OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION FOR
Contents
1. Input configuration The amplifier has two inputs both for VC and CC mode The signal applied to the analog input BNCs is converted either into a voltage command signal for the VC mode or to a current in the CC and BRIDGE mode Besides this a signal generated from the 10 turn HOLD potentiometer can be transferred into a pulse using the STEP GATE TTL input BNC This control can be also used as HOLDING potentiometer if the switch in the GATE BNC is turned off version 2 3 page 6 ELC 01X User Manual Computer control of the mode of operation In the EXT position of the MODE SELECT switch all MODEs OF OPERATION can be selected by TTL signals connected to the rear panel Output configuration The ELC 01X amplifier has two output BNCs for POTENTIAL and two output BNCs for the CURRENT signal The POTENTIAL OUTPUT FROM HEADSTAGE is a pure DC output that monitors the electrode potential directly from the headstage The signal at the POTENTIAL OUTPUT can be high and low pass filtered and amplified The CURRENT OUTPUT FROM HEADSTAGE monitors the current directly from the headstage with a scaling of 0 1V nA The current output signal at CURRENT OUTPUT can be scaled from 0 1V Na up to 1OV nA Digital displays All ELC amplifiers are equipped with two digital displays one for CURRENT nA and one for POTENTIAL mV or ELECTRODE RESISTANCE MQ The mode of operation is indicated by a row of LEDs located close to the digital displays ver2. ELC 01X amplifier 3 1 ELC 01X Components The following items are shipped with the system ELC 01X amplifier GND and optional REF connectors 2 6 mm banana plug for headstage Headstage User manual SARK 3 2 Optional Accessories Differential headstage Cell model Pipette holder Cable set O00 0 3 3 System Description The ELC 01X was optimized for extracellular recording precise single cell electrical stimulation and juxtasomal filling with patch electrodes It can be used also for intracellular recordings in CC mode The system consists of a 19 housing and a small headstage with a mounting plate or holding bar It can be used in slices or in in vivo preparations using the optional headstage with a differential input Operation modes of the amplifier The operation modes of the amplifier are selected by a rotary switch with four positions The selected mode is indicated by LEDs above EXT VC or CC are selected by a TTL pulse applied to the EXT BNC CC CURRENT CLAMP MODE used to inject current signals OFF CC Mode with all output signals turned off VC VOLTAGE CLAMP mode command potentials are applied to the electrode In addition using a toggle switch a bridge balance circuit can be activated to compensate for the electrode artifact BRIDGE mode only in CC mode The ELECTRODE RESISTANCE test mode is activated with a push button measured directly in MQ and displayed on the POTENTIAL RESISTANCE display
3. 2004 Besides sophisticated optics these techniques always require precise recording and stimulation amplifiers mostly based on the use of patch electrodes version 2 3 page 4 ELC 01X User Manual Today three methods are used for electrical recordings in vivo or in vitro LI Recordings using patch suction electrodes from single neurons o Whole cell patch clamp technique tight seal recording intracellular o Loose patch technique loose seal recording extracellular L Intracellular recordings with sharp microelectrodes _ Extracellular recordings with glass or metal electrodes The amplifiers used for such recordings are specialized on the recording of the potentials or currents generated by the neurons under investigation If these recording methods are combined with dye injection electroporation stimulation protocols etc through the recording electrode serious constraints occur and several additional devices have to be added to the experimental set up The ELC series of amplifiers fills this gap It allows intracellular extracellular voltage clamp or current clamp recordings both with sharp or patch electrodes as well as additional protocols like electroporation or juxtasomal filling The ELC amplifier is the Swiss Army Knife of modern electrophysiology It is easy to use versatile and permits a lot of sophisticated experiments with only one instrument version 2 3 page 5 ELC 01X User Manual 3
4. BNC connector TTL 115 230 V AC 60 50 Hz fuse 0 4 0 2 A slow 25 W Weight 4 0 kg version 2 3 page 29
5. Experimental chamber Sice hambe Sice differential single ended measurement measurement configuration configuration Figure 6 headstage connections A differential measurement B single ended measurement version 2 3 page 23 ELC 01X User Manual 8 2 Extracellular Voltage Measurement Extracellular measurements are usually done in the loose patch configuration or with special metal microelectrodes Recording with extracellular metal electrodes is simple The electrode is advanced into the region where the recordings will be made using a micromanipulator and the signals are filtered and amplified see chapter 5 in Lalley et al 1999 for details as required For loose patch recording the procedure is the following Barbour amp Isope 2000 Nunemaker et al 2003 Approach the cell in VC mode and apply square voltage pulses to the electrode Contact the cell and establish the seal Set the MODE OF OPERATION switch to OFF Set the required amplification of the POTENTIAL OUTPUT Set the HIGHPASS FILTER to the desired corner frequency Set the LOWPASS FILTER to the desired corner frequency UCC O vo 8 3 Extracellular Stimulation and Electroporation Cells can be stimulated using current or voltage signals Stimulation with Current _ Approach the cell in VC mode and apply square voltage pulses to the electrode _ Contact the cell establish the loose patch and disconnect the voltage signal from the COMMAND IN
