ELC-01MX manual ver2_6 - NPI Electronic Instruments
Contents
1. 20 T dntroductort mito Experie fs e edP sac 27 7 1 Recordings with the Differential Headstage 22 722 Voltabe media eae Red eade anda uncia ou vete E 23 7 3 Extracellular Stimulation and Electroporation 24 Toal Stimulation with CUEN suinkaan ERU E aE EA 24 Ti02 ABlectrOporabion win a otn 24 4 9 Simulation with oa ase e E A 24 7 3 4 Electroporation with Voltage esses eene nennen nnns 24 TA intracellular Recording oie etm teile i o occae aet D e c EE EE SEU 23 7 4 1 Current Clamp Recordin siae ea RED he Sa DAE Te uU 25 454 2 Voltage Clamp S 25 TE E 26 92 Pechnical bosco vi HD IE vce Pb yams ae PUR MAN es 30 version 2 6 page 2 1 ELC 01MX User Manual 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 please2. 103 199 208 1 Bureau I Shepherd G M G amp Svoboda K 2004 Precise Development of Functional and Anatomical Columns in the Neocortex Neuron 42 789 801 1 Duque A and L Zaborszky 2006 Juxtacellular Labeling of Individual Neurons In Vivo From Hlectrophysiology to Synaptology in Zaborszky L F G Wouterlood amp J L Lanciego eds Neuroanatomical Tract Tracing 3 3 Molecules Neurons and Systems Springer New York LJ Joshi S amp Hawken M J 2006 Loose patch juxtacellular recording in vivo A method for functional characterization and labeling of neurons in macaque J Neurosci Methods 156 37 49 LI Khaliq Z M amp Raman I M 2005 Axonal Propagation of Simple and Complex Spikes in Cerebellar Purkinje Neurons J Neurosci 25 454 463 1 Klausberger 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 L Nunemaker C S DeFazio 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 LI Nevian T amp Helmchen F 2007 Calcium indicator loading of neurons using single cell electroporation Pflugers Arch 454 675 88 version 2 6 page 26 ELC 01MX User Manual LJ Pinau
3. 18 HOLD POT mV switch and 19 HOLD POT mV potentiometer 14 COMM INP 10 mV connector BNC connector for an external command in VC mode sensitivity 10 mV i e 100 mV signal at this connector generates a command of 10 mV or in VCx10 mode sensitivity 1 mV i e 100 mV signal at this connector generates a command of 100 mV The signal form remains unchanged 16 AMPL trim pot T CONST trim pot Trim pots for compensating for the electrode capacity in VC mode A square shaped command pulse in VC will be distorted by the electrode capacity The shape of the command can be influenced by these trim pots in order to get the command pulse as square as possible AMPL trim pot Trim pot for setting t FAST in the electrode capacity compensation circuit T CONST trim pot Trim pot for setting C FAST in the electrode capacity compensation circuit Note CAPACITY COMPENSATION works only for the electrode capacity not for the capacity of the cell membrane Therefore capacitive transients of the cell are always present when square shaped pulses are applied in VC mode 17 HOLD POT mV switch Switch for setting the polarity of the holding potential in VC VCx10 mode or for the GATED stimulus see also 10 In zero position the holding potential gated stimulus is disabled 18 HOLD POT mV potentiometer Control for setting the amplitude of the continuous holding potential or for setting the amplitude of the
4. 2010 Another significant advance from classical in vivo recording methods such as Lalley et al 1999 is the use of this method in novel applications under in vivo conditions An excellent example of these kinds of technological advances are the studies of Helmchen and Stosiek Helmchen et al 2002 Stosiek et al 2003 Brecht et al 2002 Nevian amp Helmchen 2007 which used patch clamp recording together with two photon microscopy Assays have also been developed that enable both monitoring and manipulation of single cells under in vivo conditions Brecht et al 2004 Besides sophisticated optics these techniques always require precise recording and stimulation amplifiers that usually must work with patch electrodes High resolution in vivo recordings with ELC amplifiers are meanwhile a standard in many labs Geis et al 2011 Resinger et al 2011 and by using miniature headstages recordings from free moving animals are possible Epsztein et al 2011 J version 2 6 page 7 ELC 01MX User Manual Today three methods are used to record electrically in vivo or in vitro 1 Recordings using patch suction electrode from single neurons 1 Whole cell patch clamp technique tight seal recording intracellular L1 Loose patch technique loose seal recording extracellular 1 Intracellular recordings with sharp microelectrodes L4 Extracellular recordings with glass metal electrodes The amplifiers normally used for these types of measureme
