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SEC-10LX Manual - NPI Electronic Instruments

1. Important The average membrane potential can be changed only through the VOLTAGE COMMAND INPUT If changes are necessary please select a short time constant 1 or 10 s CURRENT CLAMP INPUT The current clamp input CURRENT STIMULUS BNC connectors is connected in the VCcCC mode in a way that fast current stimuli can be applied to the electrode The condition for such recordings is a ratio of gt 1 1000 between current pulse duration and VCcCC time constant Slow long lasting current signals or DC such as the HOLDING current will be removed by the action of the VCcCC system In the fast VC mode the current clamp input is disconnected automatically In this way using the VCcCC mode fast current stimuli can be used to monitor conductance changes LITERATURE 1 Peters F D Czesnik A Gennerich amp D Schild 2000 Low frequency voltage clamp recording of voltage transients at constant average command voltage J Neurosci Meth Vol 99 129 135 2 Sutor B S Greiner Fischer B Schlosser 2000 Pharmacologically isolated NMDA EPSPs recorded at resting membrane potential of rodent neocortical neurons Soc Neurosci Abstr Vol 26 Part 1 p 352 3 Sutor B and H R Polder 2001 Slow Voltage Clamp A technique which allows switched current clamp recordings of synaptic potentials at voltage clamped holding potentials Pfliig Arch 441 R221 me JI for the Life Sciences CALIBRATION of the x0 1 RANGE LOW VOLTAGE HEADST
2. D Electronic Instruments for the Life Sciences _ pAs e made OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF THE SEC 10LX SINGLE ELECTRODE SYSTEMS WITH APPENDICES SEC Cell Model Tuning Capacity Compensation in SEC Amplifier Systems SEC EXT Headstage for Extracellular Recordings with SEC Systems Calibration of SEC Amplifiers with x10 Headstage Calibration of SEC Amplifiers with x0 1 Low Voltage Headstage Synchronization of Two or More SEC Amplifier Systems SEC Systems with VCcCC mode SEC Systems with DHC mode SEC Systems with Linear Mode VERSION 3 5 npi 2010 npi electronic GmbH Hauptstrasse 96 D 71732 Tamm Germany Phone 49 0 7141 9730230 Fax 49 0 7141 9730240 support npielectronic com http www npielectronic com 1 Safety Regulations VERY IMPORTANT Instruments and components supplied by npi electronic are NOT intended for clinical use or medical purposes e g for diagnosis or treatment of humans or for any other life supporting system npi electronic disclaims any warranties for such purpose Equipment supplied by npi electronic must be operated only by selected trained and adequately instructed personnel For details please consult the GENERAL TERMS OF DELIVERY AND CONDITIONS OF BUSINESS of npi electronic D 71732 Tamm Germany 1 2 3 4 5 GENERAL This system is designed for use in scientific laboratories and must be operated by trained staff only
3. 1998 Cardiac Gap Junction Channels Physiology Regulation Patophysiology and Pharmacology Karger Basel Dietzel I D D Bruns H R Polder and H D Lux 1992 Voltage Clamp Recording in Kettenmann H and R Grantyn eds Practical Electrophysiological Methods Wiley Liss New York Draguhn A Pfeiffer M Heinemann U and Polder H R 1997 A simple hardware model for the direct observation of voltage clamp performance under realistic conditions J Neurosci Methods 78 105 113 Finkel AS Gage PW 1985 Conventional Voltage Clamping With Two Intracellular Microelectrodes In Smith TG Lecar H Redman SJ Gage PW eds Voltage and Patch Clamping With Microelectrodes Chapter 4 The William and Wilkins Company Baltimore pp 47 94 Finkel AS Redman SJ 1985 Optimal Voltage Clamping With Single Microelectrode In Smith TG Lecar H Redman SJ Gage PW eds Voltage and Patch Clamping With Microelectrodes Chapter 5 The William and Wilkins Company Baltimore pp 95 120 Ferreira H H and Marshall M W 1985 The biophysical basis of excitability Cambridge University Press Cambridge Frohr F Orttenburger F 1981 Introduction to Electronic Control Engineering Siemens Aktiengesellschaft Berlin Munich Johnston D amp Brown T H 1983 Interpretation of Voltage Clamp Measurements in Hippocampal Neurons J Neurophysiol 50 464 486 Juusola M 1994 Measuring complex admittance and receptor current by single elect
4. The use of control theory for the design of voltage clamp systems a simple and standardized procedure for evaluating system parameters J Neurosci Methods 109 97 109 3 Richter D W Pierrefiche O Lalley P M amp Polder H R 1996 Voltage clamp analysis of neurons within deep layers of the brain J Neurosci Methods 67 121 131 4 Juusola M 1994 Measuring complex admittance and receptor current by single electrode voltage clamp J Neurosci Methods 53 1 6 5 Weckstrom M Kouvaleinen E amp Juusola M 1992 Measurement of cell impedance in frequency domain using discontinuous current clamp and white noise modulated current injection Pflugers Arch 421 469 472 6 Muller A Lauven M Berkels R Dhein S Polder H R amp Klaus W 1999 Switched single electrode amplifiers allow precise measurement of gap junction conductance Amer J Physiol Cell 276 4 C980 C988 7 Kapur A Yeckel M F Gray R amp Johnston D 1998 L Type calcium channels are required for one form of hippocampal mossy fiber LTP J Neurophysiol 79 2181 2190 8 Torkkeli P H Sekizawa S amp French A S 2001 Inactivation of voltage activated Na currents contributes to different adaptation properties of paired mechanosensory neurons J Neurophysiol 85 1595 1602 For more information please contact support npielectronic com Version 1 11 page 9 me J for the Life Sciences
5. 121 131 Sala F and Sala S 1994 Sources of error in single electrode voltage clamp techniques a computer simulation study J Neurosci Methods 53 189 197 Spruston N Jaffe D B Williams S H and Johnston D 1993 Voltage and space clamp errors associated with the measurement of electrotonically remote synaptic events J Neurophysiol 70 781 802 36 Spruston N and Johnston D 1992 Perforated patch clamp analysis of the passive membrane properties of three classes of hippocampal neurons J Neurophysiol 67 508 529 Staley K J Otis T S and Mody I 1992 Membrane properties of dentate gyrus granule cells Comparison of sharp microelectrode and whole cell recordings J Neurophysiol 67 1346 1358 Silver R A Traynella S F and Cull Candy S G 1992 Rapid time course miniature and evoked excitatory currents at cerebellar synapses in situ Nature 355 163 166 Williams S H and Johnston D 1991 Kinetic properties of two anatomically distinct excitatory synapses in hippocampal CA3 pyramidal neurons J Neurophysiol 66 1010 1020 Wilson W A and Goldner M M 1975 Voltage clamping with a single microelectrode J Neurobiol 6 411 422 37 Book chapters Armstrong C M and Gilly W F 1992 Access resistance and space clamp problems associated with whole cell patch clamping In Methods in enzymology Vol 207 Academic Press San Diego CA USA Dietzel I D Bruns D Polder H R an
6. 200 200 400 voltage mV 600 800 1000 Tuning Capacity Compensation capacity overcompensated voltage 100 150 voltage mV 200 4 250 capacity undercompensated voltage 300 100 150 200 voltage mV 250 300 350 3 5 4 45 5 5 5 6 6 5 time ms capacity well compensated voltage 400 Figure 4 Tuning of the coarse capacity compensation Time course of the signal at ELECTRODE POTENTIAL OUTPUT is shown holding current 1 nA duty cycle 14 switching frequency 2 kHz A model cell was connected electrode resistance 100 MQ Version 1 11 Tuning Capacity Compensation 120 capacity overcompensated 100 T Ka R a AAN AKA B a E ah e A aa potential current 100 150 200 20 300 voltage mV TARE a ANAUE UE TUNA E N time ms 120 406 capacity undercompensated 80 60 potential current 40 voltage mV O 44 AU nag 0 50 100 150 200 250 300 time ms capacity well compensated 1 00 Z ee ee Baa D ee a eT kaka Aa Jaa Jama AA ee oo So ao potential current 0 50 100 150 200 250 300 time ms Figure 5 Capacity compensation of the electrode in the bath electrode resistance 100 MQ Current stimulus 1 nA duty cycle 1 4 switchin
7. Fig 10b Determine electrode resistance or page 13 Decrease test pulse to 10 pA Approach cell until voltage trace Changes Fig 10c Release pressure from pipette if seal does not form apply suction Watch voltage signal until gigaseal is established Fig 10d Apply stronger suction to pipette until whole cell configuration is established Fig 10d Read membrane potential If necessary apply holding current as needed to ob tain the required value Correct bridge balance page 13 switch to CC mode If a change in voltage occurs correct CAP COMP using page 15 Watch control os cilloscope Intracellular Recording Step by Step e Start with amplifier in the CC mode Switch to BR mode a TT 100 pA 2 5 mV t10 nA 10 msec 25 mV Immerse the microelectrode into the bath to the same depth as during the experiment Tune potential offset page 12 Apply constant current Check the headstage capacity compen sation and correct if necessary page 14 Turn the holding current off Apply test pulse to e g 100 pA 1 nA Fig 11a Tune bridge balance page 13 Voltage signal should be flat Fig 11b Read electrode resistance page 13 Approach the cell The electrode is close to the cell if electrode resistance increases bridge balance appears un dercompensated if extracellular APs are visible or if the
8. i 40 kHz a jn po pa pamen Si karan ian po m i Lf Lf i Jor f kai us M a jerang an a i in ae dan mT 0 18 V 4 nA 1 M i e 2 O Lt Tus sampling Figure 1 Microelectrode artifact settling Compensation of stray capacities with a SEC 05 amplifier The upper trace shows the comparison between the standard capacity compensation and the fast compensation of the SEC systems After full compensation the settling time of the microelectrode is reduced to a few microseconds allowing very high switching frequencies here 40 kHz middle and lower trace The microelectrode was immersed 3 mm deep in cerebrospinal fluid Microelectrode resistance 45 MQ current 1 nA duty cycle 25 SwF switching frequency Original data kindly provided by Prof Diethelm W Richter Goettingen For details see 3 Version 1 11 page 2 Tuning Capacity Compensation 20 mi Se Oo conce ODI Con peeled i 10 rri pe from EC during vohage sep 40 pra CO mode Wil mode h f membre potential rcoscded by a th oeoa second akan anakan kh a Lanka paraha N rai an nae SO mi Sep copacitance undercomnper ated Da opima Commenechion iy 2 5 eri lt memntecne poetic seconded bry a TO rri send aide 10 ms an ii i pohentiol output from SEC during vothage shan Figure 2 Errors resulting from wrong compensation of the electrode capacity Original data
9. 1999 Weng S Lauven M Schaefer T Polontchouk L Grover R amp Dhein S 2002 Pharmacological modification of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation FASEB J 16 1114 1116 Simultaneous recordings with two SEC amplifiers Haag J and A Borst 1996 Amplification of high frequency synaptic inputs by active dendritic membrane processes Nature Vol 379 639 641 Haag J and Borst A 2001 Recurrent Network Interactions Underlying Flow Field Selectivity of Visual Interneurons J Neurosci 21 15 5685 5692 Haag J and Borst A 2002 Dendro Dendritic Interactions between Motion Sensitive Large Field Neurons in the Fly J Neurosci 22 8 3227 3233 Simultaneous intracellular recordings during voltammetric measurements Kudernatsch M Sutor B Cholinergic modulation of dopamine overflow in the rat neostriatum a fast cyclic voltammetric study in vitro Neuroscience Letters 181 107 112 1994 Schl sser B Kudernatsch M B Sutor B ten Bruggencate G 1995 d m and k opioid receptor agonists inhibit dopamine overflow in rat neostriatal slices Neuroscience Letters 191 126 130 Staining visualization imaging and infrared video microscopy Dodt H U and W Zieglgansberger 1994 Infrared videomicroscopy a new look at neuronal structure and functure Trends in Neurosciences Vol 19 No 11 453 458 Kapur A M Yeckel and D Johnston 2001 Hippocampal mo