6. Ground connector Input resistance CC Current range Electrode parameter controls OFFSET CAPACITY COMPENSATION BIAS Bridge balance 0 100 MQ Electrode resistance test Sensitivity 1 mV MQ Display 12 V 15 V Size 23 x 70 x 26 mm grounded Size 70 mm x 50 mm length 150 mm 9 mm Size 70 mm x 17 mm x 3 mm BNC with driven shield 2 4 mm connector gt 10 Q internally adjustable 120 nA max 100 MQ feedback standard 1 2 uA max 10 MQ feedback 12 uA max 1 MQ feedback range 100 mV ten turn control range 0 30 pF ten turn control range 100 pA ten turn control adjustable with ten turn control application of square current pulses 1 nA 3 Y2 digit XXXX MQ activated by key switch same as POTENTIAL display Bandwidth and speed response CC mode optimal capacity compensation Full power bandwidth Ret 0 MQ Outputs Output impedance Max voltage Current output Current output sensitivity Current display Current filter Potential output x1 Potential output Potential output gain Potential output resolution in AC gt 30 kHz rise time 10 90 lt 10 us Ret 100 MQ lt 5 us Ret 10 MQ 50 Q 12 V BNC connector sensitivity 0 1 10 V nA Rotary switch 0 1 0 2 0 5 1 2 5 10 V nA 3 digits XXX X nA resolution 100 pA 1 pole corner frequency internally set to 5 kHz BNC connector sensitivity 1 V V BNC connector sensitivit
7. OIABES se scsonssaics cvedacgaensetds os dseaatenecducas ant saagdus dada sacennstdunsdeaceneedes 25 8 4 Intracellular Recording vrsne niena a EE a E TS 25 Current Clamp RE CORI G i a r a e E A aA EE EEA ATN 25 OTE TUT AIS 250 ee a e lg a a a Red eae ae aloes 26 10 Technical Data itca teas cea inck eadecs sepead eae hedaate xgsieg isiin aE E aiai 28 version 2 3 page 2 ELC 01X User Manual 1 Safety Regulations VERY IMPORTANT Instruments and components supplied by npi electronic are NOT intended for clinical use or medical purposes e g for diagnosis or treatment of humans or for any other life supporting system npi electronic disclaims any warranties for such purpose Equipment supplied by npi electronic must be operated only by selected trained and adequately instructed personnel For details please consult the GENERAL TERMS OF DELIVERY AND CONDITIONS OF BUSINESS of npi electronic D 71732 Tamm Germany GENERAL This system is designed for use in scientific laboratories and must be operated only by trained staff General safety regulations for operating electrical devices should be followed AC MAINS CONNECTION While working with 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 sys
8. VC mode does not function properly with sharp microelectrodes i e electrodes with more than 10 MQ resistance Although VC mode experiments can be performed in whole cell configuration with patch electrodes npi do not recommend this because of the missing series resistance compensation and capacity compensation in VC mode The VC mode of the ELC 01X amplifier is intended to be used primarily for approaching the cell and forming the loose seal Current Clamp Recording The ELC 01X can be used like a standard bridge amplifier LJ Set the MODE OF OPERATION switch to CC and the BRIDGE MODE switch to the upper position The BRIDGE MODE LED lights up _ Compensate the electrode artifact using the BRIDGE BALANCE potentiometer _ After impaling the cell readjust the bridge L If needed set an appropriate holding current using the HOLDING CURRENT potentiometer and the HOLDING CURRENT polarity switch L Apply stimuli to the cell using the STIMULUS INPUT 10 nA V BNC connector version 2 3 page 25 ELC 01X User Manual 9 Literature General Recording Methods and Voltage Clamp Technique L Dietzel I D Bruns D Polder H R and Lux H D 1992 Voltage Clamp Recording in Kettenmann H and R Grantyn eds Practical Electrophysiological Methods Wiley Liss NY Li Lalley P M Moschovakis A K and Windhorst U 1999 Electrical Activity of Individual Neurons in Situ Extra and Intracellular Recording in U Windho
9. circuit 8 1 Recordings with the Differential Headstage optional Extracellular measurements are mostly done in slices or in vivo in noisy environments where distortions of the recorded signal caused by other instruments and the animal itself are very common Additionally extracellular signals are very small and have to be amplified enormously The drawback is that noise is amplified as well Therefore the headstage of the ELC 01X can be equipped with a differential input that minimizes noise pick up Differential means that the signal for the amplifier is the difference between the positive PEL and negative REF input of the headstage This results in canceling of all common mode signals i e which both electrodes record e g noise For differential measurements both inputs of the headstage REF and Per are connected to microelectrodes using cables with grounded enclosure or electrode holders Pet is connected to the measuring electrode and REF to the reference electrode The experimental chamber is grounded by an Ag AgCl pellet or an AGAR bridge connected to GND of the headstage see Figure 6 If differential measurement is not required single ended measurement configuration see Figure 6 the REF input must be connected to ground GND The amplifier is in an undefined state if the REF is left open and can go into saturation making reliable measurements impossible for more details see Lalley et al 1999 vento