5. A K 2011 Intracellular Determinants of Hippocampal CAI Place and Silent Cell Activity in a Novel Environment Neuron 70 109 120 April 14 2011 L Geis C Weishaupt A Gr newald B Wultsch T Reif A et al 2011 Human Stiff Person Syndrome IgG Induces Anxious Behavior in Rats PLoS ONE 6 el16775 doi 10 1371 journal pone 0016775 version 2 6 page 28 ELC 01MX User Manual LJ Lee D Shtengel G Osborne JE Lee AK 2014 Anesthetized and awake patched whole cell recordings in freely moving rats using UV cured collar based electrode stabilization Nature Protocols 9 2784 95 doi 10 1038 nprot 2014 190 In vivo recording with miniature headstage using ELC amplifiers LJ Epsztein J Brecht M amp Lee A K 2011 Intracellular Determinants of Hippocampal CAI Place and Silent Cell Activity in a Novel Environment Neuron 70 109 120 April 14 2011 LJ Lee D Shtengel G Osborne JE Lee AK 2014 Anesthetized and awake patched whole cell recordings in freely moving rats using UV cured collar based electrode stabilization Nature Protocols 9 2784 95 doi 10 1038 nprot 2014 190 1 Tang Burgalossi A Ebbesen CL Ray S Naumann R Schmidt H Spicher D Brecht 2014 Pyramidal and stellate cell specificity of grid and border representations in layer 2 of medial entorhinal cortex Neuron 84 1191 7 doi 10 1016 j neuron 2014 11 009 LI Tang Q Brecht M Burgalossi A Juxtacellular recording and morphological ident
6. Control to compensate for the electrode potential OFFSET ten turn potentiometer symmetrical i e 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 its 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 15 clamped to its 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 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 3 CUR OUTP SENSITIVITY swi
7. GATED stimulus see also 10 in VC mode range 1000 mV or in VCx10 mode range 10 V In zero position the holding gated potential is disabled version 2 6 page 14 ELC 01MX User Manual Extracellular recording unit The extracellular recording unit consist of 20 HP FILTER Hz switch and 21 POT OUTP SENSITIVITY switch Hint Set the MODE OF OPERATION switch to OFF for extracellular low noise recording 19 HP FILTER Hz switch Switch for setting the corner frequency of the high pass filter attenuation 6 dB octave in AC recording mode DC Recordings are done by switching the HP FILTER Hz switch to DC Hint During AC recording a DC signal is always provided at the POTENTIAL OUTPUT x1 BNC connector 13 20 POT OUTP SENSITIVITY switch Switch for scaling the POTENTIAL OUTPUT at the POTENTIAL OUTPUT connector 11 Scaling factors x10 x100 x1000 Important The POT OUTP SENSITIVITY switch is only related to the POTENTIAL OUTPUT BNC 11 and is always active i e not only in extracellular recordings but also in CC BRIDGE and VC VCx10 mode 21 Ret BRIDGE ON switch Switch for activation of either the electrode resistance test circuit switch set to or the BRIDGE balance circuit switch set to BRIDGE ON in CC mode Both settings are indicated by LEDs 26 The BRIDGE balance circuit compensates for the electrode artifact and allows accurate intracellular measurements of the membrane
8. and mains ground protective earth can be connected by a wire using the ground plugs on the rear panel of the instrument It is not possible to predict whether measurements will be less or more noisy with the internal ground and mains ground connected We recommend that you try both arrangements to determine the best configuration 2 7 2 EPMS E 07 The 19 cabinet is connected to the CHASSIS connector at the rear panel It can be connected to the SYSTEM GROUND SIGNAL GROUND on the rear panel of the instrument see Figure 4 The chassis can be linked to PROTECTIVE EARTH by connecting it to the PWR 03D with the supplied 6 pole cable and by interconnecting the GROUND and PROTECTIVE EARTH connectors on the rear panel of the PWR 03D see Figure 3 Best performance is generally achieved without connection of the chassis to protective earth SIGNAL HASS GROUND c 5 Important Always adhere to the appropriate safety measures Figure 4 Rear panel connectors of the EPMS E 07 2 8 Technical Data 2 8 1 EPMS 07 EPMS E 07 and EPMS H 07 19 rack mount cabinet for up to 7 plug in units Dimensions high 1U 1 3 4 44 45 mm 254 mm deep 2 8 2 EPMS 07 and EPMS H 07 Power supply 115 230 V AC 60 50 Hz fuse 2 1 A slow 45 60 W 2 8 3 EPMS E 07 External power supply PWR 03D 115 230 V AC 60 50 Hz fuse 1 6 0 8 A slow Dimensions of external power supply W x Dx H 225 mm x 210 mm x 85 mm 2 8 4 EPMS 03 Pow
9. be applied by gating a reset stimulus in CC or VC mode version 2 6 page 25 ELC 01MX User Manual 8 Literature General Recording Methods and Voltage Clamp Technique LJ Dietzel I D Bruns D Polder and Lux D 1992 Voltage Clamp Recording in Kettenmann H and R Grantyn eds Practical Electrophysiological Methods Wiley Liss NY LJ Lalley P M Moschovakis A and Windhorst U 1999 Electrical Activity of Individual Neurons in Situ Extra and Intracellular Recording in U Windhorst and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin New York 1 Ogden DC 1994 Microelectrode Techniques The Plymouth Workshop Handbook Second Edition The Company of Biologists Limited Cambridge LJ Polder H R M Weskamp K Linz amp R Meyer 2004 Voltage Clamp and Patch Clamp Techniques Chapter 3 4 272 323 in Dhein Stefan Mohr Friedrich Wilhelm Delmar Mario Eds Practical Methods in Cardiovascular Research Springer Berlin Heidelberg and New York 2004 1 Windhorst U and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin Heidelberg New York Juxtasomal Filling Loose Patch Techniques General 1 Auger C amp Marty A 2000 Topical Review Quantal currents at singlesite central synapses J Physiol 526 1 3 11 1 Barbour B amp Isope P 2000 Combining loose cell attached stimulation and recording J Neurosci Methods