10. CURRENT OUTPUT SENSITIVITY 0 1 V nA corresponds 0 01 VnA older instruments SEC 10L 0 125 V nA corresponds 0 0125 V nA 0 2 V nA corresponds 0 02 V nA 0 5 V nA_ corresponds 0 05 V nA 1 V nA corresponds 0 1 V nA 2V nA corresponds 0 2 V nA 5 V nA corresponds 0 5V nA 10 V nA corresponds 1 V nA CURRENT DISPLAY shows correct current XX X nA Maximum is 199 9 nA BRIDGE BALANCE XX X MQ 50 corresponds 5 MO Maximum is 99 9 MQ ELELCTR RESISTANCE DISPLAY XXX MQ 005 corresponds 5 MQ Maximum is 999 MQ Important If high resistance electrodes are used the capacity compensation must be set properly for exact determination of REL Example HOLDING CURRENT 100 corresponds to 10 nA the DISPLAY will show 10 0 nA INPUT at 1V nA BNC 1 V or INPUT at 0 1V nA BNC 10 V corresponds to 10 nA the DISPLAY will show 10 0 nA voltage at CURRENT OUTPUT is then CURRENT OUTPUT SENSITIVITY 0 1V nA 0 1 V CURRENT OUTPUT SENSITIVITY 1 V nA 1V CURRENT OUTPUT SENSITIVITY 10 VmnA 10V me Ji for the Life Sciences SEC EXT Headstage for Extracellular Recordings with npi SEC Systems The SEC EXT headstage extends the range of operation of SEC amplifiers to the field of extracellular recordings It has a differential high impedance input stage with capacity compensation for the non inverting input INPUT and a gain of ten This input stage is followed by a high pass filter with six corner frequencies 1 3 10 30 100 300 Hz e DC
11. SYNCHRONIZATION OF TWO OR MORE SEC AMPLIFIER SYSTEMS For recordings with two or more switched mode amplifiers in the same preparation it is necessary to synchronize the current injection and voltage recording timing protocols to avoid artifacts and excessive noise This is done by the synchronization inputs and outputs at the rear panel of the instruments based on a master slave arrangement The MASTER instrument provides the clock frequency from which the switching frequency is generated internally for the SLAVE instruments The MASTER instrument has a BNC connector marked SYNC OUTPUT TTL To this output the SLAVE instruments are connected by means of standard BNC cables The SLAVE instruments have a SYNC INPUT TTL BNC connector and a toggle switch marked INTERN EXTERN In the position EXTERN the instrument is used with the clock frequency of the MASTER instrument 1 e in SLAVE mode In the position INTERN the instrument can be used independently of the MASTER instrument Warning If this switch is in the EXTERN position and no signal is connected to the SYNC INPUT BNC the switched modes of the amplifier VC and CC are not working no switching frequency In the EXTERN position and with the MASTER instrument connected in both switched modes VC CC the switching frequency is controlled by the MASTER amplifier In this case current injection and sampling of current and potential signals is synchronous therefore all artifac
12. e after internal temperature has reached steady state values In most cases a warm up period of 20 30 minutes is sufficient HANDLING Please protect the device from moisture heat radiation and corrosive chemicals Warning 2 Introduction Safety precautions To prevent fire or shock hazard do not expose the unit to rain or moisture When working with the SEC systems manufactured by mpi electronic always adhere to the appropriate safety precau tions for operation of electronic devices Always use a 3 prong grounded outlet with a protected line plug Disconnect the mains power plug before opening the unit or when replacing the fuse or changing the line voltage Replace fuse with specified type only Refer all servicing to npi elec tronics SEC 05H high voltage headstage The SEC 05H headstage has a 150 V output compliance and is equipped with a driven shield electrode connector After turning on the unit ensure that the interior contact and the electrode plug shield and the shield of the cable connected to it can not be touched accidentially It is also ex tremely important to turn the unit off be fore changing or adjusting the electrode Static electricity hazard As all npi headstages are equipped with very sensitive FET amplifiers they must be handled with care FET amplifiers are sus ceptible to damage by static electricity Therefore the user should always touch a grounded metal surface before cha
13. fourth the switching frequency This results in a very sensitive control of the membrane potential with a steady state error of less than 1 and a fast response of the clamp to command steps The use of discontinuos current and voltage clamp in combination with high switching frequencies in npi s SEC systems yields five major advantages 1 The large recording bandwidth makes it possible to record even fast signals accu rately 2 High clamp gains up to 100 A V can be used in the voltage clamp mode 3 It is possible to voltage clamp very small cells with relatively short membrane time constants 4 Series resistance effects can be com pletely eliminated allowing for a correct membrane potential control even with high resistance electrodes 5 The true membrane potential is recorded in the voltage clamp mode whereas continuous feedback VC amplifiers only reflect the command potential Getting Started General System Description Modes of Operation All SEC systems consist of a 19 rack mountable unit with a built in power supply and a headstage which should be placed close to the recording site The recording electrode is connected to the headstage either via a SUBCLIC connector and a short cable or via a BNC connector and a suction electrode holder All electrode connectors use a driven shield approach to minimize the capacitive effect of the connecting cable Furthermore all low voltage headstages are equippe
14. kindly provided by Ajay Kapur For details see 7 Tuning Procedure see also chapter Getting Started pages 14 15 First part basic setting In SEC systems the capacity compensation of the electrode is split into two controls the coarse control in the headstage and a the fine control at the front panel of the amplifier The aim of the first part of the tuning procedure is to set the coarse capacity compensation at the headstage so that an optimal wide range of CAP COMP at the amplifier is achieved 11 Insert the electrode into the electrode holder and connect it to the amplifier 4 Immerse the electrode as deep as it will be during the experiment into the bath solution OY Set the CAP COMP control at the amplifier potentiometer 24 at the front panel to a value around 2 and turn COARSE CAPACITY COMPENSATION at the headstage to the leftmost position Selecta DUTY CYCLE as desired 24 at the front panel 4 Connect the BNC connector ELECTRODE POTENTIAL OUTPUT at the rear panel to an oscilloscope and trigger with the signal at BNC connector SWITCHING FREQUENCY also at the rear panel The oscilloscope should be in external trigger mode The time base of the oscilloscope should be in the range of 250 us OY Set the amplifier in CC mode and select the lowest switching frequency 1 to 2 kHz 4 Apply positive or negative current to the electrode using the HOLDING CURRENT control potentiometer 21 at the front panel 44 You
15. on the headstage Use a cell model for test measurement Connect the headstage to the HEADSTAGE INPUT 6 5 Turn the POWER switch on and let the amplifier warm up for at least 30 minutes Reset the OSCILLATION SHUT OFF LED green and select the BR mode All controls DISPLAY should be on low values and OFFSET should be in the range of 5 mV Adjust HEADSTAGE BIAS CURRENT to zero and nullify the potential offset using the OFFSET control page 16 In order to measure the resistance in the R mode place the electrode in the bath not in a cell 8 Apply a current step BR mode or CC mode to the CURR STIMULUS INPUT and adjust either the BRIDGE ilg 3 ti IE SE E BALANCE BR mode see page 13 or the cap comp CC mode see page 15 Now the system is ready for use 12 Getting Started a Tuning Procedures Adjusting the Bridge Balance overcomp correct undercomp 7 amp my 20 msot Fig 4a Adjustment of the bridge balance Lower lane current stimulus upper lanes potential output overcomp undercomp 1nd 20 msec Fig 4b Adjustment of the bridge balance after establishing the whole cell configuration Lower lane current stimulus upper lanes potential out put Patch Clamp Electrode Selection cS eee TT a ae eens soa ens ce PER eae eee sanan a Be a ao ees BAN k e SER kin rin r Tu tee
16. rear of the instrument are as square as possible Fig 5 middle By increasing the SWITCHING FREQUENCY D no considerable change in the amplitude and shape of these pulses should occur Adjusting the CAP COMP overcomp undercomp 560 pA 20 msec Fig 6 Adjustment of the capacity compensation Lower lane current stimulus upper lanes poten tial output in whole cell configuration The cap comp setting must be adjusted before and during an experiment After adjusting the CC setting headstage see p 14 switch off the HOLDING CURRENT bring toggle switch in mid die position and apply square pulses positive and negative of a few nA intracellular recordings or 10 to 100 pA patch clamp recordings and 5 to 10 msec duration to one of the CURRENT STIMULUS INPUT BNCs or The signals from the POTENTIAL OUTPUT and CURRENT OUTPUT BNCs are monitored on a Storage scope Set the SWITCHING FREQUENCY D and DUTY CYCLE to the desired values The switching frequency should be at least 12 to15 kHz Now turn the CAP COMP control clockwise until there is no artifact on the POTENTIAL OUTPUT BNC Fig 6 The shape of the injection pulses on the ELECTRODE POTENTIAL OUTPUT rear panel must be still square Fig 5 If not the CC control must be readjusted or the switching frequency is too high Getting Started 15 Headstage Bias Current Adjust ment 16 Getting Started The zero
17. should see a signal at the oscilloscope similar to those in Figure 3 Turn the COARSE CAPACITY COMPENSATION carefully clockwise until the signal becomes as square as possible lower diagram in Figure 3 Important If you use a model cell e g to train yourself in adjusting the capacity compensation the capacity of the model cell is always present Thus you will get an approximately square shaped signal with a slight slope as shown in Figure 4 lower panel 1 Increase the switching frequency to at least 15 kHz The amplitude and shape of the signal should not change considerably Version 1 11 page 3 Version 1 11 voltage mV voltage mV voltage mV 200 gt 100 100 200 4 300 4 400 500 Tuning Capacity Compensation capacity overcompensated voltage 600 50 100 4 150 4 200 4 250 4 3 5 4 49 5 55 6 6 5 7 time ms capacity undercompensated voltage 300 50 5 50 100 150 200 4 250 300 4 350 4 400 3 5 4 49 9 5 5 6 6 5 7 time ms capacity well compensated voltage 450 3 5 4 49 5 g9 6 6 5 7 time ms Figure 3 Tuning of the coarse capacity compensation with an electrode resistance 100 MQ in the bath Time course of the signal at ELECTRODE POTENTIAL OUTPUT is shown holding current 1 nA duty cycle 1 4 switching frequency 2 kHz page 4