10. electrodes for loose patch clamp and or stimulation or electroporation respectively The headstage is also capable of intracellular recordings with sharp electrodes in CC mode or extracellular recordings The use of metal electrodes is possible as well A differential headstage see Optional accessories in chapter 3 2 for measurements in vivo is also available For details contact npi 7 1 _Headstage Elements Reference electrode connector connector nel sleceoniaternbll D 71732 Tamm REF A 4 www npielectronic com ji AMPLIFIER HEADSTAGE Cable to tne NPE RFB GND i a Electrode amplifier holder i at Mounting plate optional Eeuna connector Figure 5 ELC 01X headstage PEL BNC connector for the electrode holder REF Connector for the reference electrode differential headstage only GND Ground connector TYPE Type of amplifier BA gt Bridge amplifier ELC gt ELC amplifier R FB Value of feedback resistor 10M 10 MQ 100 M 100 MQ Headstage cable to amplifier Mounting plate The electrode filled with electrolyte is inserted into an electrode holder optional that fits into the BNC connector of the headstage or into an electrode holder adapter The electrical connection between the electrolyte and the headstage is established using a carefully chlorinated silver wire Chlorinating of the silver wire is very important since contact of silver to the electrolyte leads to electrochemical potentials causing varying o
11. rotate the holder counter clockwise using a screw driver 16 LINE SELECT switch Switch for selecting the line voltage Switch to the right for 230 V to the left for 115 V The selected voltage is indicated on the switch Caution Before turning on the instrument make sure that the correct line voltage is selected 17 Mains connector Plug socket for the mains power plug Important Check line voltage before connecting the ELC amplifier to power Always use a three wire line cord and a mains power plug with a protection contact connected to ground Disconnect mains power plug when replacing the fuse or changing line voltage Replace fuse only by appropriate specified type Before opening the cabinet unplug the instrument version 2 3 page 15 ELC 01X User Manual SEAL TEST SIGNALS connectors optional The SEAL resistance is determined similar to the Rez test 10 mV square pulses with 15 Hz are applied to the pipette and the resulting current is measured The resistance is calculated according to Ohm s law and is indicated on the CURRENT DISPLAY The maximum SEAL resistance that can be displayed is 19 99 GQ The value of the SEAL resistance is also monitored at 20 see below 18 COMMAND MONITOR TTL connector BNC connector providing a TTL 5V signal synchronous to the 10 mV test pulses 19 SEAL TEST INPUT TTL connector Starts seal test remotely see also 36 Figure 1 20 OUTPUT SEAL 0 1V GQ connect
12. weak but synchronously active thalamocortical synapses Science 312 1622 1627 L Hoshi H Liu W L Massey S C amp Mills S L 2009 ON inputs to the OFF layer bipolar cells that break the stratification rules of the retina J Neurosci 29 8875 8883 L Fuentealba P Begum R Capogna M Jinno S Marton L F Csicsvari J Thomson A Somogyi P amp Klausberger T 2008 Ivy cells a population of nitric oxide producing slow spiking GABAergic neurons and their involvement in hippocampal network activity Neuron 57 917 929 LJ Fuentealba P Tomioka R Dalezios Y Marton L F Studer M Rockland K Klausberger T amp Somogyi P 2008 Rhythmically active enkephalin expressing GABAergic cells in the CAI area of the hippocampus project to the subiculum and preferentially innervate interneurons Journal of Neuroscience 28 10017 10022 Extracellular recording using ELC amplifiers LI Strenzke N Chanda S Kopp Scheinpflug C Khimich D Reim K Bulankina A V Neef A Wolf F Brose N Xu Friedman M A amp Moser T 2009 Complexin I is required for high fidelity transmission at the endbulb of held auditory synapse Journal of Neuroscience 29 7991 8004 version 2 3 page 27 ELC 01X User Manual 10 Technical Data Headstage Input voltage range Operating voltage Enclosure Mounting plate on request Holding bar Dovetail Electrode connector
13. whole cell configuration are not automatically cancelled Important Command potentials are not affected Potential out shows zero Note This procedure has to be done at the beginning of the experiment as soon as the pipette has contact to the bath solution i e before approaching a cell 32 CAP COMP potentiometer Potentiometer for the capacity compensation of the electrode version 2 3 page 13 ELC 01X User Manual 4 Description of the Rear Panel Fs ls 00 AAR 6066 DO Or E Figure 2 ELC 01X rear panel view MONITORING OUTPUTS connectors 1 FILTER CURRENT connector Not installed 2 CURRENT SENSITIVITY connector BNC connector providing a voltage monitoring the position of the CURRENT OUTPUT SENSITIVITY switch 1 V to 7 V 1V STEP 3 LP FILTER POTENTIAL connector BNC connector providing a voltage monitoring the position of the POTENTIAL LOWPASS FILTER switch 8 V to 7 V 1V STEP 4 HP FILTER POTENTIAL connector BNC connector providing a voltage monitoring the position of the POTENTIAL HIGHPASS FILTER switch 1 V to 4 V 1V STEP 5 POTENTIAL SENSITIVITY connector BNC connector providing a voltage monitoring the position of the POTENTIAL OUTPUT GAIN switch 1 V to 7 V 1V STEP RANGE connector 6 HEADSTAGE connector BNC connector for remote control of a switchable headstage ELC SWI optional A TTL HI 5 V signal switches the feedback resist
