connector for BIDIRECTIONAL COMMUNICATION between CellWorks and TEC 03X CW BIDIRECTIONAL COMMUNICATION is performed using two digital ports of a PCI 6503 digital I O board NI linked to this connector see also CellWorks Manual 2 Ai2 Ai7 connectors BNC input connectors for Ai2 Ai7 of
99. t al 1992 Heteromeric NMDA receptors molecular and functional distinction of subtypes Science 256 1217 1221 L Rettinger J amp Schmalzing G 2003 Activation and desensitization of the recombinant P2X1 receptor at nanomolar ATP concentrations J Gen Physiol 121 451 461 L Rettinger J amp Schmalzing G 2004 Desensitization masks nanomolar potency of ATP for the P2X1 receptor J Biol Chem 279 6426 6433 L Schoepfer R A F ll and H R Polder 1996 EggWorks A New Control Software for the Entire Experimental Setup in Elsner N and H U Schnitzler G ttingen Neurobiology Report 1996 Thieme Verlag Stuttgart LI Schoepfer R G Buchholz J Planck and H R Polder 2000 CellWorks A Control Software for the Entire Experimental Setup in Jamal R and H Jaschinski eds Virtuelle Instrumente in der Praxis H thig Verlag Heidelberg LI Schroder Lang S Schwarzel M Seifert R Strunker T Kateriya S Looser J Watanabe M Kaupp U B Hegemann P amp Nagel G 2007 Fast manipulation of cellular cAMP level by light in vivo Nat Meth 4 39 42 LI Sinkkonen S T Mansikkamaki S Moykkynen T Luddens H Uusi Oukari M amp Korpi E R 2003 Receptor subtype dependent positive and negative modulation of GABA A receptor function by niflumic acid a nonsteroidal anti inflammatory drug Mol Pharmacol 64 753 763 Recordings from Muscle Cells Giant Fibers L Bhattacharya A
100. t is red an error had occurred during the last test and a number appears in the field Last Error Click the button Error Codes to open a window with explanations about this error code version 4 8 page 45 TURBO TEC 03X User Manual 9 Positioning of Electrodes The position of the electrodes plays an important role in tuning the clamp speed The position of the current electrode is especially crucial for homogeneous charging of the membrane capacitance one limiting factor of clamp speed If the current electrode is placed just at the edge of the oocyte i e with a small penetration depth the part of the membrane close to the electrode will be charged more quickly than the membrane at the other side of the oocyte Thus the voltage controlled by the clamp is different This leads to a capacitive transient with a slow tail see Figure 18 right side Placing the current electrode central in the oocytes i e with a large penetration depth leads to a homogeneous charging of the membrane In this case the capacitive transient can be kept short by critical compensating Rs see Figure 18 left side Im Vi Im Joy co S T im ff me No m is f Rg comp Critical Critical Rg comp I Rg comp m More Rg comp Figure 18 penetration depth of the current electrode and consequences for the clamp EA Current Figure 19 demonstration of electrode positioning Figure 19 shows the side view of positioning the el
101. the National Instruments board Note Ai and Ail are not available at the rear panel of the TEC 03X CW These channels are used for recording current Ai0 and voltage signals Ail from the amplifier and therefore these signals are internally connected to AiO current and Ail voltage 3 TRIG OUT Output BNC connector providing a TTL 5 V trigger if a pulse routine or snapshot in CellWorks is activated see Figure 7 This signal indicates that CellWorks is waiting for a trigger signal before starting pulses and recording version 4 8 page 23 TURBO TEC 03X User Manual Start Start Data Acquisition Data Acquisition Begin of Begin of Wait for Trigger Wait for Trigger 5V TRIG OUT Py 0v Figure 7 timing of the TRIG OUT signal 4 A18 Ail5 9 pole male SUBD connector providing A18 Ail5 of the National Instruments board 5 POWER OUTPUT 12V 0 5A 9 pole female connector for devices controlled by digital pulses from CellWorks e g valves of a perfusion system Specification 12 V 0 5 A sufficient for most pinch valve manifolds e g ALA VM8 PG 6 TRIG IN TTL Input BNC connector for triggering CellWorks If the user has started a pulse or snapshot protocol in CellWorks via double click and CellWorks is configured to wait for a trigger a TTL pulse applied to this input will cause CellWorks to start the protocol 7 TO DATA ACQUISITION BOARD connector 68 pole SCSI co