18. that of the standard headstage All current related signals must be divided by 10 Setup Step by Step Unpacking The following items should be included with your amplifier Headstage Headstage cable Electrode cable Bath ground Power cord User s manual If any of these items are missing please contact npi electronics immediately After unpacking the SEC amplifier and accessories check each piece for any signs of shipping damage Please contact the delivering carrier and npi electronics im mediately if there is any damage All npi shipments are insured against shipping damages It is advisable to keep the ship ping box in the event that the unit must be returned for servicing Getting Started 11 1 a 1 Connect a digital or analog timing and d Connectin SA Setting Up and Connecting a pulse unit A to the respective Inputs VOLTAGE COMMAND INPUT and CURRENT STIMULUS INPUT Q 2 Connect a storage oscilloscope B to the POTENTIAL OUTPUT amp and to the CURRENT OUTPUT triggered with the trigger signal from the timing unit You might want to connect an analog to digital converter and a computer to the same connectors by using a T connector Connect a normal oscilloscope E to the ELECTRODE POTENTIAL Vel con nector on the rear panel This oscillo scope is triggered by the SWITCHING FREQUENCY signal rear panel Connect the electrode cable and the bath ground to the respective connectors
19. whole cell patch clamp recordings The low voltage headstage has the capability to inject a maximum current of ap proximately 120 nA into a resistance of 100 MQ Considering the duty cycle 1 2 1 4 1 8 in the discontinuous modes of operation the maximum effec tive range of current is 60 nA 30 nA and 15 nA into 100 MQ The high voltage headstage is designed for recording larger currents up to the pA range e g from Xenopus oocytes This headstage has the capability to in ject 1 2 pA 600 nA 300 nA or 150 nA in the discontinuous modes into a resis tance of 100 MQ or alternatively 12 WA 6 WA 3 WA 1 5 HA in the discon tinuous modes into 10 MQ The cur rent range of the high voltage headstage is 10 times higher than that of the stan dard headstage Therefore all current related signals have to be multiplied by ten The x10 low voltage headstage can record currents larger those that can be measured using the standard version This headstage is normally operated us ing suction low resistance electrodes and it has an output current range of 1pA 10MQ The calibration is the same as for the high voltage headstage The x0 1 low voltage headstage is used for low noise recording of small currents 2 10 pA via whole cell patch clamp technique or for making recordings from very small cells This headstage has an output current range of 15nA into 100 MQ and the noise and bias current are 10 times lower than
20. 113 Eder M Zieglgansberger W amp Dodt H U 2002 Neocortical long term potentiation and long term depression site of expression investigated by infrared guided laser stimulation J Neurosci 22 7558 7568 Marsicano G Wotjak C T Azad S C Bisognok T Rammes G Casciok M C Hermann H Tang J Hofmann C Zieglg nsberger W Di Marzok V amp Lutz B 2002 The endogenous cannabinoid system controls extinction of aversive memories Nature 418 530 533 Nakazawa K Quirk M C Chitwood R A Watanabe M Yeckel M F Sun L D Kato A Carr C A Johnston D Wilson M A amp Tonegawa M A 2002 Requirement for Hippocampal CA3 NMDA Receptors in Associative Memory Recall Science 297 211 218 Rammes G Steckler T Kresse A Schutz G Zieglgansberger W and Lutz B 2000 Synaptic plasticity in the basolateral amygdala in transgenic mice expressing dominant negative cAMP response element binding protein CREB in forebrain Eur J Neurosci 12 2534 2546 Intra and extracellular recording Sillaber I Rammes G Zimmermann S Mahal B Zieglgansberger W Wurst W Holsboer F amp Spanagel R 2002 Enhanced and Delayed Stress Induced Alcohol Drinking in Mice Lacking Functional CRH1 Receptors Science 296 931 933 Perforated Patch Hanganu I L Kilb W amp Luhmann H J 2002 Functional synaptic projections onto subplate neurons in neonatal rat somatosenso
21. 12 Eisenberg R S and Engel E 1970 The spatial variation of membrane potential near a small source of current in a sperical cell J Gen Physiol 55 736 757 Engel E Barcilon V and Eisenberg R S 1972 The interpretation of current voltage relations recorded from a spherical cell with a single microelectrode Biophys J 12 384 403 Gorman A L F and Mirolli M 1972 The pasive electrical properties of the membrane of a molluscan neurone J Physiol 227 35 49 Hamill O P Marty A Neher E Sakmann B and Sigworth F J 1981 Improved patch clamp techniques for high resolution current recording from cells and cell free membrane patches Pfliigers Arch 391 85 100 Henze D A Cameron W E and Barrionuevo G N 1996 Dendritic morphology and its effects on the amplitude and rise time of synaptic signals in hippocampal CA3 pyramidal cells J Comp Neurol 369 331 344 35 Jackson M B 1992 Cable analysis with the whole cell patch clamp Theory and experiment Biophys J 61 756 766 Johnston D 1981 Passive cable properties of hippocampal CA3 pyramidal neurons Cell Mol Neurobiol 1 41 55 Johnston D and Brown T H 1983 Interpretation of voltage clamp measurements in hippocampal neurons J Neurophysiol 50 464 486 Kawato M 1984 Cable properties of a neuron model with non uniform membrane resistivity J Theor Biol 111 149 169 Mainen Z F and Sejnowski T J 1996 Influence of dendr
22. 6 Polder H R and 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 Neuroscience Methods 109 97 109 Richter D W Pierrefiche O Lalley P M and Polder H R 1996 Voltage clamp analysis of neurons within deep layers of the brain J Neurosci Methods 67 121 131 Windhorst U and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin Heidelberg New York 1999 Selection of switching frequency electrode time constant capacity compensation Juusola M 1994 Measuring complex admittance and receptor current by single electrode voltage clamp J Neurosci Meth 53 1 6 Weckstr m M Kouvaleinen E Juusola M 1992 Measurement of cell impedance in frequency domain using discontinuous current clamp and white noise modulated current injection Pfliigers Arch 421 469 472 Comparison of recording methods sharp electrode whole cell perforated patch Jarolimek W and U Miseld 1993 4 Aminopyridine induced synaptic GABA B currents in granule cells of the guinea pig hippocampus Pfliigers Arch 425 491 498 Kapur M F Yeckel R Gray and D Johnston 1998 L Type Calcium Channels Are Required for One Form of Hippocampal Mossy Fiber LTP J Neurophysiol 77 2181 2190 Coating of sharp microelectrodes for VC recordings Juusola M Seyfarth E A and French A S 1997 Fast coating of glass capil
23. 7141 601266 support npielectronic com http www npielectronic com Passive Cell Model User Manual 1 Introduction The cell model is designed to be used to check the function of the instrument either 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 incase of trouble to check which part of the setup does not work correctly e g to find out whether the amplifier is broken or if something is wrong with the electrodes or holders etc This cell model consist only of passive elements 1 e resistors that simulate the resistance of the cell membrane and the electrodes and capacitances that simulate the capacitance of the cell membrane see Figure 2 A switch allows to simulate two different cell types a small cell with 100 MQ membrane resistance and 100 pF membrane capacitance or a large cell with 20 MQ and 500 pF The headstage of the amplifier can be connected to one of two different types of electrodes see below 2 Cell Model Description BNC jack RE 5MQ Ground GND connector SEC CELL MODEL npi 100M CELL 100p CELL membrane switch 20M 500p R 100 MQ SUBCLICK connector Figure 1 SEC passive cell model version 2 2 page 2 Passive Cell Model User Manual Ret BNC connector for the patch electrode resistance 5 MQ GND ground connector CELL switch for cell membrane representing a mem
24. AGE SEC SYSTEM This headstage has an output current range of 15nA into maximum 100 MQ and the noise and bias current are reduced by a factor of 10 compared to the standard headstage Therefore it is recommended for whole cell patch clamp recordings although it can be used also with high resistance sharp microelectrodes All current related signals have to be divided by 10 e CURRENT DISPLAY XXX pA no decimal point 100 100 pA e HOLDING CURRENT XXX pA 100 100 pA e Input sensitivity BNC labeled 1 nA V has now 0 1 nA V 1 e 1V 100 pA BNC labeled 0 1 nA V has now 0 01 nA V 1 e 1V 10 pA e Output sensitivity 1V nA lOOV nA e BRIDGE BALANCE XXX x10 MQ Ret mode display KAKAK MQ i e 101s 100 MQ The potential input and output signals are not affected me Jl for the Life Sciences Calibration of SEC Amplifiers with x10 Headstage User s Manual page 10 GENERAL The current range of the x10 low voltage headstage is the following BR 1 2 uA into 10 MQ max output voltage is 12 V switched modes 600 nA duty cycle 50 300 nA 25 and 150 nA duty cycle 12 5 All current related inputs and outputs must be multiplied by a factor of ten This is valid for all four modes of operation Rr BR CC VC Potential related signals are not affected CURRENT STIMULUS INPUT 1 nA V corresponds 10 nA V 0 1 nA V corresponds nA V GATE XX X nA max 99 9 nA SEC10 only HOLDING XX X nA max 99 9 nA
25. ENCY kHz 37 CURRENT OUTPUT 38 41 CURRENT STIMULUS INPUT 42 REMOTE 43 HEADSTAGE INPUT 27 panel see REAR PANEL ELEMENTS GND EARTH connectors VC COMMAND INPUT e 30 40 mV input BNC connector See 19 e 32 10 mV input BNC connector See 19 e 33 STEP GATE TTL input BNC connector See 19 POTENTIAL OUTPUT BNC connector monitoring potential with a gain of ten the recorded SWITCHING FREQUENCY kHz The selected switching frequency see 17 is displayed with a 3 digit display In the linear modes B and R modes it must show zero CURRENT OUTPUT BNC connector on the front panel monitoring the effective average current passed through the electrode CURRENT STIMULUS INPUT e 38 STEP GATE TTL input BNC connector See 21 e 40 1 nA Volt input BNC See 21 e 410 1 nA V input BNC See 21 REMOTE BNC connector for activating the PENETRATION UNIT remotely See also 10 HEADSTAGE INPUT connector The headstages are connected via a flexible cable and a 12 pole connector to the mainframe CAUTION Please always adhere the appropriate safety regulations see SAFETY REGULATION chapter When connecting or disconnecting the headstages from the 19 cabinet connector please turn power off REAR PANEL ELEMENTS 28 SWITCHING OUTPUTS MODE OF OPERATION CURR SENSITIVITY BNC FREQUENCY MONITOR CURRENT SWITCHING OUTPUTS see Fig 3 These outputs provide signals for tuning of the switched operati
26. ETRATION UNIT This unit is used to clean the tip of the electrode and to facilitate the penetration of the cell membrane e The unit can be operated by a remote switch connected to the REMOTE BNC 42 active LOW e The duration can be set by the DURATION control 10 e It can be turned off by the mode select OFF PULSE I BUZZ rotary switch 27 e PULSE mode application of DC pulses In the B bridge mode or C switched current clamp mode square pulses are applied to the electrode to clean the tip of the electrode or to facilitate cell penetration e The PULSE parameters are set by two controls and a switch AMPLITUDE control 12 polarity switch 12 and FREQUENCY control 11 13 BRIDGE BALANCE 14 VC OUTPUT LIMITER 15 VC GAIN 16 DUTY CYCLE 24 e Iaa In this mode DC currents are applied to the electrode The amplitude and polarity are also set by the respective controls AMPLITUDE polarity switch e BUZZ CAPACITY COMPENSATION mode overcompensation of the capacity compensation effective in all four modes of operation R B C V modes CAUTION Once an appropriate cell is found always turn off the PENETRATION UNIT OFF position of switch 27 BRIDGE BALANCE MQ In the B bridge mode the electrode resistance is compensated with this control ten turn potentiometer clockwise calibrated in MQ 100 MQ 10 MO switch With this switch the range of the BRIDGE BALANCE control is set 10 MQ