14. PUT 10 mV connector LJ Set the MODE OF OPERATION switch to CC J Set the HOLDING CURRENT to zero L For stimulation Apply the stimulus signal to the STIMULUS INPUT 10 nA V connector or Adjust the stimulus amplitude with the CURRENT STEP potentiometer and set the stimulus polarity using the switch aside Gate the preset stimulus with a TTL signal linked to the STEP GATE INPUT TTL BNC connector Electroporation with Current Electroporation can be done using the stimulation procedure but usually the applied current is much higher and the stimulus duration is shorter version 2 3 page 24 ELC 01X User Manual Stimulation with Voltage L Approach the cell in VC mode and apply square voltage pulses to the electrode L Contact the cell and establish the loose patch _ For stimulation apply a voltage signal of the required amplitude and duration to the COMMAND INPUT 10 mV connector or Adjust the stimulus amplitude with the POTENTIAL STEP potentiometer and set the stimulus polarity using the switch aside Gate the preset stimulus with a TTL signal linked to the STEP GATE INPUT TTL BNC connector Electroporation with Voltage Electroporation can be done using the stimulation procedure but usually the applied voltage is much higher and the stimulus duration is shorter 8 4 Intracellular Recording Intracellular current clamp CC recordings can be performed with patch or sharp microelectrodes Note
15. The 1 KQ resistor simulates the resistance of the bath solution This can be used to train cancellation of offsets using the bridge balance and using the capacity compensation Simulation of SEAL formation L Set switch 4 Figure 3 to the upper position L Set switch 5 Figure 3 to SEAL position The 1 GQ resistor simulates the SEAL resistance when forming a GIGASEAL in patch clamp experiments Simulation of intracellular recording Intracellular recordings can be mimicked with one of two cells with different properties Use the 50 MQ electrode connector 3 Figure 3 for an experiment with sharp electrodes or the 10 MQ electrode connector 1 Figure 3 for simulating an experiment with patch electrodes LI Switch the CELL membrane switch see 4 Figure 3 to the desired position CELL 1 or CELL 2 L Turn all controls at the amplifier to low values less than 1 and the OFFSET in the range of 5 zero position and the OSCILLATION SHUTOFF in the DISABLED position L Turn POWER switch of the amplifier on Now you can adjust the amplifier see below and apply test pulses to the cell model The lower position of the CELL membrane switch CELL 1 simulates a cell with a resistance of 50 MQ and a capacitance of 22 pF In the middle position CELL 2 a cell membrane with 200 MQ and 100 pF is simulated version 2 3 page 20 ELC 01X User Manual 7 Headstage The ELC 01X comes with a headstage for connecting suction
16. ance in the switchable headstage from 1 MQ to 10 MQ or from 1 MQ to 100 MQ To which resistor is indicated by a box to the left MODE SELECT connectors All MODEs OF OPERATION can be selected by TTL signal connected to the rear panel see below if the MODE OF OPERATION switch 34 Figure 1 is in EXT position This is very convenient when switching often between electroporation and recording because this can be done automatically by the data acquisition system using TTL signals version 2 3 page 14 ELC 01X User Manual 7 Rs connector Not installed 8 Ret connector BNC connector for remote control of the electrode resistance test A TTL HI 5 V signal can be connected here to select the electrode resistance test remotely 9 BR connector BNC connector for remote control of the bridge mode A TTL HI 5 V signal can be connected here to select the bridge mode remotely 10 OFF connector BNC connector to switch the ELC 01X in OFF mode remotely with a TTL HI 5 V signal 11 x10 MODE connector Not installed 12 VC CC connector BNC connector for remote control of the VC CC mode of operation A TTL signal can be connected here to select the mode of operation remotely HI VC LO CC 13 GROUND connector Banana plug providing internal ground see below 14 CHASSIS connector Banana plug providing mains ground see below 15 FUSE holder Holder for the line fuse For changing the fuse
17. ausberger T Marton L F Baude A Roberts J D Magill P J amp Somogyi P 2004 Spike timing of dendrite targeting bistratified cells during hippocampal network oscillations in vivo Nature Neuroscience 7 41 47 Li Nunemaker C S DeFazio R A amp Moenter S M 2003 A targeted extracellular approach for recording long term firing patterns of excitable cells a practical guide Biol Proced Online 5 53 62 www biologicalprocedures com L Pinault D 1996 A novel single cell staining procedure performed in vivo under electro physiological control morpho functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin J Neurosci Methods 65 113 136 LI Rathenberg J Nevian T amp Witzemann V 2003 High efficiency transfection of individual neurons using modified electrophysiology techniques J Neurosci Methods 126 91 98 LI Roberts W M amp Almers W 1992 Patch Voltage Clamping with Low Resistance Seals Loose Patch Clamp In Rudy B amp Iversen L E eds Ion Channels Methods in Enzymology 207 Academic Press San Diego version 2 3 page 26 ELC 01X User Manual LY Strickholm A 1961 Impedance of a Small Electrically Isolated Area of the Muscle Cell Surface J Gen Physiol 44 1073 1088 Tracer injection juxtasomal filling and extracellular recording L Bruno R M amp Sakmann B 2006 Cortex is driven by
18. de in XXXX mV 1999 mV max or the electrode resistance in XXXX MQ 1999 MQ max 22 mV LED The unit of the display 19 is indicated by the mV LED 23 ELECTRODE RESISTANCE TEST button and MQ LED Push button activating the Rex test circuit The unit of the display 19 is indicated by the MQ LED version 2 3 page 11 ELC 01X User Manual 24 MODE OF OPERATION switch and LEDs La Switch for selecting the MODE OF OPERATION VC the amplifier operates in Voltage Clamp mode OFF all outputs of the amplifier are switched OFF and the amplifier is set to CC mode Ret test works CC the amplifier operates in Current Clamp mode EXT the amplifier is set to CC mode VC BR or Rex test modes can be selected by application of a TTL HIGH gt 2 5 V signal to the respective BNC at the rear panel The MODE OF OPERATION that is currently activated is indicated by the LEDs above the switch BRIDGE unit The BRIDGE unit consists of 25 BRIDGE BALANCE potentiometer 26 BRIDGE MODE ON LED and 27 BRIDGE MODE ON switch 25 BRIDGE BALANCE potentiometer Potentiometer for balancing the BRIDGE circuit that eliminates electrode artifacts in CC mode 10 MQ turn range 100 MQ 26 BRIDGE MODE ON LED LED that indicates that the amplifier operates in BRIDGE mode 27 BRIDGE MODE ON switch Switch for activating the BRIDGE mode 28 CURRENT nA display Display for the current at the electrode in XXX X nA ie 10 0