10. 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 offset potentials at the electrode deterioration of the voltage measurement etc for details see Kettenmann and Grantyn 1992 An automated chlorinating apparatus ACI 01 is available from npi for optimal chlorinating of sliver wires please contact npi for details Ground provides system ground and is linked to the bath via an agar bridge or a Ag AgCl pellet The headstage is attached to the amplifier with the headstage cable see Figure 6 and an 8 pole connector The headstage is mounted to a insulated mounting plate that fits to most micromanipulators A holding bar or dovetail is available on request Note The shield of the BNC connector is linked to the driven shield output and must not be connected to ground The headstage enclosure is grounded Caution Please always adhere to the appropriate safety precautions see chapter 1 Please turn power off when connecting or disconnecting the headstage from the HEADSTAGE connector version 2 6 page 17 ELC 01MX User Manual 5 2 Headstage Bias Current Adjustment Caution It is important that this tuning procedure is performed ONLY after a
11. potential und application of current stimuli The BRIDGE is adjusted using the BR BAL potentiometer 23 22 BR BAL potentiometer If current is passed through the recording electrode in BRIDGE mode the potential deflection caused at the electrode resistance is compensated with this control ten turn potentiometer clockwise calibrated in MQ range 100 MQ i e 100 10 MQ 23 MODE OF OPERATION switch 5 position rotary switch to set the MODE OF OPERATION The following modes are possible EXT VC or CC are selected remotely by a TTL pulse 5 V applied to the MODE SELECT TTL connector 8 HI 21 V selects VC LO 0 8 V selects CC CC Current Clamp mode allows recording and stimulation in current clamp The electrode artifact is not compensated unless the BRIDGE mode is activated and adjusted see also 22 and 23 OFF CC Mode with all input signals turned off Can be used for extra and intracellular recordings is functional VC VOLTAGE CLAMP mode allows recording and stimulation in voltage patch clamp Series resistance and t slow not compensated VCx10 High range VC mode up to 12 V used for stimulation and electroporation version 2 6 page 15 ELC 01MX User Manual 24 POTENTIAL RESISTANCE display Display showing the POTENTIAL at the electrode in XXXX mV or the electrode resistance in XXX MQ see also 22 25 Status LEDs LEDs indicating the unit of the POTENTIAL RESISTANC
12. the instrument or 19 cabinet TEMPERATURE DRIFT WARM UP TIME analog electronic systems are sensitive to temperature changes Therefore all electronic instruments containing analog circuits should be used only in a warmed up condition 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 6 page 3 ELC 01MX User Manual 2 EPMS 07 Modular Plug In System 2 1 General System Description Operation The npi EPMS 07 is a modular system for processing of bioelectrical signals in electrophysiology The system is housed in a 19 rack mount cabinet 3U has room for up to 7 plug in units The plug in units are connected to power by a bus at the rear panel The plug in units must be kept in position by four screws M 2 5 x 10 The screws are important not only for mechanical stability but also for proper electrical connection to the system housing Free area must be protected with covers 2 2 5 07 Housing The following items are shipped with the EPMS 07 housing V EPMS 07 cabinet with built in power supply Mains cord V Fuse 2 A 1 A slow inserted V Front covers Figure 1 Left front view of empty 5 07 housing In order to avoid induction of electromagnetic noise the power supply unit the power switch and the fuse are located
13. E display or the selected MODE OF OPERATION of the amplifier respectively 26 BIAS trim pot Trim potentiometer for adjusting the output BIAS current of the headstage see also chapter 5 2 5 Headstage The ELC 01MX comes with a headstage for connecting suction electrodes for loose patch clamp or whole cell recordings and or stimulation or electroporation respectively or sharp electrodes for extracellular or intracellular recordings A differential headstage see Optional accessories in chapter 4 2 or a miniature headstage for measurements in vivo is also available For details contact 1 Reference electrode connector differential headstage eciroae con nector electronic GmbH 4 Eun com ki n AMPLIFIER HEADSTAGE zs Cable to TYPE RFB I N Om aa ROSE BA EL x amplifier olaer optional Mounting plate Ground connector Figure 6 Headstage of 01 version 2 6 page 16 ELC 01MX User Manual 5 1 Headstage Elements 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 holding bar or dovetail available on request The electrode filled with electrolyte is inserted into an electrode holder optional that