27. 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 is to be operated only at 115 230 Volt 60 50 Hz AC Please check for appropriate line voltage before connecting any system to mains Always use a three wire line cord and a mains power plug with a protection contact connected to ground protective earth Before opening the cabinet unplug the instrument Unplug the instrument when replacing the fuse or changing line voltage Replace fuse only with an appropriate specified type STATIC ELECTRICITY Electronic equipment is sensitive to static discharges Some devices such as sensor inputs are equipped with very sensitive FET amplifiers which can be damaged by electrostatic charge and must therefore be handled with care Electrostatic discharge can be avoided by touching a grounded metal surface when changing or adjusting sensors Always turn power off when adding or removing modules connecting or disconnecting sensors headstages or other components from the instrument or 19 cabinet TEMPERATURE DRIFT WARM UP TIME 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 1
28. Getting Started Capacity compensation adjustment is per formed using the ELECTRODE POTENTIAL signal connector at the rear panel This signal is displayed on a regular ie non storage oscilloscope which is triggered by the SWITCHING FREQUENCY signal connector at the rear panel Correct tun ing of the capacity compensation is essen tial for proper switched mode operation CC and VC The SEC 05 recording system has two controls for capacity compensation one located on the front panel CAP COMP ten step control and one located at the headstage CC The CC control at the headstage adapts the bandwidth to the time constant of the electrode This control has to be readjusted only if the set up arrange ment or the type of electrode has been changed The CAP comp control on the front panel is the conventional feed back compensation This control is used for CC tuning during an experiment To adjust the capacity compensation on the headstage immerse the electrode in the bath as deep as necessary during the ex periment Then apply positive and negative currents with the HOLDING CURRENT con trol at the lowest switching frequency in the CC mode A clear signal on the os cilloscope should be visible Fig 5 The CAP COMP control is on a low position 1 3 on the dial Now the CC control headstage is turned on clockwise until the signals on the oscilloscope connected to the ELECTRODE POTENTIAL OUTPUT on the
29. L pulse 5 V is applied to the BNC connector under the DHC switch the SEC is switched to VC mode The COMMAND INPUT for voltage clamp is disabled and the command potential is provided by the sample and hold electronic e g the command potential represents the last membrane potential before switching to VC mode In practice the investigator needs additionally a spike detector and a timing unit The spike detector detects an action potential and triggers with a possible delay set by the timing unit the transition from CC mode to VC mode Literature Dietrich D Clusmann H amp Kral T 2002 Improved hybrid clamp resolution of tail currents following single action potentials J Neurosci Meth 116 55 63
30. N f SS HEADSTAGE THRESHOLD AMPLITUDE BIAS CURRENT 0 PULSE lia B DISABLED OFF BUZZ SWITCHING FREQUENCY STEP SIZE nA ELECTRODE CLEAR ON OFFSET COMP CURRENT DURATION eee CURRENT STEP GATE TIL 1nAN O 1NAN TIL OUTPUT CURRENT STIMULUS INPUT REMOTE HEADSTAGE INPUT FRONT PANEL VIEW OF THE SEC 10LX AMPLIFIER SYSTEM GD GDA GA wD D amp amp amp CONTROLS AND CONNECTORS FRONT PANEL ELEMENTS 1 POWER 2 VC ERROR 3 INTEGRATOR TIME CONSTANT 4 POTENTIAL FILTER 5 6 DISPLAYS 22 POWER This switch turns on the power supply The line fuse line voltage selector and power cable connector are located on the rear panel VC ERROR This analog display shows the error in the VC voltage clamp mode command minus recorded potential The desired range of operation is around zero INTEGRATOR TIME CONSTANT In the most left position the integrator is turned off i e the VC controller has only proportional characteristic By turning the knob clockwise the integrator is set on 1 e the VC controller has a PI characteristic proportional and integral which reduces the error considerably theoretically to zero The time constant of the integrator is set with a ten turn potentiometer 3 clockwise time constant is decreased effect of integrator is increased When using the integrator step commands applied to the input can cause overshoots which can be red
31. acustic monitor sig nal changes Penetrate cell via buzzing or capacitance overcompensation After penetration the V response Fig lid to the test pulse should reflect the time constant and the cell membrane re sistance Read membrane potential and apply holding current to get the desired value if necessary Correct bridge balance page 13 If change in voltage occurs correct CAP COMP using page 15 Watch control oscilloscope Start recording in the CC or VC mode Step by Step Instructions 21 elelel i SRi URRENT FILTER Hz CURRENT OUTPUT O LATION P RA CURRENT nA CC BR 1k 1 3K k SENG III SHUT OFF 700 500 300 200 0 5 2 p 0 2 O 6 10K 50 20 20K HOLDING CURRENT nA 1 8 1 4 1 2 V nA MODE OF OPERATION SEC 1 OLX npi SWITCHING FREQUENCY kHz PI CONTROLLER INTEGRATO POTENTIAL FUER Hz POWER R Jl TIME CONSI ne Nk POTENTIAL RESISTANCE 700 3k 500 5k 200 10k 0 1 ms 100 13k a ME OFF a an O ae 50 We 20 HOLDING POTENTIAL mV COMPUTER V K CONTROL 10 OFF VC OUTPUT LIMITER ON DUTY CYCLE STEP SIZE mV VOLTAGE CLAMP GROUND b 40mV 10mV STEP GATE TIL x10mV VC COMMAND INPUT POTENTIA OUTPUT 100 MQ TURN 10 MO TURN AAN BRIDGE BALANCE Ra
32. al Imax Imax do not work Important This headstage is sensitive to static discharges It is equipped with very sensitive FET amplifiers which can be damaged with electrostatic charge and must therefore be handled with care Damage 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 Always turn power off when connecting or disconnecting headstages from the 19 cabinet For more information please contact support npielectronic com www npielectronic com NDI Instruments for the Life Sciences SEC SYSTEMS WITH LINEAR x1 AND x10 MODE General Description The linear mode of the SEC amplifier is an unswitched operation mode of the SEC working in voltage clamp VC and current clamp CC In contrast to standard patch clamp amplifiers the electrode voltage is nevertheless measured also in VC However due to current flow during voltage measurement this measurement is distorted by the series resistance This is the reason why the linear mode should be used only for recordings where only little current flows In the linear mode the background noise of the amplifier is substantially reduced Therefore the linear mode is predestined for low noise recordings in VC and CC mode The linear mode allows also loose patch or macro pa
33. analog input BNC 40mV e 33 GATE TTL 25 STEP SIZE mV analogue to the current GATE INPUT 38 STEP SIZE nA 26 MODE OF OPERATION Push button selector of the four operating modes the selected mode of operation is indicated by a green LED at the respective push button e R ELECTRODE RESISTANCE TEST MODE see POTENTIAL monitor e B BRIDGE MODE see BRIDGE BALANCE control 13 e C CURRENT CLAMP MODE using discontinuous current injection e V VOLTAGE CLAMP MODE using discontinuous feedback The mode of operation can be selected also by TTL signals connected to the respective BNC connectors at the rear panel CURRENT STIMULUS INPUT The current injected through the electrode in the current clamp modes B and C modes is the sum of the following input signals e 21 HOLDING CURRENT nA With this control a constant current can be generated ten turn potentiometer clockwise calibrated in nA The polarity is selected with a 0 toggle switch near the control 22 OFFSET 23 AUDIO 24 RISE TIME 25 STEP SIZE mV 26 STEP SIZE nA 27 OFF PULSE Ina BUZZ 28 CAP COMP 29 GROUND 26 e 40 Analog input BNC connector 1 nA V e 41 Analog input BNC connector 0 1 nA V All analog inputs have an ON OFF switch e 38 STEP GATE TTL 26 STEP SIZE nA With this input a current step set in nA with a digital potentiometer can be generated with a positive digital pulse 3 15V The polarity is sel
34. at ned i i A eat ce ve The tuning of the bridge balance is per formed with the help of the POTENTIAL OUTPUT signal which is displayed on a storage oscilloscope The following adjust ments are performed with the electrode immersed in the bath as deep as necessary during the experiment To adjust the bridge balance switch to the BR mode and apply current to the CURR STIMULUS INPUT Tum BRIDGE BALANCE until the potential trace on the oscilloscope connected to POTENTIAL OUTPUT is flat Fig 4a The potentio meter reading shows the electrode resis tance Sometimes the bridge balance has to be readjusted after establishing an intracellular recording or the whole cell configuration Fig 4b Before performing a patch clamp experi ment the electrodes should be tested in the BR mode by applying positive and negative current pulses Electrodes with significant rectification cannot be used for voltage clamping The current carrying capability of the electrode can be estimated by increasing the current amplitude The current gener ated in the VC mode should not exceed this amount and must be limited using the YC OUTPUT LIMITER Getting Started 13 Capacity Compensation Adjust ment r r r vo assem l j Lo p PERRET Cad te NE t mat Ni i 1 B k y_n i 8 anal 1 144 1 HP Fig 5 Adjustment of capacity compensation Using the CC Control Headstage 14
35. avoiding series resistance errors in Elsner N and H Wassle eds G ttingen Neurobiology Report 1997 Thieme Verlag Stuttgart Polder H R and 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 Neuroscience Methods 109 97 109 Richter D W Pierrefiche O Lalley P M and Polder H R 1996 Voltage clamp analysis of neurons within deep layers of the brain J Neurosci Methods 67 121 131 Smith TG Lecar H Redman SJ Gage PW ed Voltage and Patch Clamping With Microelectrodes The William and Wilkins Company Baltimore 1985 Weckstr m M Kouvaleinen E Juusola M 1992 Measurement of cell impedance in frequency domain using discontinuous current clamp and white noise modulated current injection Pfliigers Arch 421 469 472 Wilson W A and Goldener M M 1975 Voltage Clamping with a Single Microelectrode J Neurobiol 6 41 1 422 31 Selected Literature About the npi SEC 05 10 Single Electrode Clamp Systems Recording Methods and Voltage Clamp Technique Dietzel I D D Bruns H R Polder and H D Lux 1992 Voltage Clamp Recording in Kettenmann H and R Grantyn eds Practical Electrophysiological Methods Wiley Liss NY Misgeld U W M ller and H R Polder 1989 Potentiation and Supression by Eserine of Muscarinic Synaptic Transmission in the Guinea Pig Hipocampal Slice J Physiol 409 191 20
36. brane of either 100 MQ and 100 pF or 20 MQ and 500 pF Rr SUBD connector for the sharp electrode resistance 100 MQ SIMULATION OF WHOLE CELL RECORDING SIMULATION OF SHARP MICROELECTRODE RECORDING DRIVEN SHIELD Do not connect to GROUND 5 MOhm 100 MOhm BNC CONNECTOR SUBCLIC CONNECTOR 1900 MOhm 2a MOhm GROUND SEC 7 BA IS 7 BRAMP CELL MODEL Figure 2 schematic diagram of the passive cell model 3 Connections and Operation 3 1 Checking the Configuration with the Cell Model _J Turn POWER switch of the amplifier off a For simulation of an experiment using a suction electrode I Connect the BNC jack of the cell model to the BNC connector MICROELECTRODE of the headstage b For simulation of an experiment using a sharp electrode _J Connect SUBCLICK connector of the cell model to the BNC connector at the headstage For a and b 1 Connect GND of the cell model to GND of the headstage I Leave REF untouched Ld Switch the CELL membrane switch see Figure 1 to the desired position Ld Turn all controls at the amplifier to low values less than 1 and the OFFSET in the range of 5 version 2 2 page 3 Passive Cell Model User Manual _J Turn POWER switch of the amplifier on Now you can adjust the amplifier and apply test pulses to the cell model Connection to the BNC jack gives access to the cell via an electrode with 5 MQ resistance Connection to SUBD adapter simulates access to the cell v