19. deaavaeesdasecenne oiaeascateneate suze te toaesvass 9 4 Description of the Rear Panel csccieswcacctssccsseasengettedevestaasenegsesy eeacguduced ads fees esa stacestesaecuasseees 14 Grounding eer as ar E da E EE AET EEEa ta bequaadun guna saeavonnnanaatedy puaedeawnes 16 oR AED Taa KA G E E ON D sa E E E E A E EE 17 6 Passive Cell Modelen ei i e E E EAE BA E ee E 18 6 1 Cell Model Description isinen ieii i i E ai ts dese 18 6 2 Connections and Operation essssssessseeessseessetssetsseesseeessteesstessetsseessetesseeesseesseessreeseet 19 Teg PS AES CAG nene ina E a ee needa E ae 21 Poles ISAC STASE EATE a1 yc setae ots era ies icc cath asBicu dans eu ctiva seueee nies E better vesed 21 7 2 Headstage Bias Current Adjustment cccecccccesccecssececssececesccecssececsscescnsecessceeenaes 22 8 Introduction into Experiments sesessseseseeesssetssresseesseressseesstesstesseesseeesseesstesstesseesseeeesete 23 8 1 Recordings with the Differential Headstage optional eee eeeeeeseeeesneeeenteeeensees 23 8 2 Extracellular Voltage Measurement sseseeeseeeseseessesssesresseseresressesnresressesnresrreseeseeesee 24 8 3 Extracellular Stimulation and Electroporation ssssssssesesssesessseesseesseesseeeseressseessees 24 Stimulation With C Trent ssesisisicctsiressorisii eiersiden 24 El ctrop rati n with Currente i E E EARE E E E 24 SLUT AOI With Voltage eenei a e ia E TEETE EES 25 Electroporation with V
20. ed current stimulus eal respectively 5 CURRENT STEP nA potentiometer Potentiometer for setting the amplitude of the holding current or the gated current stimulus 100 10 nA range 100 nA The polarity of the current stimulus is set by 4 6 HOLD GATE switch Switch for setting the function of the CURRENT STEP GATE Potentiometer 5 sets a gated stimulus current HOLD Potentiometer 5 sets a holding current in CC mode OFF Potentiometer 5 is disabled 7 STIMULUS INPUT 10 nA V connector version 2 3 page 9 ELC 01X User Manual 8 STEP GATE INPUT TTL connector BNC connector for gating a CURRENT STEP in CC mode or a VOLTAGE STEP in z4 VC mode As long as the voltage linked to this BNC is HIGH i e gt 2 5 V a current Stimulus or command potential is generated by the amplifier Amplitudes of the stimuli are set by potentiometers 5 or 11 respectively eur it 9 COMMAND INPUT 10 mV connector 7 OJ BNC connector for the COMMAND potential in VC mode scaling 10 mV 10 potential polarity switch My Switch for setting the polarity of the holding potential or the gated COMMAND 4 potential respectively 11 POTENTIAL STEP mV potentiometer Potentiometer for setting the amplitude of the holding potential or the gated command potential 100 100 mV range 1000 mV The polarity of the current stimulus is set by 10 12 HOLD GATE switch Switch for setting the function of t
21. ed during transport or 2 to train personnel using the instrument or 3 in case of trouble to check which part of the setup does not work correctly e g to find out whether the amplifier or headstage is damaged or something is wrong with the electrodes or holders etc The passive cell model consists only of passive elements i e resistors that simulate the resistance of the cell membrane and the electrodes and capacitances that simulate the capacitance of the cell membrane A switch allows simulation of two different cell types a cell with 50 MQ and 22 pF CELL 1 represents an astrocyte like cell or a small cell with 200 MQ membrane resistance and 100 pF membrane capacitance CELL 1 represents a neuron like cell Electrode immersed into the bath or SEAL formation can be mimicked as well The headstage of the amplifier can be connected to one of two different types of electrodes see below 6 1 Cell Model Description ELC MOD GROUND Rey 4 1kQ JOMQ CELL 2 100pF GND J 5 4 CELL 1 i 50MQ 1GQ 22pF SOMQ npi electronic GmbH D 71732 Tamm Germany www npielectronic com Figure 3 passive cell model version 2 3 page 18 ELC 01X User Manual 1 3 connectors for the headstage 1 electrode resistance 50 MQ 3 electrode resistance 10 MQ GND ground connector to be connected to GND jack of the headstage 4 CELL switch for cell membrane representing a membrane of eithe
22. ffset potentials at the electrode deterioration of the voltage measurement etc for details see Kettenmann and Grantyn 1992 For optimal chlorinating of sliver wires an automated chlorinating apparatus ACI 01 is available contact npi for details version 2 3 page 21 ELC 01X User Manual GND provides system ground and is linked to the bath via an agar bridge or a Ag AgCl pellet The headstage is attached to the amplifier with the headstage cable and an 8 pole connector The headstage can be mounted directly to a micromanipulator using the mounting plate or a holding bar Important The shield of the BNC connector is linked to the driven shield output and must not be connected to ground The headstage enclosure is grounded Caution Please always adhere to the appropriate safety precautions see chapter 1 Please turn power off when connecting or disconnecting the headstage from the HEADSTAGE connector 7 2 _Headstage Bias Current Adjustment Caution It is important that this tuning procedure is performed ONLY after a warm up period of at least 30 minutes The ELC 01X is equipped with a voltage to current converter with a very high output impedance which is connected to the recording electrode The zero current of this unit is tuned with the BIAS current potentiometer The tuning procedure should be performed regularly at least once a month since the bias current changes over time The tuning procedure is performed usi