14. Electronic Instruments for the Life Sciences pease ade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION FOR THE ELC 01MX ELC AMPLIFIER MODULE for EXTRA amp INTRACELLULAR RECORDING SINGLE CELL STIMULATION and ELECTROPORATION LJ ELC O1MX 7 E Ed IN 0 bad E 27 ea S SAT 5 S NX i SRI 7 9 T D NY 3 9 LP FILTER 6 T VERSION 2 6 npi 2015 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 01MX User Manual Table of Contents Regula ONS aii oO RE EUR MASA E eU RUE dU MAP MA INN 3 2 TEPNIS 07 NIOdulat PIug In zS ys CCN cae eot ecoute toe Pa ed edd tu Cominus oio dott 4 2 1 General System Description Operation Re eene ted 4 2 2 EPMS 07 HOUSING asc ei bend toten da eda laute dose 4 2 3 uus n 4 2 2 JEPMSSB O7 EIOUSIDS cis isis tnde eo bos on Ro terre re RS I mant eem Ms Poss ee tan 4 DiS SEPM S 0 M 5 20 TOW FRO M 5 eta LEID CD E 6 2 Ll JBPNMSSOYIBPNIS 09 USE USER ERU MU RE Io RUIT 6 22 em etn cle e mtb IRIURE EM EE c eae 6 2 58 Techincabjatiss uncta dmi sags tases auld C
15. at the rear of the housing see Figure 2 right 2 3 EPMS H 07 Housing In addition to the standard power supply of the EPMS 07 the EPMS H 07 has a built in high voltage power supply This is necessary for all MVCS MVCC modules the HVA 100 HV TR150 and HVC 03M modules The output voltage depends on the modules in use 2 4 EPMS E 07 Housing The following items are shipped with the EPMS E 07 housing EPMS E 07 cabinet External Power supply PWR 03D Power cord PWR 03D to EPMS E 07 Mains chord Fuse 1 6 A 0 8 A slow inserted Front covers lt 44 54 SN version 2 6 page 4 ELC 01MX User Manual The EPMS E 07 housing is designed for low noise operation especially for extracellular and multi channel amplifiers with plugged in filters It operates with an external power supply to minimize distortions of the signals caused by the power supply 2 5 EPMS 03 The following items are shipped with the EPMS 03 housing V EPMS 03 cabinet with built in power supply Mains cord Y Fuse 034 A 0 2 A slow inserted V Front covers DPA 2FS OFFSET ON 1 ZERO ADJ z 7 LOW PASS Hz SU ge ie 401 300 10 L PNE TE gt Figure 2 Left front view of EPMS 03 housing Right rear panel detail of EPMS 03 and EPMS 07 housing In order to avoid induction of electromagnetic noise the power supply unit the power switch and the fuse are loca
16. consult the GENERAL TERMS OF DELIVERY AND CONDITIONS OF BUSINESS of npi electronic D 71732 Tamm Germany 1 2 3 4 5 GENERAL This system is designed for use in scientific laboratories and must be operated by trained staff only General safety regulations for operating electrical devices 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
17. del to GND of the headstage Important When using a differential headstage optional the REF connector at the headstage must not be left open It must be connected to ground Simulation of electrode in the bath 1 Set switch 4 Figure 8 to the lower position 1 Set switch 5 Figure 8 to GROUND position The 1 resistor simulates the resistance of the bath solution This can be used to train cancellation of offsets using the bridge balance and using the capacity compensation Simulation of SEAL formation 1 Set switch 4 Figure 8 to the lower position 1 Set switch 5 Figure 8 to SEAL position The 1 GQ resistor simulates the SEAL resistance when forming a GIGASEAL in patch clamp experiments Simulation of intracellular recording Intracellular recordings can be mimicked with one of two cells with different properties Use the 50 MQ electrode connector 1 Figure 8 for an experiment with sharp electrodes or the 10 MQ electrode connector 3 Figure 8 for simulating an experiment with patch electrodes 1 Switch the CELL membrane switch see 4 Figure 8 to the desired position CELL 1 or CELL 2 1 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 1 Turn POWER switch of the EPMS housing on Now you can adjust the amplifier see below and apply test pulses to the cell model The upper position the CELL me
18. display 25 kHz rise time 10 90 15 us 100 MQ lt 8 us 5 MQ 50 Q 12 V BNC connector sensitivity V nA Rotary switch range 0 1 1 10 3 digits XX XX nA resolution 10 pA 3 pole BESSEL filter other options available attenuation 18 dB octave corner frequency 3 kHz BNC connector sensitivity 1 mV mV page 30 Potential output Potential low pass filter Potential high pass filter Digital displays Display mV MQ Display current Inputs Input impedance analog Input range Input impedance digital TTL Input TTL Current stimulus input Step gate input Gated stimulus Polarity Voltage command input EPMS 07 SYSTEM Power Requirements ELC 01MX User Manual BNC connector range 10 100 1000 3 pole BESSEL filter other options available attenuation 18 dB octave corner frequency 3 kHz 1 pole BESSEL filter other options available attenuation 6 dB octave corner frequencies Hz 1 10 50 100 500 3 V5 digits XXXX mV or XXX MQ 3 0 digits XX XX nA 100 12 10 range 0 5 V LO 0 8 HI gt 1 V via BNC connectors sensitivity 10 nA V via BNC connector TTL with ten turn control of holding current resolution 100 pA range 100 nA Or with ten turn control of holding potential in VC mode resolution 1 mV range 1 V in VCx10 mode resolution 10 mV range 10 V selectable with toggle switch via BNC con