37. current of the headstage is tuned using the HEADSTAGE BIAS CURRENT control This tuning procedure must be performed before starting a recording ses sion A high value resistor or a cell model is re quired for tuning The amplifier must be in the BR mode The procedure cannot be performed with an electrode since there are always unknown potentials involved tip potential junction potentials 1 To trigger the oscillation shut off unit LED red the amplifier is caused to oscillate by over compensating the CAP comp The DISABLED RESET switch must be in middle position to avoid incoming signals from the CURRENT STIMULUS UNITS 2 The electrode output on the headstage is connected to ground via a 10 100 ki resistor and the offset on the potential DISPLAY is tuned to 0 mV with the OFFSET control After tuning the offset connect a large re sistor about 100 MQ as if an electrode were attached 1 The potential now appearing on the po tential DISPLAY is related to the bias current of the headstage according to Ohm s Law Cancel out this voltage us ing the HEADSTAGE BIAS CURRENT potentiometer D 2 Now both DISPLAYS and should read 000 Potentiometers and are usually close to 5 An offset of 001 to 002 on the displays can occur due to the limited resolution of the displays unbalanced offsets and thermal drifts This small deviation is generally neg ligible but if very small current
38. d Lux H D 1992 Voltage clamp recording In Kettenmann H and Grantyn R eds Practical electrophysiological methods Wiley Liss NY Eisenberg R S and Johnson E A 1970 Three dimensional electrical field problems in physiology In Butler J V A and Noble D eds Progress in Biophysics and Molecular Biology Vol 20 Pergamon Press Oxford p 1 Finkel A S and Gage P W 1985 Conventional voltage clamping with two intracellular microelectrodes In Smith T G Lecar H Redman S J and Gage P W eds Voltage and patch clamping with microelectrodes Chapter 4 The William and Wilkins Company Baltimore p 47 Finkel A S and Redman S J 1985 Optimal voltage clamping with single microelectrode In Smith T G Lecar H Redman S J and Gage P W eds Voltage and patch clamping with microelectrodes Chapter 5 The William and Wilkins Company Baltimore p 95 Jack J 1979 An introduction to linear cable theory In Scmitt F O and Worden F G eds The Neurosciences Fourth Study Program MIT Press Cambridge p 423 Marty A and Neher E 1995 Tight seal whole cell recording In Sakmann B and Neher E eds Single channel recording 2nd edition Plenum Press New York Ogden D and Stanfield P 1994 Patch clamp techniques for single channel and whole cell recording In Ogden D ed Microelectrode techniques 2nd edition The Company of Biologists Ltd Cambridge Rall W 1977 Core co
39. d with a ground connector and a connector provid ing the driven shield signal The SEC 05H high voltage headstage is equipped with a ground connector only To cover all the needs of electrophysio logical research npi electronic s SEC sys tems have four modes of operation Besides the discontinuous modes CC current clamp mode and VC voltage clamp mode all systems also have two linear modes of operation the bridge mode BR compensation of the electrode resis tance by a linear bridge circuit and an automatic electrode resistance test mode Rel The VC CC and BR modes are se lected using a rotary switch which has a fourth position for external control of the system EXT The Rel mode is activated by a toggle switch Some functions of the SEC 05 systems can be controlled by digital signals EXT posi tion of the MODE switch In the stan dard unit this setting allows the operator to switch from CC to VC by means of an ex ternal TTL pulse applied to the MODE SELECT BNC connector 6 Additional functions controlled by an ex ternal digital computer can be accessed using the npi interface card optional Getting started 9 Available Headstages 10 Getting Started The SEC 05 system has four different head stages to chose from 1 The low voltage headstage is the stan dard version for intracellular recordings using high resistance microelectrodes or low resistance suction electrodes for
40. e green yellow connector the internal ground is connected to the yellow connector POWER FUSE LINE VOLTAGE SELECTOR The power chord is connected by a standardized coupling which comprises also the fuse voltage selector and a line filter With 230V AC the fuse must be 0 63A slow with 115V AC it must be 1 25A slow CAUTION see also Safety Regulations e Always use a three wire line cord and a mains power plug with a protection contact connected to ground e Before opening the disconnect mains power plug e Disconnect mains power plug when replacing the fuse or changing line voltage e Replace fuse only with appropriate specified type see Appendix Synchronization of Two or More SEC Amplifier Systems cabinet REFERENCES General Brennecke R and Lindemann B 1971 A Chopped Current Clamp for Current Injection and Recording of Membrane Polarization with Single Electrodes of Changing Resistance T I T Journal of Life Sciences 1 53 58 Brennecke R Lindemann B 1974 Theory of a membrane voltage clamp with discontinuous feedback through a pulsed current clamp Rev Sci Instrum 45 184 188 Brennecke R and Lindemann B 1974 Design of a fast voltage clamp for biological membranes using discontinuous feed back Rev Sci Instrum 45 656 661 Brown T H and Johnston D 1983 Voltage Clamp Analysis of Mossy Fiber Synaptic Input to Hippocampal Neurons J Neurophysiol 50 487 507 Dhein St
41. e as possible Fig 7c 5 An overshoot Fig 7b can be adjusted using the RISE TIME compensation Getting Started 17 Sample Recordings Recordings Using a Cell Model The following r cordings were made using the npi SEC cell model la 1b 20 mV 10 nA 10 msec 10 msac 2a Fig 8 Behavior of the test cell model in the VC 1b and CC modes 2b for a series of voltage la or current steps 2a Simulation of a patch clamp experiment in whole cell configuration 18 Sample Recordings la ib 10 nA 10 mV 10 msec 10 msec to 5 mY 10 msec 10 msec Fig 9 Behavior of the test cell model in VC 1b and CC modes 2b for a series of voltage la or current steps 2a Simulation of an intracellular recording 19 Step by Step Instructions Whole Cell Recording Step by Step e Start with amplifier in the CC mode e Switch to BR mode amp a ee 100 pa 2 5 mV 10 mV b mV 25meec Start recording in CC or VC mode 20 Step by Step Instructions Apply positive pressure to the patch pipette Immerse the patch pipette into the bath to the same depth as during the experi ment Tune potential offset page 12 Apply constant current Check headstage capacity compensa tion and correct if necessary page 14 Turn holding current off Apply test pulse to e g 100 pA Fig 10a Tune bridge balance page 13 Voltage signal should be flat
42. ected with a 0 toggle switch OFFSET Control to zero the output of the electrode preamplifier ten turn potentiometer 5 0 mV up to 400 mV offset can be compensated AUDIO MONITOR Output volume of audio monitor voltage to frequency conversion of the recorded potential RISE TIME Sometimes it is necessary to limit the rise time of a voltage clamp pulse especially in connection with PI controllers to avoid overshooting of the potential See also 3 STEP SIZE Control for the amplitude of the voltage command step elicited by using the STEP GATE TTL 33 Polarity is selected by a 0 switch near the control See also 19 STEP SIZE nA With this digital potentiometer control the amplitude of a current step set in nA generated by using the STEP GATE TTL 38 input The polarity is selected with a 0 toggle switch near the control See also 21 MODE SWITCH for the PENETRATION unit See also 10 CAPACITY COMPENSATION Fine adjustment for the compensation of input capacitance ten turn potentiometer clockwise up to 20 pF can be compensated Coarse compensation control on headstage CC and tuning procedure see CAPACITY COMPENSATION chapter GROUND plug This connector is linked to the internal system ground which has no connection to the 19 cabinet and the mains ground to avoid ground loops Ground connectors are also on the rear 30 33 VC COMMAND INPUT 34 POTENTIAL OUTPUT x10mV 36 SWITCHING FREQU
43. een the ability to record routinely with high switching frequencies in the range of tens of kilohertz regardless of the microelectrode resistance 1 Principles of the dSEVC technique are found in 1 2 Looking back In the early eighties when the design of the SEC 1L system was started single electrode clamping began to gain importance beside the two classical intracellular methods bridge recording or whole cell patch clamp recording The great advantage compared to the whole cell recording method using a patch amplifier was the elimination of series resistance due to the time sharing protocol No current flow during voltage recording means no interference from the series resistance regardless of its value Voltage clamp recordings became possible with sharp microelectrodes in deep cell layers The historical weak point of this method was the low switching frequency due to the fact that stray capacities around the microelectrode could not be compensated sufficiently The SEC systems provided a solution for this problem With their improvements on capacity compensation electronics they could be used with switching frequencies of tens of kHz even with high resistance microelectrodes What are the technical principles that make possible such high switching frequencies In SEC systems a special protocol is used to rapidly compensate the microelectrode Figure 1 shows the compensation scheme of a sharp microelectrode immersed 3 mm in cerebrospinal fl
44. ent and rec ord the potential in an alternating mode which is why the technique is called dis continuous SEVC This ensures that no current passes through Ra during potential recording and completely eliminates access resistance artifacts After each injection of current the potential gradient at the electrode tip decays faster than the potential added at the cell mem brane during the same injection The mo mentary membrane potential is measured after the potential difference across R has completely dropped The difference be tween this value and the command potential determines the size of the next current in jection The discontinuous current and voltage signals are then smoothed and read at the CURRENT OUTPUT and the POTENTIAL OUTPUT connectors 1 Circled numbers refer to the parts on the front panel of the unit illustrated toward the end of the manual m Principle of Operation SH2 currant output record ccs Iyi potential output Vel Fig 2a Model circuit of npi s SEC systems See text for details current inject current free sample timing Vm Fig 2b Principle of SEVC operation Figure 2 illustrates the basic circuitry and operation of the SEC 05 discontinuous single electrode voltage clamp A single microelectrode penetrates the cell or 1s connected to the cell interior in the whole cell configuration of the patch clamp tech nique The recorded voltage is buffered by an x1 ope