23. g the power cord of the instrument into a grounded outlet Connect the headstage to the HEADSTAGE connector 1 Figure 1 at the ELC 01X Connect a cell model see chapter 6 Connect a digital analog timing unit or a stimulation device to STIMULUS INPUT or to GATE TTL if you intend to use a gated stimulus Connect a store oscilloscope or a data acquisition system to the POTENTIAL OUTPUT and to the CURRENT OUTPUT triggered from the stimulation device Set the desired gain at the POTENTIAL OUTPUT GAIN switch 18 Figure 1 and the CURRENT OUTPUT SENSITIVITY switch 29 Figure 1 Before using the ELC 01X always make the basic settings to avoid oscillations Basic settings m m m Turn all controls to low values less than 1 and the OFFSET and BIAS controls in the range of 5 zero position see chapter 3 4 Set the MODE OF OPERATION switch 24 Figure 1 to CC Turn POWER switch on Now the ELC 01X is ready for an initial check with the cell model version 2 3 page 17 ELC 01X User Manual 6 Passive Cell Model The ELC 01X can be ordered with a passive cell model as an optional accessory An active cell model is also available on request for ref see Draguhn et al 1997 The passive cell model is designed for use with single electrode amplifiers BA series ELC series to check the function of the instrument in the following circumstances 1 just after unpacking to see whether the instrument has been damag
24. he POTENTIAL STEP GATE Potentiometer 11 sets a gated command potential HOLD Potentiometer 11 sets a holding potential in VC mode OFF Potentiometer 11 is disabled 13 POTENTIAL OUTPUT FROM HEADSTAGE V connector BNC connector providing the potential output signal directly from the headstage in Volt 14 POTENTIAL OUTPUT connector O BNC connector providing the potential output signal scaling is set by POTENTIAL OUTPUT GAIN switch 18 15 GROUND connector o Connector providing system GROUND which is not connected to PE Protective Earth 16 POWER switch POWER Push button to switch the amplifier ON pushed or OFF released version 2 3 page 10 ELC 01X User Manual 17 AUDIO monitor volume control gt a Control for setting the volume of the internal speaker connected to POTENTIAL POTENTIAL OUTPUT FILTER unit The POTENTIAL OUTPUT FILTER unit consists of 19 HIGHPASS Hz filter switch and 20 LOWPASS Hz filter switch 18 HIGHPASS Hz filter switch 4 position switch for setting the corner frequency of the one pole POTENTIAL HIGHPASS filter 300 to 600 Hz in DC position the HIGHPASS filter is disabled 19 LOWPASS Hz filter switch 16 position switch for setting the corner frequency of the four pole POTENTIAL LOWPASS filter 20 Hz to 20 kHz 20 POTENTIAL OUTPUT GAIN Switch for setting the amplification of the POTENTIAL OUTPUT at 14 Display for the potential at the electro
25. i D Electronic f Instruments for the Life Sciences ease wade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION FOR THE ELC 01X AMPLIFIER FOR EXTRACELLULAR RECORDING AND ELECTROPORATION VERSION 2 3 npi 2014 npi electronic GmbH Bauhofring 16 D 71732 Tamm Germany Phone 49 0 7141 9730230 Fax 49 0 7141 9730240 support npielectronic com http www npielectronic com ELC 01X User Manual Table of Contents 1 Safety PRUE AUL OTN Soft Mat lp Ba iin nd neo doe Dehua ao soe ed ea seas Pn nal ean geste 3 De ATMO CUCU CO a SE iucdeadt otens ng gedeta uaa sealed aeadc sa geese Gatien Gis daads edeas nas dee ede pean dvheean 4 3J ELC QLIX amplifiki eener Gee naa ieecaen Ay a E E a AS N E EE aus 6 Sole ELC 0IX Components en a a N S 6 e O aO E A E T A nase ests E ETET esas 6 3 3 System DES CM ptl oN paseri pt es e e tea oraes Wis a E E 6 Operation modes of the amplifier scccccscccassscvssassesdecedssseaeaeesdaasassias ence soaaeasoseactosnnes 6 Inp tconf rura Oe a a a a EE AA 6 Computer control of the mode of operation s ssesesseseesesssesressessrerreesetsresresseeseeseeesee 7 OQutp t CONF PUTA OM isss ecseri areni aoe E R EEE EES ATES rE S e Ea aSa 7 Digitaldisplay S i irse Sees ssenlanneantvactienieeiareePeeantedahayasasdicasdded gies e ES 7 3 4 Front Panel View of the ELC O1X Amplifier 0 00 eccsesceceeececeeeeeeseeceeseeeeeeaeees 8 3 5 Description of the Front Pane js lt 2 c toscneCocsncdescesudo
26. is 10 nA 199 9 nA max 29 CURRENT OUTPUT SENSITIVITY switch Switch for selecting the amplification of the current output signal in V nA 30 BIAS trim pot H Trim pot for cancellation of the BIAS current range 100 pA version 2 3 page 12 ELC 01X User Manual 31 OFFSET potentiometer Control to compensate for the electrode potential OFFSET ten turn potentiometer symmetrical i e 0 mV 5 on the dial in CC mode range 100 mV or to zero the pipette current in VC mode In CC mode any offset caused by electrode tip potential liquid junction potential etc is cancelled by subtracting this potential value from the electrode i e an offset of 10 mV is cancelled by subtracting 10 mV using this potentiometer In VC mode an offset at the electrode would lead to current flow through the electrode because without a COMMAND the electrode will be clamped to 0 mV and the VC circuit generates a current to achieve this In order to avoid this current flow the electrode has to be clamped to it s offset potential i e a potential has to be added to the COMMAND In the scenario mentioned above the electrode has to be clamped to 10 mV Then the electrode is clamped to it s offset potential and no current will flow If the OFFSET is correctly compensated in CC mode there is automatically no current flow when approaching the cell in VC mode However liquid junction potentials occurring after establishing the