19. easurement Extracellular measurements are usually done in a loose patch configuration or with special metal microelectrodes Recordings with extracellular metal electrodes is simple The electrode is advanced in 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 For loose patch recording the procedure is the following Barbour amp Isope 2000 Nunemaker et al 2003 1 Approach the cell in VC mode and apply square voltage pulses to the electrode Contact the cell and establish the loose patch Set the MODE OF OPERATION switch 23 Figure 5 to OFF Set the required amplification of the POTENTIAL OUTPUT using 20 Figure 5 Select DC or filtered AC coupling with switch 19 Figure 5 If needed apply the LP filter 3 KHz to the output signal see 11 Figure 5 L C D D wv version 2 6 page 23 ELC 01MX User Manual 7 3 Extracellular Stimulation and Electroporation Cells can be stimulated using current or voltage signals 7 3 1 Stimulation with Current 1 Approach the cell in VC mode and apply square voltage pulses to the electrode 1 Contact the cell establish the loose patch and disconnect the voltage signal from COMM INP 10 mV 14 Figure 5 Set the MODE OF OPERATION switch 23 Figure 5 to CC 1 Set the holding current to zero using switch 5 Figure 5 L1 For stimulation Apply the stimulus si
20. eplenishment of synaptic vesicles in hair cells Nat Neurosci 13 869 876 LJ Reisinger E Bresee C Neef J Nair R Reuter K Bulankina A Nouvian R Koch M B ckers J Kastrup L Roux I Petit C Hell SW Brose N Rhee JS K gler S Brigande JV amp Moser T 2011 Probing the Functional Equivalence of Otoferlin and Synaptotagmin 1 in Exocytosis J Neurosci 31 4886 4895 1 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 1 Subramaniam M Althof D Gispert S Schwenk J Auburger G Kulik A Fakler B Roeper J 2014 Mutant a synuclein enhances firing frequencies in dopamine substantia nigra neurons by oxidative impairment of A type potassium channels Journal of Neuroscience 34 13586 99 doi 10 1523 J NEUROSCI 5069 13 2014 Intracellular recording using ELC amplifiers 1 Albertson AJ Williams SB Hablitz JJ 2013 Regulation of epileptiform discharges in rat neocortex by HCN channels Journal of Neurophysiology 110 1733 43 doi 10 1152 jn 00955 2012 1 Bhumbra GS Bannatyne BA Watanabe M Todd AJ Maxwell DJ Beato M 2014 The recurrent case for the Renshaw cell Journal of Neuroscience 34 12919 32 doi 10 1523 JNEUROSCI 0199 14 2014 1 Epsztein J Brecht M amp Lee
21. er supply 115 230 Volts AC 60 50 Hz fuse 0 4 0 2 A slow Maximum current supply 500 mA Dimensions 3U high 1U 1 3 4 44 45 mm 254 mm deep 265 mm wide version 2 6 page 6 ELC 01MX User Manual 3 Rationale Loose patch recordings or loose seal recordings Roberts amp Almers 1992 are used to record from single excitable cells without damage i e without a direct access to the cell interior This technique was described in a seminal paper 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 twenty years before the introduction of tight seal recording by Neher and Sakmann Review in Sakmann amp Neher 1995 The loose seal has a resistance of a few ten to a few hundred 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 this technique has the great advantage that the same electrode can be reused for recording from several cells since its beginnings several attempts have been made to make such precise extracellular methods accessible to various preparations An excellent overview can be fo
22. g electroporation with up to 12 V with a 100 MQ electrode version 2 6 page 24 ELC 01MX User Manual 7 4 Intracellular Recording Intracellular recordings can be performed in the whole cell patch configuration and with sharp microelectrodes 7 4 1 Current Clamp Recording The ELC 01MX can be used like a standard bridge amplifier 1 Set the MODE OF OPERATION switch 23 Figure 5 to CC and switch 21 Figure 5 to BR ON 1 After impaling the cell readjust the bridge using the BR BAL potentiometer 22 Figure 5 1 If needed set an appropriate holding current using the HOLD CUR nA potentiometer 4 Figure 5 and the holding current polarity switch 1 Apply stimuli to the cell using the STIM INPUT 10 nA V BNC connector 7 Figure 5 7 4 2 Voltage Clamp Recording The ELC 01MX can also be used like a simple patch clamp amplifier 1 Approach the cell in VC mode and apply square voltage pulses to the electrode 1 Contact the cell set a holding potential using the HOLD POT mV potentiometer 18 Figure 5 and establish the whole cell patch clamp configuration 1 If necessary adjust the shape of the voltage signal using the VC COMP trim pots 16 Figure 5 1 Apply stimuli to the cell using the COMM INP 10 mV BNC connector 14 Figure 5 Important Voltage patch clamp experiments are possible only with low resistance patch pipettes not with high resistance sharp micro electrodes Note Stimuli can also