102. thout ports one adapter for the current electrode holder TEC MOD passive cell model TEC MOD A active cell model Headstages with four current ranges version 4 8 page 71 TURBO TEC 03X User Manual Index A Abbreviations 5 ose oe eee 4 Absolute value optimum 52 AV Oe Method eese cette reist detecta 52 B bandwidth sosta e pis dite des 10 Basic setting Sininen innii 30 Bias Current Adjustment 33 breakoiit BOK 5o DO Ue 43 C cellamodel aoo oe p bte Mmi uie 30 COMNECION Ss soso deste arent 32 CUS SCHIPELON Jod estuve ie stie it ec ndes 31 operation iie recette ertet ree eed 32 CelIWOotKs 35 eth ctt 8 42 clamp performance sss 51 closed loop system esses 50 COMPONENTS iate coti se dite 8 Control tlie Or uuo tet os 50 current clamp mode OVETVICW ui ed NR dod edu 10 Current Injection and Measurement 10 D data acquisition boards 42 I rp 43 E Electrical connections 20 electrodes capacity compensation 36 GUISE Ps oai b oed pe ia EN 35 POSIVONINE soccer Govt eee eke 46 resistance test escetisiecatande sidecases nes 35 equivalent circuit amplifier 9 F TIgdi res scc cot ane ae ae E a 8 front panel items sesesess 15 front panel view eoe eves 14 H headstages CODTIe UOTIS usc
103. timum 32 MOMMA cs oes esteiet ac de la ditum 12 T ji 12 voltage clamp mode 10 technical data uie vett eie s 69 block diagram ise 11 test and tuning procedures 33 Control CIFCUTES us breed eo e 11 time constants soos eo ete fucrat bon eed 55 Iiproyetmehls co ed iter ms 12 tip potential eoo cst PRORA 35 model circuit for oocyte 47 Trouble Shooting 49 OVEL VIEWS 2 cece Roth tessa R E 10 V theory MN M 50 TGTITIB oue ue E sea avoid teoedeyu 38 52 voltage clamp control modes 11 PASC ps eoo Lu a Be 12 version 4 8 page 73
104. tput voltage range 15 V DISPLAY switch selected XXX mV AUDIO MONITOR Pitch correlated with potential signals OSCILLATION SHUT OFF Turns off current injection and capacity compensation function indicated by red green LED disabled off reset switch threshold set with linear control 0 1200 mV ELECTRODE RESISTANCE TEST both electrodes 100 mV MQ obtained by application of square current pulses 10 nA display XX X MQ selected automatically CURRENT OUTPUTS Uncompensated output signal sensitivity 0 1 V u A output impedance 50 Q output voltage range 15 V Compensated filtered output sensitivity 0 1 10 V uA 0 1 0 2 0 5 1 2 5 10V uA steps selected by rotary switch with low pass Bessel filter output impedance 50 Q Sensitivity monitor 1 47 Volt 1V switch position output impedance 50 Q DISPLAY X XX uA CURRENT OUTPUT FILTERS One pole standard version or four pole TEC 03X BF low pass Bessel filter 16 corner frequencies 20 50 100 200 300 500 700 1k 1 3k 2k 3k 5k 8k 10k 13k 20k Hz Frequency monitor 8 7 V 1 Volt switch position output impedance 50 Q CURRENT CLAMP standard current headstage Inputs l1 uA V Input resistance 2100 KQ HOLD X XX uA ten turn digital control with 0 switch maximum 10 uA Noise Potential output 100 uV pp Current output 200 pA pp with 1 MQ resistance and 10 kHz bandwidth internal four pole Bessel filters