27. lls without damage i e without a direct access to the cell interior The first recordings were made around 1960 from muscles cells by Alfred Strickholm long time before tight seal recording was invented by Erwin Neher and Bert Sakmann twenty years later A method has been developed permitting measurement of membrane impedance and current as a function of transmembrane potential at small electrically isolated regions of the muscle cell surface without microelectrode impalement Strickholm 1961 The loose seal has a resistance of a few ten to a few hundred MQ and it creates an electrically isolated access to a single neuron This isolated area can be used for precise recording stimulation or drug and dye application on the single cell level without damaging the cell Babour amp Isope 2000 In contrast to tight seal recordings the same electrode can be reused for recording from several cells which is a great advantage Since its beginnings several attempts have been made to make such precise extracellular methods accessible to various preparations A nice overview can be found in the chapter by Roberts amp Almers Roberts amp Almers 1992 Over the years the method was extended to cultured neurons and brain slice preparations and also for in vivo recordings Bureau et al 2004 The method is particularly well suited for long term recording with little damage to the recorded neuron Nunemaker et al 2003 It can be used both f
28. ng high value resistors and or a cell model It cannot be performed with an electrode since there are always unknown potentials involved tip potential junction potentials _ Disconnected all input signals except the headstage Put the HOLD OFF GATE switch 6 Figure 1 to position OFF _ Connect the Per connector of the headstage to ground Note This cannot be done with the cell model Please use a wire to connect the input of the BNC connector on the headstage to GND of the headstage Do not use the shield of the BNC connector since it is connected to driven shield _ Tune the OFFSET to zero using the OFFSET control J Remove the wire and attach the cell model or a resistor with a value of about 5 to 10 MO across the same connection L The value displayed at the POTENTIAL DISPLAY is related to the BIAS current of the headstage according to Ohm s Law Cancel this voltage by tuning the headstage BIAS current potentiometer until the POTENTIAL DISPLAY shows 000 version 2 3 page 22 ELC 01X User Manual 8 Introduction into Experiments The ELC 01X is capable to perform several types of experiments that are briefly introduced in the following with special focus on loose patch stimulation and recording It is assumed that the capacity of the electrode is compensated the offset of the electrode is cancelled and for intracellular recordings in BRIDGE mode electrode artifact is eliminated using the bridge balance
29. or BNC connector monitoring the value of the SEAL resistance scaling 100 mV GQ Grounding ELC instruments have two ground systems 1 the internal ground called internal GROUND represents the zero level for the recording electronics and is connected to the recording chamber and the BNC input output sockets 2 mains ground CHASSIS is connected to the 19 cabinet and through the power cable to the protection contact of the power outlet For both grounds there is an outlet on the rear panel GROUND black socket internal system ground CHASSIS green yellow socket mains ground 19 cabinet All ELC systems have a high quality toroid transformer to minimize stray fields In spite of this noise problems could occur if other mains operated instruments are used in the same setup The internal system ground GROUND sockets should be connected to only one point on the measuring ground Multiple grounding should be avoided and all ground points should originate from a central point to avoid ground loops version 2 3 page 16 ELC 01X User Manual 5 Setting up the ELC 01X The following steps should help you set up the ELC 01X correctly Always adhere to the appropriate safety measures see chapter 1 After unpacking the ELC 01X is attached to the setup by assembling the electrical connections It is assumed that first a cell model will be attached Electrical connections m Ly Ly m Turn POWER off Plu
30. or somatic and axonal recording Khaliq amp Raman 2005 Even subcellular structures such as synaptic boutons are accessible to loose patch recordings Auger amp Marty 2000 Another valuable application of this method is single cell stimulation The high resistance loose patch makes possible the application of 1 2 V stimuli to one cell only Babour amp Isope 2000 In the nineties of the last century the method of juxtacellular dye application Guxtasomal filling became popular Pinault 1996 This staining method is based on repetitive current pulse trains applied in the close vicinity of cell somata or dendrites and is meanwhile well established in the field of slice and in vivo preparations Klausberger 2004 In parallel attempts were made towards transfection of single cells by electroporation using patch pipettes DNA or other large molecules were successfully inserted through a patch pipette into living cells by using an optimized protocol application of 10 V 1 ms pulse trains Rathenberg et al 2003 Far in excess of classical in vivo recording methods Lalley et al 1999 several new approaches are used for monitoring neuronal activity under natural conditions using new techniques e g the combination of two photon excitation and patch clamp in vivo Helmchen et al 2002 Stosiek et al 2003 Brecht et al 2002 Assays have been developed that allow to monitor and manipulate single cells under in vivo conditions Brecht et al