23. gnal to the STIM INPUT 10 nA V connector 7 Figure 5 Or Set switch 9 Figure 5 to the lower position Adjust the stimulus amplitude with the holding current potentiometer 4 and set the stimulus polarity using switch 5 Figure 5 Gate the preset stimulus with a TTL signal linked to the GATE BNC connector 9 Figure 5 7 3 2 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 7 3 3 Stimulation with Voltage 1 Approach the cell in VC mode and apply square voltage pulses to the electrode L1 Contact the cell and establish the loose patch 1 For stimulation apply a voltage signal of the required amplitude and duration to the COMM INP 10 mV connector 14 Figure 5 Or Set switch 9 Figure 5 to the lower position Adjust the stimulus amplitude with the holding potential potentiometer 18 and set the stimulus polarity using switch 17 Figure 5 Gate the preset stimulus with a TTL signal linked to the GATE BNC connector 10 Figure 5 LJ If necessary adjust the shape of the voltage signal using the VC COMP trim pots 16 Figure 5 7 3 4 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 For stimuli with large amplitudes set the MODE OF OPERATION switch 23 to VCx10 enablin
24. grounded enclosure or electrode holders PEL 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 10 Important For reliable measurements the experimental chamber or animal respectively must be grounded Also Very Important When operating in VC mode the reference electrode must not have a resistance of more than 1 kQ CC mode is not affected If differential measurement is not required single ended measurement configuration see Figure 10 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 version 2 6 page 22 ELC 01MX User Manual Experimental Experimental chamber Slice chamber Slice REF Ag AgCl pellet electrode i Electrode holder Electrode holder npi electronic npi electronic GND REF REF Ground SUBCLICK Ground SUBCLICK jack connector jack connector REF i REF Cable to Cable to amplifier amplifier differential measurement configuration single ended measurement configuration Figure 10 headstage connections A differential measurement B single ended measurement 7 2 Extracellular Voltage M
25. hm s Law Cancel this voltage by tuning the headstage BIAS current trim pot 27 Figure 5 until the POTENTIAL DISPLAY shows 000 version 2 6 page 18 ELC 01MX User Manual 6 Passive Cell Model The cell model is designed to be used to check the function of the instrument either 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 i e to find out whether the amplifier is broken or if something is wrong with the electrodes or holders etc ELC MOD D SEAL 160 in Figure 7 ELC MOD passive cell model 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 cell with 200 MQ membrane resistance and 100 pF membrane capacitance CELL 2 represents an neuron like cell or 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 o 4 5 GROUND 1kQ npi electronic GmbH D 71732 Tamm Germany www npielectro
26. ification of single neurons in freely moving rats 2014 Nature Protocols 9 2369 81 doi 10 1038 nprot 2014 161 version 2 6 page 29 ELC 01MX User Manual 9 Technical Data Module for EPMS 07 system with headstage Headstage Input voltage range Operating voltage Enclosure Headstage connector Electrode connector Ground connector Reference electrode connector optional Input resistance CC Current range Electrode parameter controls OFFSET CC CAPACITY COMPENSATION VC CAPACITY COMPENSATION t fast BIAS control CC only Bridge balance 0 100 MQ 0 1000 MQ possible Electrode resistance test Sensitivity mV MQ Display Bandwidth and speed response Full power bandwidth Rex 0 Outputs Output impedance Max voltage Current output Current output sensitivity Current display Current low pass filter Potential output x1 version 2 6 12 V 15 V Size 23 x 70 x 26 mm grounded 8 pole DIN connector BNC with driven shield 2 4 mm banana jack SMB connector 10P Q 120 nA max 100 MQ feedback range 100 mV ten turn control range 0 30 pF ten turn control time constant and amplitude adjustable with trim pots min response time 30 us range 100 pA current adjustable with trim potentiometer Adjustable with ten turn control application of square current pulses 1 nA 3 digit XXX MQ activated by push button same as POTENTIAL
27. l mouse development Journal of Neurophysiology 113 578 91 doi 10 1152 jn 00586 2014 version 2 6 page 27 ELC 01MX User Manual Bhumbra GS Bannatyne BA Watanabe M Todd AJ Maxwell DJ Beato M 2014 The recurrent case for the Renshaw cell Journal of Neuroscience 34 12919 32 doi 10 1523 JNEUROSCTI 0199 14 2014 LJ Geis C Weishaupt A Hallermann S Grunewald B Wessig C Wultsch T Reif A Byts N Beck M Jablonka S Boettger M K Uceyler N Fouquet W Gerlach M Meinck H M Siren A L Sigrist S J Toyka K V Heckmann M amp Sommer C 2010 Stiff person syndrome associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition Brain doi 10 1093 brain awq253 L4 Iwai Y et al A simple head mountable LED device for chronic stimulation of optogenetic molecules in freely moving mice Neurosci Res 2011 doi 10 1016 j neures 2011 01 007 LI Neef J Gehrt A Bulankina A V Meyer A C Riedel D Gregg R G Strenzke N amp Moser 2009 The Ca channel subunit 82 regulates Ca channel abundance and function in inner hair cells and is required for hearing Journal of Neuroscience 29 10730 10740 LJ Pangrsic T Lasarow L Reuter K Takago H Schwander M Riedel D Frank T Tarantino L M Bailey J S Strenzke N Brose N Muller U Reisinger E amp Moser T 2010 Hearing requires otoferlin dependent efficient r