105. ulation of cellular cAMP level by light in vivo Nat Meth 4 39 42 LI Strutz Seebohm N Werner M Madsen D M Seebohm G Zheng Y Walker C S Maricq A V amp Hollmann M 2003 Functional analysis of Caenorhabditis elegans glutamate receptor subunits by domain transplantation J Biol Chem 278 44691 44701 L Strutz Seebohm N Seebohm G Shumilina E Mack A F Wagner H J Lampert A Grahammer F Henke G Just L Skutella T Hollmann M amp Lang F 2005 Glucocorticoid adrenal steroids and glucocorticoid inducible kinase isoforms in the regulation of GluR6 expression J Physiol 565 391 401 version 4 8 page 64 TURBO TEC 03X User Manual LI Strutz Seebohm N Seebohm G Mack A F Wagner H J Just L Skutella T Lang U E Henke G Striegel M Hollmann M Rouach N Nicoll R A McCormick J A Wang J Pearce D amp Lang F 2005 Regulation of GluR1 abundance in murine hippocampal neurones by serum and glucocorticoid inducible kinase 3 J Physiol 565 381 390 LI Strutz Seebohm N Seebohm G Korniychuk G Baltaev R Ureche O Striegel M amp Lang F 2006 Additive regulation of GluR1 by stargazin and serum and glucocorticoid inducible kinase isoform SGK3 Pflugers Arch 452 276 282 L Sinkkonen S T Mansikkamaki S Moykkynen T Luddens H Uusi Oukari M amp Korpi E R 2003 Receptor subtype dependent positive a
106. urrents Thus for successful and reliable experiments it is necessary to tune the clamp loop Only if no intracellular series resistance is considered tuning of the clamp is performed according to optimization methods It depends on the type of experiment to which method one should follow see below o Linear Optimum LO with this method only the proportional part of the PI controller is used The response to a command step is slow but produces no overshoot The response to a disturbance is also slow with a large deviation of the membrane potential Clamp accuracy is a maximum of 90 9796 Finkel and Redman 1985 Therefore this method should only be used only if it is very important to avoid overshoots of the membrane potential o Absolute Value Optimum AVO uses the PI controller and provides the fastest response to a command step with very little overshoot maximum 4 The response to a disturbance is of moderate speed and the amplitude of the deviation is only half the amplitude obtained with LO It is applied if maximum speed of response to a command step is desirable e g if large voltage activated currents are investigated o Symmetrical Optimum SO uses also the PI controller and has the best performance compensating intrinsic disturbance signals The response to a command step shows a very steep rise phase followed by a considerable overshoot maximum 43 The response to a disturbance is fast and the amplitude of th
107. ween substituted cysteine residues located in the putative ATP binding site of the P2X1 receptor J Neurosci 27 1456 1466 L Morales A J Aleu LIvorra J A Ferragut J M Gonzales Ros and R Miledi 1995 Incorporation of reconstituted acetycholine receptors from Torpedo in the Xenopus oocyte membrane Proc Natl Acad Sci USA Vol 92 pp 8468 8472 L Nawrath H Wegener J W Rupp J Habermeier A and Closs E I 2000 Voltage dependence of L arginine transport by hCAT 2A and hCAT 2B expressed in oocytes from Xenopus laevis Am J Physiol 279 C1336 C1344 L Pertovaara A Ostergard M Anko M L Lehti Koivunen S Brandt A Hong W Korpi E R amp Panula P 2005 RFamide related peptides signal through the neuropeptide FF receptor and regulate pain related responses in the rat Neuroscience 134 1023 1032 L Rettinger J amp Schmalzing G 2003 Activation and desensitization of the recombinant P2X1 receptor at nanomolar ATP concentrations J Gen Physiol 121 451 461 L Schmidt C Werner M amp Hollmann M 2006 Revisiting the postulated unitary glutamate receptor electrophysiological and pharmacological analysis in two heterologous expression systems fails to detect evidence for its existence Molecular Pharmacology 69 119 129 LI Schroder Lang S Schwarzel M Seifert R Strunker T Kateriya S Looser J Watanabe M Kaupp U B Hegemann P amp Nagel G 2007 Fast manip

TEC-03X - NPI Electronic Instruments

Contents

Download Pdf Manuals

Related Search

Related Contents