45. g frequency 2 kHz Current stimulus and electrode potential are shown Version 1 11 page 6 Tuning Capacity Compensation 800 700 600 capacity overcompensated gt PeT gi fests current F voltage gt time ms 800 4 700 capacity undercompensated S gag p kn current 2 voltage gt 4 60 100 time ms 800 700 600 capacity well compensated gt a aji gi asa current 5 voltage gt 40 45 50 99 60 100 time ms Figure 6 Capacity compensation of the electrode using a model cell electrode resistance 100 MQ current 1 nA cell membrane 100 MQ 100 pF duty cycle 14 switching frequency 2 kHz Current stimulus and membrane potential are shown Version 1 11 page 7 Tuning Capacity Compensation Second part fine tuning Now the basic setting of the CAPACITY COMPENSATION is achieved Since the electrode parameters change during the experiment especially after impaling a cell it is necessary to fine tune the CAPACITY COMPENSATION during the experiment using the CAP COMP control on the amplifier To get familiar with this connect a cell model and go through the following steps the procedure is the identical with a real cell 4 Connect POTENTIAL OUTPUT and CURRENT OUTPUT front panel to another oscilloscope LY Set SWITCHING FREQUENCY to the desired value gt 15 kHz and DUTY CYCLE to the des
46. ia an electrode with 100 MQ resistance The upper position the CELL membrane switch simulates small cell with a resistance of 100 MQ and a capacitance of 100 pF In the lower position a large cell membrane with 20 MQ and 500 pF is simulated version 2 2 page 4 Electron I Instruments for the Life Sciences APPENDIX TUNING CAPACITY COMPENSATION IN SEC AMPLIFIER SYSTEMS VERSION 1 11 NPI 2002 For accurate measurements in switched mode it is essential that the capacity of the electrode is fully compensated Important Wrong compensation of electrode capacity leads to errors in measurements done in switched mode of the amplifier see Figure 2 Microelectrode selection Electrodes must be tested before use This is done by applying positive and negative current pulses Electrodes that show significant changes in resistance rectification cannot be used for intracellular recordings By increasing the current amplitude the capability of the electrode to carry current can be estimated The test current must cover the full range of currents used in the experiment For details see 3 Switching frequency is a key parameter of discontinuous single electrode clamp dSEVC systems The switching frequency determines the accuracy speed of response and signal to noise ratio of the dSEVC system 3 6 Since its launch in 1984 one of the outstanding features of the SEC series of single electrode voltage current clamp systems has b
47. ical R values obtained with suction electrodes are around 5 to 10 MO which would result in a time constant of 0 5 to 1 ms for a cell with a membrane capacitance of 100 pF Thus the membrane needs roughly a millisecond to follow the command voltage step Intracellular elec trodes have much larger resistances 30 to 120 MQ Introduction 5 6 Introduction Besides slowing the voltage response of the cell R can also cause additional adverse effects such as error in the potential meas urement R together with membrane re sistance R forms a voltage divider Fig 1 Current flowing from the amplifier to the grounded bath of a cell preparation will cause a voltage decrease at both R and Ra If Ra lt lt Rm the majority of the voltage decrease will develop at R and thus reflect a true membrane potential If in an extreme case Ra Ra the membrane potential will follow only half of the voltage command To achieve a voltage error of less than 1 R must be more than a 100 times smaller than Rm This condition is not always easy to achieve especially if the recordings are made from small cells If conventional in tracellular electrodes are used it is virtually impossible If R is not negligible precise determination of the pure membrane po tential Ry is possible only when no current is flowing across R This is the strategy employed in npi electronic s SEC amplifier systems The SEC amplifiers inject curr
48. in the pA range is recorded it can be trimmed inter nally Ask npi technical support for details Tuning Procedures in VC mode The settings in the VC mode should be optimized for each cell to yield the best possible voltage control and recording bandwidth With the electrode in the bath it is best to start in the CC mode The a POTENTIAL OUTPUT and CURRENT ee a KIH OUTPUT signals are monitored on a storage scope The GAIN CONTROL D should be set at around 0 5 the IN me fo ooo TEGRATOR TIME CONSTANT 7 amp has to be turned off toggle switch ao es 1 Turn the HOLDING POTENTIAL until VC ERROR is around 0 Now the preadjusted VC command voltage corre ao ff Aoo sponds exactly to the actual membrane potential in CC configuration 1nA 2 Most cells survive best at membrane 10 msec potentials of 50 to 80 mV If necessary preadjust such a potential in the CC a mode injecting DC current at 3 Set VC MODE using and apply volt 1 nA tomes age steps of ca 10mV to the VC msec COMMAND INPUT BNC toggle switch ON If oscillations occur lower Fig 7 Tuning of the capacity compensation in GAIN b or readjust CAP COMP In CC VC mode a voltage command potential gult mode see page 15 put b overcompensated c correct d under compensated e current stimulus 4 Adjust GAIN and INTEGRATOR TIME CONSTANT until the voltage signal on the oscilloscope becomes as squar
49. ired value OY Set the HOLDING CURRENT to zero With the amplifier in CC mode apply square pulses of a few nA or a few tens of pA for patch recordings to the cell Negative current pulses are recommended If you apply positive current pulses be sure only to elicit ohmic responses of the cell membrane 1 e pulses should not elicit openings of voltage gated channels 4 The POTENTIAL OUTPUT should show the ohmic response of the cell membrane without an artifact as illustrated in Figure 6 and Figure 7 undercompensated compensated overcompensated Sy 5 mV 20 ms Ng _ Figure 7 Capacity compensation of the electrode inside a cell Current stimulus and membrane potential are shown Hint The results of this procedure look very similar to tuning of the bridge balance If the BRIDGE is balanced accurately no differences in the potential outputs should occur when switching between CC and BRIDGE mode Important Always monitor the OUTPUT from ELECTRODE POTENTIAL OUTPUT at the rear panel using a second oscilloscope The signals must be always square If not CAPACITY COMPENSATION has to be readjusted or the switching frequency must be lowered Version 1 11 page 8 Tuning Capacity Compensation References 1 Polder H R Swandulla D Konnerth A amp Lux H D 1984 An Improved High Current Single Electrode Voltage Current Clamp System Pflugers Arch 402 R35 2 Polder H R amp Swandulla D 2001
50. itic structure on firing pattern in model neocortical neurons Nature 382 363 366 Magistretti J Mantegazza M Guatteo E and E Wanke 1996 Action potentials recorded with patch clamp amplifiers are they genuine Trends Neurosci 19 530 534 Major G 1993 Solutions for transients in arbitrarily branching cables MI Voltage clamp problems Biophys J 65 469 491 M ller W and Lux H D 1993 Analysis of voltage dependent membrane currents in spatially extended neurons from point clamp data J Neurophysiol 69 241 247 Polder H R and Swandulla D 1990 Design and optimal tuning of single and double electrode voltage clamp systems using methods of modulus hugging Pfliigers Arch 415 S77 Polder H R Swandulla D Konnerth A and Lux H D 1984 An improved high current single electrode current voltage clamp system Pfl gers Arch 406 R43 Polder H R and 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 Neuroscience Methods 109 97 109 Rall W 1959 Branching dendritioc trees and motoneuron resistivity Exp Neurol 1 491 527 Rall W 1969 Time constants and electrotonic length of membrane cylinders and neurons Biophys J 9 1483 1508 Richter D W Pierrefiche O Lalley P M and Polder H R 1996 Voltage clamp analysis of neurons within deep layers of the brain J Neurosci Methods 67
51. lary microelectrodes for single electrode voltage clamp J Neurosci Meth 71 199 204 Capacitive transients in VC recordings Sutor B Hablitz J J 1989 Excitatory postsynaptic potentials in rat neocortical neurons in vitro I Electrophysiological evidence for two distinct EPSPs Journal of Neurophysiology 61 607 620 Leak subtraction Sutor B Zieglgansberger W 1987 A low voltage activated transient calcium current is responsible for the time dependent depolarizing inward rectification of rat neocortical neurons in vitro Pfligers Archiv 410 102 111 32 Cardiac cells double cell voltage clamp method Dhein St 1998 Cardiac Gap Junction Channels Physiology Regulation Patophysiology and Pharmacology Karger Basel Lu J J F Dalton IM D R Stokes and R L Calabrese 1997 Functional role of Ca2 currents in graded and spike synaptic transmission between leech heart interneurons J Neurophysiol 77 1779 1794 Double Cell Recordings Gap Junctions Dhein S Wenig S Grover R Tudyka T Gottwald M Schaefer T amp Polontchouk L 2002 Protein kinase Calpha mediates the effect of antiarrhythmic peptide on gap junction conductance Cell Adhes Commun 8 257 264 Miller A M Lauven R Berkels S Dhein H R Polder and W Klaus 1999 Switched single electrode amplifiers allow precise measurement of gap junction conductance American Journal of Physiology Cell Vol 276 No 4 C980 C988 April
52. lses through high resistance microelectrodes up to 120 MQ and to record membrane potentials accurately within the same cycle The system has been designed primarily to overcome the limitations related with the use of high resistance electrodes in intra cellular recordings but it can also be used with suction electrodes like those employed in conventional whole cell patch clamp experiments These electrodes allow the user to investigate even small dissociated or cultured cells as well as cells in slice prepa rations in both the voltage mode and cur rent clamp mode while the intracellular medium is being controlled by a pipette solution Npi electronic s Single Electrode Systems are independent devices that incorporate four instruments in one Very fast switching current clamp Fast and precise voltage clamp High precision bridge amplifier Electrode resistance measurement The only external components required are a digital timing unit and an oscilloscope 4 Introduction Why a Single Electrode Voltage Clamp A amplifier electrode B amplifier cell pipette ground Fig 1 Model circuits for intracellular A and patch clamp recording in the whole cell configura tion B Voltage clamp techniques permit the analy sis of ionic currents flowing through bio logical membranes at preset membrane po tentials Under ideal conditions the re corded current is directly related to the conductance cha
53. nce patch pipettes only Ringing can be avoided by setting the GAIN in VC mode not higher than 1 and by setting the capacity compensation of the electrode to very low values best close to zero Note Be always aware that the linear mode introduces a series resistance error that is dependent on the magnitude of series resistance and current that flows during measurement Important The LIN mode x1 or x10 must not be used if two SEC amplifiers work in synchronized Master Slave configuration Important BRIDGE balance and Capacity Compensation work in LIN mode and can be used to minimize artifacts during electroporation me Inst I for the Life Sciences SEC 10 SYSTEMS WITH DHC MODE General Description The Dynamic Hybrid Clamp DHC mode is used for investigations of ionic conductances in voltage clamp VC mode following action potentials in current clamp CC mode In CC mode an action potential is detected by a spike detector and triggers a timing unit This timing unit generates a TTL signal for triggering the SEC being in CC mode The SEC switches from CC mode to VC mode with the actual membrane potential as holding potential Operation The DHC mode is set through the an additional switch labeled DHC at the front panel Important The SEC must be in CC mode in order to use the DHC feature When the switch is set to DHC amplifier must be in CC mode the membrane potential is fed into a sample and hold electronic If a TT