28. lt 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 LJ Pinault D 2011 The Juxtacellular Recording Labeling Technique Chapter 4 41 77 in Vertes R P amp Stackman Jr R W Eds Electrophysiological Recording Techniques Humana Press Springer New York Dordrecht Heidelberg London L Rathenberg J Nevian T amp Witzemann V 2003 High efficiency transfection of individual neurons using modified electrophysiology techniques J Neurosci Methods 126 91 98 LJ Roberts W M amp Almers W 1992 Patch Voltage Clamping with Low Resistance Seals Loose Patch Clamp In Rudy amp Iversen L E eds Channels Methods in Enzymology 207 Academic Press San Diego LJ 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 using ELC amplifiers LJ Bruno R M amp Sakmann 2006 Cortex is driven by weak but synchronously active thalamocortical synapses Science 312 1622 1627 1 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
29. mbrane switch CELL 1 simulates a cell with a resistance of 50 MQ and a capacitance of 22 pF In the lower position CELL 2 a cell membrane with 200 MQ and 100 pF is simulated version 2 6 page 21 ELC 01MX User Manual 7 Introduction into Experiments The ELC 01MX is capable to perform several types of experiments that are briefly introduced in the following 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 circuit 7 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 OIMX 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 PEL are connected to microelectrodes using cables with
30. nector sensitivity 10 mV 115 230 V AC 60 50 Hz fuse 2 A 1A slow 45 60 W depending on the modules plugged in Dimensions 19 rack mount cabinet 3U high 1U 1 3 4 44 45 mm version 2 6 page 31
31. nic com Figure 8 ELC MOD passive cell model version 2 6 page 19 ELC 01MX 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 either 50 MQ and 22 pF CELL 1 or 200 MQ and 100 pF CELL 2 5 In GROUND lower position the electrodes are connected to ground via a 1 KQ resistor In SEAL upper position are connected to a 1 GQ resistor simulating the formation of a GIGASEAL with a patch electrode Whole cell Patch sharp Microelectrode Driven Shield BNC connector o BNC connector Rg 10 MQ Rg 50 MO 2 CELL 1 GROUND REAL Rm l Cm Cm Rm 1 kQ 1 GQ 200 MQ 100 pF 22 pF 50 MQ Z Figure 9 Schematic diagram of passive cell model 6 2 Connections and Operation Checking the configuration L4 Turn POWER switch of the EPMS housing off a For simulation of an experiment using a patch pipette 1 Connect the BNC jack labeled 10 MQ of the cell model to the BNC connector of the headstage version 2 6 page 20 ELC 01MX User Manual b For simulation of an experiment using a sharp electrode 1 Connect the BNC jack labeled 50 of the cell model to the BNC connector at the headstage For a and b 1 Connect GND of the cell mo
32. nts are specialized for the recording of the potentials or currents generated by the neurons under investigation If these recording methods are combined with dye injection electroporation stimulation or other electrical techniques that require multiple use of the recording electrode additional constraints are imposed on the experimental setup that may require the acquisition of additional components 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 and amperometry voltammetry methods The ELC amplifier is the Swiss Army Knife of modern electrophysiology It is not only easy to use but it is also sufficiently versatile to enable the use of a single amplifier for many sophisticated experiments version 2 6 page 8 ELC 01MX User Manual 4 ELC 01MX 4 1 ELC 01MX Components The following items are shipped with the ELC 01 MX system Amplifier module for the EPMS 07 system V GND 2 6 mm banana plug and optional REF SMB connectors for headstage V Headstage V User manual 4 2 Optional Accessories Differential headstage Miniature headstage Cell model Pipette holder 4 3 System Description The ELC 01IMX was designed for intra and extracellular recording precise single cell electrical stimulation as well as juxtasomal filling with pa
33. onse oue MU Ma Mem 6 2 8 1 EPMS 07 EPMS E 07 and EPMS H 07 essere 6 2 0 2 BPNIS 07 and BENISSEIUT tiens eo ER Ope URSUS E c Eds 6 2 8 ERMS EOT seo t Dt vbi nude O rrr te 6 P EBD VASO 9 c 6 J Rallonalexc doses eM CL MC 7 S MUBOLUNUp Gm 9 d BEC ODMDOSGOmDOBOIS vcio a 9 2 ONES E S NIS E EO D D QT 9 97 deed edes de ce ERE eaae duse emu wide nds 9 4 3 1 Operation nodes of the amplifier used 9 432 InputconbbourattOonb ana e 10 4 3 3 Computer control of the mode of 10 AS OM MPU COMME UTA ON odios tu ter niti abu eate uda euin dudes 10 2 39 rr tue D d Ud 10 4 4 Description of the Front Panel eee eoe ebore PAS Resa eda Seer eR Se etu pres 11 I Head tate E EE tam aor eunuch ves 16 Sd Headstase BIemelits ie ons kr EPA Mu MEM ob UE ERR PER ce 17 5 2 Headstase Bias Current Adjustment dole doe aeo eae eoe eq Ix ob E E euge eia 18 9 Passive Coll NIOeL ue tete ht Ein eas in ve khe m dns 19 Cell Model Descriptio 19 02 Connections and OPIO arenes oo