31. r 50 MQ and 22 pF CELL 1 or 200 MQ and 100 pF CELL 2 5 In GROUND upper position the electrodes are connected to ground via a 1 KQ resistor In SEAL lower position are connected to a 1 GQ resistor simulating the formation of a GIGASEAL with a patch electrode b Whole cell Patch Sharp Microelectrode Driven Shield BNC connector c BNC connector aN CELL 2 CELL 1 O R GROUND REAL Rm ae Cm L Cm Rm 1 KQ 1 GQ 200 MQ 100 pF 22 pF 50 MQ Figure 4 Schematic diagram of the passive cell model 6 2 Connections and Operation Checking the configuration _ Turn POWER switch of the amplifier off a For simulation of an experiment using a suction electrode _ Connect the BNC jack labeled 10MQ of the cell model to the BNC connector Pex of the headstage version 2 3 page 19 ELC 01X User Manual b For simulation of an experiment using a sharp electrode LI Connect the BNC jack labeled 50MQ of the cell model to the BNC connector Pex at the headstage For headstages with SMB connector use the supplied SMB to BNC adapter For a and b LI Connect GND of the cell model to GND of the headstage Important When using the differential headstage optional the REF connector must not be left open It must be connected to ground Simulation of electrode in the bath L Set switch 4 Figure 3 to the upper position L Set switch 5 Figure 3 to GROUND position
32. rst and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin New York L Ogden DC 1994 Microelectrode Techniques The Plymouth Workshop Handbook Second Edition The Company of Biologists Limited Cambridge LI Polder H R M Weskamp K Linz and R Meyer 2004 Voltage Clamp and Patch Clamp Techniques Chapter 3 4 pp 272 323 in Dhein Stefan Mohr Friedrich Wilhelm Delmar Mario Eds Practical Methods in Cardiovascular Research Springer Berlin Heidelberg and New York 2004 L Windhorst U and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin Heidelberg New York Juxtasomal Filling Loose Patch Techniques General L Auger C amp Marty A 2000 Topical Review Quantal currents at singlesite central synapses J Physiol 526 1 3 11 L Barbour B amp Isope P 2000 Combining loose cell attached stimulation and recording J Neurosci Methods 103 199 208 L Bureau I Shepherd G M G amp Svoboda K 2004 Precise Development of Functional and Anatomical Columns in the Neocortex Neuron 42 789 801 L Joshi S amp Hawken M J 2006 Loose patch juxtacellular recording in vivo A method for functional characterization and labeling of neurons in macaque V1 J Neurosci Methods 156 37 49 L Khaliq Z M amp Raman I M 2005 Axonal Propagation of Simple and Complex Spikes in Cerebellar Purkinje Neurons J Neurosci 25 454 463 L Kl
33. sion 2 3 page 7 ELC 01X User Manual 3 4 Front Panel View of the ELC 01X Amplifier 0 00 0 D 3 z 2 Figure 1 ELC 01X front panel view version 2 3 page 8 ELC 01X User Manual 3 5 Description of the Front Panel Basically the front panel is functionally divided into two halves the right half has controls for CC and BR modes and the left half for VC mode and extracellular recording Each element has a number in bold that is related to that in Figure 1 The number is followed by the name in uppercase letters written on the front panel and the type of the element in lowercase letters Then a short description of the element is given 1 HEADSTAGE connector Connector for the headstage with optional differential input REF of the headstage must be connected to ground single ended measurement or to the bath differential measurement 2 CURRENT OUTPUT connector BNC connector providing the CURRENT OUTPUT signal scaling is set by CURRENT OUTPUT SENSITIVITY switch 29 Note The current output is filtered by a one pole filter with a corner frequency of 5 kHz Other corner frequencies are possible on request Please contact npi electronic 3 CURRENT OUTPUT FROM HEADSTAGE 0 1V nA connector Kz BNC connector providing the CURRENT OUTPUT signal directly from the headstage scaling is 0 1V nA 4 current polarity switch oA Switch for setting the polarity of the holding current or the gat
34. tem 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 i e after internal temperature has reached steady state values In most cases a warm up period of 20 30 minutes is sufficient HANDLING Please protect the device from moisture heat radiation and corrosive chemicals version 2 3 page 3 ELC 01X User Manual 2 Introduction Loose patch recordings or loose seal recordings Roberts amp Almers 1992 are used to record from single excitable ce
35. y 10 1k V V Rotary switch 10 20 50 100 200 500 1k 50 uV version 2 3 page 28 ELC 01X User Manual Potential LP filter attenuation corner frequencies Hz Potential HP filter attenuation corner frequencies Hz Telegraph potential LP filter Telegraph potential HP filter Telegraph potential output sensitivity Telegraph current output sensitivity Digital displays Display mV MQ Display current Inputs Input impedance analog Input range Input impedance digital TTL Input range TTL Current stimulus input CC Gated stimulus Polarity Voltage command input VC Gated stimulus VC Polarity Step gate input Dimensions 19 rackmount cabinet 19 483 mm 10 250 mm 3 5 88 mm Power r eq uirements 4 pole BESSEL filter other options available 24 dB octave 20 50 100 200 300 500 700 1k 1 3k 2k 3k 5k 8k 10k 13k 20k 1 pole filter other options available 6 dB octave DC 300 400 600 8 7 V 1V step 1 4 V 1V step 1 7 V 1 V step 1 7 V 1 V step 3 digits XXXX mV or XXXX MQ 3 2 digits XXX X nA 100 KQ 12 V 10 KQ 0 5 V via BNC connectors sensitivity 10 nA V with ten turn control of holding current resolution 100 pA range 100 nA selectable with toggle switch via BNC connectors sensitivity 10 mV with ten turn control of holding potential resolution 1 mV range 1 V selectable with toggle switch via
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