54. nd edition The Company of Biologists Limited Cambridge Polder H R 1984 Entwurf und Aufbau eines Ger tes zur Untersuchung der Membranleitfahigkeit von Nervenzellen und deren Nichtlinearitat nach der potentiostatischen Methode Voltage Clamp Methode mittels einer Mikroelektrode Diplomarbeit Technische Universitat M nchen Rudy B and Iverson L E eds 1992 Ion channels In Methods in enzymology Vol 207 Academic Press San Diego CA USA Sahm IMI W H and Smith M W eds 1984 Optoelectronics manual 3rd edition General Electric Company Auburn NY USA Sakmann B and Neher E eds 1995 Single channel recording 2nd Edition Plenum NY Smith T G Jr Lecar H Redmann S J and Gage P W eds 1985 Voltage and patch clamping with microelectrodes American Physiological Society Bethesda The Wiliams amp Wilkins Company Baltimore Windhorst U and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin Heidelberg New York 1999 39 ic Instruments r the Life Sciences OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION FOR THE PASSIVE CELL MODEL FOR SINGLE ELECTRODE PATCH CLAMP AND BRIDGE AMPLIFIERS Aa gt lZ m oO N mi E e A N 2y E m 9 S g 7 85 oO oo os Sf FF 6 2 z Se eR AT NG teeta VERSION 2 2 npi 2002 npi electronic GmbH Hauptstrasse 96 D 71732 Tamm Germany Tel 49 0 7141 601534 Fax 49 0
55. nductor theory and cable properties of neurons In Kandel E R ed Handbook of Physiology Section I The Nervous System Volume I Part I American Physiological Society Bethesda p 39 Sigworth F J 1995 Electronic design of the patch clamp In Sakmann B and Neher E eds Single channel recording 2nd edition Plenum Press New York 38 Whole books Boulton A A Baker G B and Vanderwolf C H eds 1990 Neurophysiological techniques Basic methods and concepts Humana Press Clifton New Jersey Cole K S 1968 Membranes ions and impulses University of California Press Berkely CA Ferreira H G and Marshall M W 1985 The biophysical basis of excitability Cambridge University Press Cambridge Frohr F 1985 Electronic control engineering made easy An introduction for beginners Siemens AG Berlin and Munich Horowitz P and Hill W 1989 The art of electronics Cambridge University Press NY Jack J J B Noble D and Tsien R W 1975 Electric current flow in excitable cells Claredon Press Oxford Kettenmann H and Grantyn R eds 1992 Practical electrophysiological methods Wiley Liss New York Neher E 1974 Elektrische Me technik in der Physiologie Springer Verlag Berlin Numberger M and Draguhn A eds 1996 Patch Clamp Technik Spektrum Akad Verl Heidelberg Berlin Oxford Ogden D C ed 1994 Microelectrode techniques The Plymouth Workshop Handbook 2
56. nges in the membrane and thus gives an accurate measure of the ac tivity of ion channels and electrogenic pumps The membrane potential is generally kept at a preselected value command or holding potential Ionic currents are then activated by sudden changes in potential e g volt ape pated ion channels by transmitter release at synapses e g electrical stimula tion of fiber tracts in brain slices or by external application of an appropriate ago nist Sudden command potential changes like those used to activate voltage gated Na K or Ca currents are especially challenging because the membrane will adopt the new potential value only after its capacitance Cm has been charged There fore the initial transient current following the voltage step should be as large as pos sible to achieve rapid membrane charging In conventional patch clamp amplifiers this requires a minimal resistance between the amplifier and the cell interior a sumple consequence of Ohms law AU RI for a given voltage difference AU the current I is inversely proportional to the resis tance R In this context R is the access or series resistance R between the electrode and the cell interior The time constant for charging a cell is t Ra Cm Series resis tance times membrane capacitance R is largely determined by certain elec trode properties e g pipette resistance and the connection between the electrode and the cell Typ
57. nging or adjusting the electrode If not used the in put of the headstage is should always be grounded either using an appropriate con nector or by wrapping aluminum foil around the headstage Table of Contents Introduction 4 Welcome 5 Why a Single Electrode Voltage Clamp 7 Principle of Operation Advantages of the npi System Getting Started 9 General System Description Modes of Operation 10 Available Headstages 11 Unpacking 12 Setting Up and Connecting 13 Tuning Procedures Adjusting the Bridge Balance 14 Capacity Compensation Adjustment Tuning the CC Control 15 Adjusting the Cap Comp 16 Headstage Bias Current Adjustment 17 Tuning Procedures in VC Mode Sample Recordings 18 Recordings Using a Cell Model 20 Whole Cell Recording 21 Intracellular Recording Description of Components 22 Front Panel 29 Rear Panel 30 Literature introduction 3 Welcome Thank you for purchasing an SEC Single Electrode Amplifier Npi electronic s SEC Single Electrode Systems are based on the newest develop ments in the field of modern electronics and control theory These versatile cur rent voltage clamp amplifiers permit x tremely rapid switching between current injection and current free recording of true intracellular potentials The use of modern high voltage operational amplifiers and a new improved method of capacity compensation makes it possible to inject very short current pu
58. on modes V and C modes In the V and C modes these signals are necessary for tuning the Capacity compensation see following chapter e SW FREQUENCY MONITOR BNC connector monitoring the selected switching frequency 5V pulses used to trigger the oscilloscope which displays the switching pulses e ELECTRODE POTENTIAL Vp BNC connector monitoring the electrode potential i e the response of the electrode to the discontinuous current injection MODE OF OPERATION TTL BR CC VC REL These inputs can be used to select the mode of operation by means of TTL pulses from a digital computer or timing unit see also 20 CURRENT SENSITIVITY MONITOR This BNC connector provides eight output voltages 1 7V IV per switch position corresponding to the seven positions of the CURRENT OUTPUT SENSITIVITY switch See also 7 FREQUENCY MONITOR BNC The position of the CURRENT FILTER switch 8 is monitored at the FREQUENCY MONITOR BNC 8 7 Volt 1 Volt switch position 29 POTENTIAL GROUND PROTECTIVE EARTH POWER SYNC MODE CONNECTORS SWITCH The position of the POTENTIAL FILTER switch 4 is monitored at the FREQUENCY MONITOR BNC 8 7 Volt 1 Volt switch position GROUND PROTECTIVE EARTH Connectors see also 29 In order to avoid ground loops the internal zero ground signal of the instrument is not connected to the mains ground and the cabinet The cabinet and mains ground are connected to th
59. on within each duty cycle 1 8 to 1 2 and read out as cur rent output Is Si then switches to the voltage recording position input to the CCS is 0 The potential at Al decays rap idly due to the fast relaxation at the compensated electrode capacitance Ex act capacitance compensation is essential to yield an optimally flat voltage trace at the Introduction 7 Advantages of the Npi System 8 Introduction end of the current free interval when Veen is measured The cellular membrane potential however will relax much slower due to the large uncompensated membrane capaci tance The interval between two current injection pulses must be long enough to allow for complete lt 1 settling of the electrode potential but short enough to minimize the loss of charges at the cell membrane level minimal relaxation of Veen At the end of the current free period anew V sample is taken and a new cycle begins Thus both current and potential output are based on discontinuous signals which are stored during each cycle in the sample and hold amplifiers SH and SH2 The signals will be optimally smooth at maximal switching frequencies Npi electronic s SEC amplifiers are the only systems that use a PI controller to avoid artificial recordings known to occur in other single electrode clamp systems clamping of the electrode The PI con troller design increases the clamp gain to as much as 100 A V in frequencies less than one
60. output POTENTIAL OUTPUT of the SEC The direct output is connected to the POTENTIAL channel of the SEC system 1 e the signal is passed through the OFFSET compensation stage magnified by ten and filtered by the POTENTIAL FILTER The overall gain for the DC output is 100 x10 input stage x 10 SEC potential magnification e AC output high pass output CURRENT OUTPUT of the SEC The output of the high pass filter stage is fed into the CURRENT channel of the SEC system i e it is passed through the CURRENT OUTPUT SENSITIVITY stage where it is amplified overall gains 10 12 5 20 50 100 200 500 1000 The amplified signal is filtered by the CURRENT FILTER Important The SEC system must be in BRIDGE B mode All inputs must be turned off or disconnected The BRIDGE balance control must be on 000 to avoid incoming disturbances The following systems and front panel elements are working CAP COMPENSATION Capacity compensation control for the non inverting input POTENTIAL OUTPUT DC output x100 OFFSET Offset control for the DC output POTENTIAL FILTER Low pass Bessel filter for the DC output POTENTIAL DISPLAY Shows electrode potential x10 100 are 10 mV CURRENT OUTPUT AC high pass output x10 x 1000 CUR OUTPUT Gain stage 10 1000 for the AC output SENSITIVITY CURRENT FILTER Low pass Bessel filter for the AC output OSCILLATION as described in the SEC manual SHUTOFF PENETRATION BUZZ as described in the SEC manu
61. rational amplifier Al At this point the potential V A1 is the sum of the cell s membrane potential and the volt age gradient which develops when current injected at the access resistance Due to our unique compensation circuitry the voltage at the tip of the electrode decays extremely fast after each injection of current and therefore allows for a correct assessment of Vn after a few microseconds At the end of the current free interval when the elec trode potential has dropped to zero the sample and hold circuit SH1 samples Vm and holds the value for the remainder of the cycle Figs 2 a and b Vsi The differ ential amplifier A2 compares the sampled potential with the command potential Vem The output of this amplifier be comes the input of a controlled current source CCS if the switch 1 is in the cur rent passing position The gain of this cur rent source increases as much as 100 A V due to a PI proportional integral con troller and improved electrode capacity compensation In Fig 2a S1 is shown in the current passing position when a square pulse of current is applied to the electrode When the current passes the electrode a steep voltage gradient develops at the electrode resistance Veen is only slightly changed due to the slow charging of the cell capacitance The amplitude of the in jected current is sampled in the sample and hold amplifier SH2 multiplied by the frac tional time of current injecti
62. rode voltage clamp J Neurosci Meth 53 1 6 30 Kettenmann H and R Grantyn eds Practical Electrophysiological Methods Willey Liss New York 1992 Misgeld U M ller W Polder HR 1989 Potentiation and suppression by eserine of muscarinic synaptic transmission in the guinea pig hippocampal slice J Physiol 409 191 206 M ller A M Bachmann R Berkels S Dhein H R Polder and W Klaus 1998 Switched single electrode amplifiers allow precise measurement of gap junction conductance American Journal of Physiology in press Ogden DC 1994 Microelectrode electronics In Ogden D C ed 1994 Microelectrode techniques The Plymouth Workshop Handbook 2nd edition The Company of Biologists Limited Cambridge Polder HR 1984 Entwurf und Aufbau eines Ger tes zur Untersuchung der Membranleitfahigkeit von Nervenzellen und deren Nichtlinearit t nach der potentiostatischen Methode Voltage Clamp Methode mittels einer Mikroelektrode Diplomarbeit M Sc EE thesis Technische Universitat Miinchen Polder HR Swandulla D Konnerth A and Lux HD 1984 An Improved High Current Single Electrode Current Voltage Clamp System Pfliigers Archiv 402 R35 Polder HR Swandulla D 1990 Design and optimal tuning of single and double electrode voltage clamp systems using methods of modulus hugging Pfliigers Archiv 415 S77 Polder H R R Schliephacke W St hmer and H Terlau 1997 A new switched mode double electrode clamp amplifier
63. ry cortex J Neurosci 22 7165 7176 34 Selected Literature Voltage amp Patch Clamp Techniques Publications in scientific journals Armstrong C M and Chow R H 1987 Supercharging A method for improving patch clamp performance Biophys J 52 133 136 Bekkers J M and Stevens C F 1996 Cable properties of cultured hippocampal neurons determined from sucrose evoked miniature EPSCs J Neurophysiol 75 1250 1255 Blanton M G Lo Turco J J and Kriegstein A R 1989 Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex J Neurosci Meth 30 203 10 Brennecke R and Lindemann B 1974 Theory of a membrane voltage clamp with discontinuous feedback through a pulsed current clamp Rev Sci Instrum 45 184 188 Bush P C and Sejnowsky T J 1993 Reduced compartmental models of neocortical pyramidal cells J Neurosci Methods 46 159 166 De Schutter E and Bower J M 1994 An active membrane model of the cerebellar purkinje cell II Simulation of synaptic responses J Neurophysiol 71 401 419 Draguhn A Pfeiffer M Heinemann U and Polder H R 1997 A simple hardware model for the direct observation of voltage clamp performance under realistic conditions J Neurosci Methods 78 105 113 Edwards F A Konnerth A Sakmann B and Takahashi T 1989 A thin slice preparation for patch clamp recordings from neurons of the mammalian nervous system Pfliigers Arch 414 600 6
64. s been decreased by incorporation of electronic circuits with large time constants 1 10000 s In addition through the current clamp input fast current stimuli e g for conductance measurements can be applied Operation The VCcCC mode is controlled through two front panel elements located in the VC part of the front panel a toggle switch marked on off and a rotary switch to set the time constants 1 10 100 1000 optional 5000 and 10000 sec for the low pass filter To start using the VCcCC mode the amplifier must be tuned accurately in the fast VC mode toggle switch off The holding potential control must be set on the desired value or a holding potential signal must be provided from an external device e g computer This holding potential will be the preset membrane potential for the VCcCC mode Under these conditions PSPs or other changes of the membrane potential will be voltage clamped If the toggle switch is set on the VCcCC mode is started Depending on the preset time constant fast changes of the membrane potential will not be voltage clamped any more This is a condition that corresponds to an accurate current clamp Fast changes of the membrane potential are monitored on the potential output slow changes are compensated by the VCcCC circuit The time constant should be selected in a way that the signals under investigation are not altered by the VCcCC please compare with current clamp recordings
65. ssy fiber activity evokes Ca2 release in CA3 pyramidal neurons via a metabotropic glutamate receptor pathway Neuroscience 107 1 59 69 R hrig G Klausa G and Sutor B 1996 Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurons Journal of Physiology 490 1 pp 31 49 Single S and A Borst 1998 Dendritic Integration and Its Role in Computing Image Velocity Science Vol 281 1848 50 33 Single S and Borst A 2002 Different Mechanisms of Calcium Entry Within Different Dendritic Compartments J Neurophysiol 87 1616 1624 Performance test with active cell model Draguhn A Pfeiffer M Heinemann U and Polder H R 1997 A simple hardware model for the direct observation of voltage clamp performance under realistic conditions J Neurosci Methods 78 105 113 Hybrid Clamp Dietrich D Clusmann H and T Kral 2002 Improved hybrid clamp resolution of tail currents following single action potentials J Neurosci Meth 116 55 63 LTP LDP Investigations Blank T Nijholt I Eckart K and Spiess J 2002 Priming of long term potentiation in mouse hippocampus by corticotropin releasing factor and acute stress implications for hippocampus dependent learning J Neurosci 22 3788 94 Dodt H Eder M Frick A and Zieglgansberger W 1999 Precisely localized LTD in the neocortex revealed by infrared guided laser stimulation Science 286 110
66. tch recordings and can be used to approach the cell and form a gigaseal in VC mode The LIN x10 mode can be used for iontophoresis or electroporation 1 e juxtacellular non invasive filling of cells with or single cell transfection with DNA The stimulus amplitude range in CC or BRIDGE mode is also enhanced to max 120 nA Operation The linear mode is set through the Linear Mode switch at the front panel When the switch is set to the middle position the amplifier 1s in switched VC or CC or in BRIDGE mode CC Setting the switch to x1 or x10 lets the amplifier work in linear mode either without or with x10 amplification LJ Linear Mode x1 x10 switch KI The amplifier operates in linear unswitched mode see below current and or voltage are not enhanced LIN LED lights green x10 The amplifier operates in linear electroporation mode Command voltage in VC or current stimulus in CC or BRIDGE mode are multiplied by the factor of ten This allows to apply stimuli of max 120 nA In this operation mode the LIN LED lights red and the voltage output at POTENTIAL OUTPUT x10mV BNC connector is set to xImV middle In the middle position of this switch the amplifier works in switched or BRIDGE mode The LIN LED does not light Important In LIN x10 the voltage output POTENTIAL OUTPUT x10 mV BNC connector is set to xI mV i e 1 Vis 1 V and not 100 mV as in LIN mode x1 Important The linear mode must be used with low resista
67. ts are suppressed Important All synchronized instruments must use the same duty cycle Literature e Dhein St Double Cell Voltage Clamp in Cardiac Gap Junctions Karger Verlag Basel 1998 e M ller A M Bachmann H R Polder S Dhein R Berkels and W Klaus 1998 Measurement of Gap Junction Conductance with Switched Single Electrode Voltage Clamp Amplifiers No Effect of Series and Input Resistance Pfl gers Archiv 435 R238 e M ller A M Lauven R Berkels S Dhein H R Polder and W Klaus 1999 Switched single electrode amplifiers allow precise measurement of gap junction conductance American Journal of Physiology Cell Vol 276 No 4 C980 C988 April 1999 For more information please contact support npielectronic com www npielectronic com Electronic Instruments for the Life sciences SEC SYSTEMS WITH VCcCC MODE General Description The Voltage Clamp controlled Current Clamp VCcCC or slow voltage clamp SLOW VC mode is used for performing accurate current clamp recordings in the presence of membrane potential oscillations The npi single electrode current and voltage clamp amplifiers npi SEC 05 10 series have been modified in a way that slow membrane potential oscillations are exactly controlled by the voltage clamp module without affecting faster responses e g postsynaptic potentials PSPs and action potentials APs The response speed of the voltage clamp feed back circuit ha
68. turn 1 e 100 10 MQ or 100 MQ turn 1 e 100 100 MQ VC OUTPUT LIMITER Current Limit Under certain experimental conditions it is necessary to limit the current in the voltage clamp mode e g in order to prevent blocking of the electrode or to protect the preparation This is possible with an electronic limiter that sets the current range between 0 and 100 VC GAIN This control sets the gain of the VC controller range 1OOnA V 10uUA V The gain must be as high as possible DUTY CYCLE 1 8 1 4 1 2 In discontinuous modes V and C modes this switch sets the ratio between current injection and potential recording mode 12 5 25 or 50 of each switching period 17 SWITCHING FREQUENCY 18 HEADSTAGE BIAS CURRENT 19 VOLTAGE COMMAND INPUT 20 MODE OF OPERATION 21 CURRENT STIMULUS INPUT 25 SWITCHING FREQUENCY In switched modes V and C modes the switching frequency for the discontinuous current injection is set with this control ca IkHz 50kHz The selected frequency is shown on the display 36 HEADSTAGE BIAS CURRENT With this 10 turn control the output current of the headstage can be tuned to zero see following chapters VOLTAGE COMMAND INPUT see also CURRENT STIMULUS INPUT 21 The command signal for the voltage clamp mode V mode is a sum of following input signals e 19 HOLDING POTENTIAL mV with a 0 switch for selecting the polarity e 32 analog input BNC 10mV e 30
69. uced with the RISE TIME control 24 POTENTIAL FILTER Low pass Bessel filter for the POTENTIAL OUTPUT see also CURRENT FILTER 8 The setting of the filter is monitored at FREQUENCY MONITOR POTENTIAL at the rear panel DISPLAYS 5 POTENTIAL RESISTANCE display for the recorded potential in mV B C V modes or the electrode resistance in MQ R mode 6 CURRENT nA display for the membrane current in nA 7 CURRENT OUTPUT SENSITIVITY 8 CURRENT FILTER 9 OSCILLATION SHUT OFF 10 12 PENETRATION 23 CURRENT OUTPUT SENSITIVITY This switch sets the sensitivity of the current output 0 1 10 V nA seven position rotary switch The setting of OUTPUT SENSITIVITY is monitored at CURRENT SENSITIVITY MONITOR at the rear panel CURRENT FILTER 20Hz 20 kHz 16 position rotary switch e 4 pole tunable Bessel filter 24dB oct with 16 corner frequencies selected by a rotary switch e The following 16 frequencies can be set 20 50 100 200 300 500 700 1k 1 3k 2k 3k 5k 8k 10k 13k 20k Hz The setting of the filter is monitored at FREQUENCY MONITOR CURRENT at the rear panel OSCILLATION SHUT OFF Disconnects current injection and capacity compensation if parasitic oscillations occur e A red green LED shows the state of the system red shut off triggered e THRESHOLD Sets the threshold for shut off activation e DISABLED RESET switch resets or disables oscillation shut off unit CELL PEN
70. uid Here the increase in speed can be seen clearly Recordings under such conditions and possible applications have been presented in several papers e g 3 Version 1 11 page Tuning Capacity Compensation Criteria for the selection of the switching frequency Which are the most important criteria for the selection of the switching frequency This question was analyzed in detail by M Weckstrom and colleagues 4 5 They presented a formula that describes the conditions for obtaining reliable results during a switching single electrode clamp fe gt 3fsw fw gt 2fs fs gt 2fr gt fm fe upper cutoff frequency of the microelectrode Tee switching frequency of the dSEVC f sampling frequency of the data acquisition system f upper cutoff frequency of the lowpass filter for current recording Tit upper cutoff frequency of the membrane Example 6 With the time constant of 1 3 us recorded for the electrodes used in this study is 80 160 kHz the selected switching frequency of the dSEVC was 30 50kHz calculated range is 25 53 kHz data were sampled at 10 kHz and the current signals have been filtered at 5 kHz These settings are currently used for recordings in many labs The principle of operation in switched mode is shown below Sw npi fast compensation pama o meem ang aaa f aa Kanaan Cn pa AAN AA NG 10 kHz a ce ae 4 A i ni f 5 kal a 0 18 V t Tee SANG a a eS compensation re 4 kih ANU Y sarmbpure

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