34. pass filter All filters can be bypassed 4 3 5 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 version 2 6 page 10 ELC 01MX User Manual 4 4 Description of the Front Panel 1 01 ni mv MO OFF gc BR EXT VCx10 VC OFF CC EXT Q Q Q Q POTENTIAL RESISTANCE VODE Q OPERATION 1k 8 pc 100 10 orat 18 SENSITIVITY PIP HOLD 0 1 CUR OUTP PONI SENATTIVITY HP FILTER Hz HEADSTAGE POTENTIAL POTENTIAL CURRENT GATE MODE OUTPUT 1 OUTPUT OUTPUT TTL SELECT TTL O Figure 5 ELC 01MX front panel view In the following description of the front panel elements each element has a number that is related to that in Figure 5 The number is followed by the name in uppercase letters written on the front panel and the type of the element in lowercase letters Then a short description of the element is given Some elements are grouped in functional units e g extracellular recording unit and are described as units regardless of the order of numbers 1 CURRENT nA display Display showing the current flowing through the electrode in XX XX nA max 19 99 nA version 2 6 page 11 ELC 01MX User Manual 2 PIP HOLD OFFSET potentiometer
35. plied to the electrode as long as the TTL signal connected here is HI and the ON switch is set to the upper position ON The amplitude of the stimulus is set by the HOLD CUR nA potentiometer 4 in CC mode or the HOLD POT mV potentiometer 18 in VC mode The polarity of the stimulus is set by the HOLD CUR nA polarity switch 5 Setting the ON switch to the lower position disables the GATED stimulus and enables the setting of a holding current in CC or BRIDGE mode or a holding potential in VC or VCx10 mode 10 CURRENT OUTPUT connector LP FILTER ON switch BNC connector providing the current flowing through the electrode in nA V The scaling is dependent on the setting of the CUR OUTP SENSITIVITY switch 3 Setting the LP FILTER ON switch to the upper position ON enables the built in low pass filter corner frequency 3 kHz attenuation 18 dB octave i e the CURRENT OUTPUT connector provides the filtered current with the scaling set by switch 3 In the lower position the built in low pass filter is disabled 1 the CURRENT OUTPUT connector provides the unfiltered current with the scaling set by switch 3 11 POTENTIAL OUTPUT connector LP FILTER ON switch BNC connector providing the potential at the electrode in mV The scaling is dependent on the setting of the POT OUTP SENSITIVITY switch 21 Setting the LP FILTER ON switch to the upper position ON enables the built in low pass filter corner fre
36. producing slow spiking GABAergic neurons and their involvement in hippocampal network activity Neuron 57 917 929 _J 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 CA1 area of the hippocampus project to the subiculum and preferentially innervate interneurons Journal of Neuroscience 28 10017 10022 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 Journal of Neuroscience 29 8875 8883 1 Subramaniam M Althof D Gispert S Schwenk J Auburger G Kulik A Fakler B Roeper J 2014 Mutant a synuclein enhances firing frequencies in dopamine substantia nigra neurons by oxidative impairment of A type potassium channels Journal of Neuroscience 34 13586 99 doi 10 1523 JNEUROSCI 5069 13 2014 Transfection using ELC amplifiers 1 Stan A Pielarski K N Brigadski T Wittenmayer N Fedorchenko O Gohla A Lessmann V Dresbach T amp Gottmann K 2010 Essential cooperation of N cadherin and neuroliginc in transsynaptic control of vesicle accumulation Proc Natl Acad Sci U S A 107 11116 11121 Extracellular recording using ELC amplifiers 1 Arancillo M White JJ Lin T Stay TL Sillitoe RV 2015 In vivo analysis of Purkinje cell firing properties during postnata
37. quency 3 kHz attenuation 18 dB octave i e the POTENTIAL OUTPUT connector provides the filtered potential with the scaling set by switch 21 In the lower position the built in low pass filter is disabled i e the POTENTIAL OUTPUT connector provides the unfiltered current with the scaling set by switch 21 12 BUZZ push button Push button to activate the BUZZ circuit In order to facilitate the penetration of the cell membrane or to clean the tip of the electrode a BUZZ circuit is provided which is based on oscillations caused by overcompensating the capacitance compensation system The overcompensation of capacitance compensation yields very powerful high frequency oscillations The BUZZ circuit is active as long as the button is pressed 13 POTENTIAL OUTPUT x1 connector BNC connector providing the potential at the electrode in V The scaling is fixed to x1 and therefore independent on the setting of the POT OUTP SENSITIVITY switch 20 This connector is normally used to monitor voltage pulses in the VCx10 mode see also 23 or else to provide a DC signal if the high pass filter 19 Figure 5 is used version 2 6 page 13 ELC 01 MX User Manual 14 COMM INP 10 mV connector Please see below Voltage Clamp Unit 15 HEADSTAGE connector Connector to attach the HEADSTAGE via a flexible cable Voltage Clamp Unit The voltage clamp unit consists of 14 COMM INP 10 mV connector 16 17 AMPL and T CONST trim pots
38. sing a toggle switch a bridge balance circuit can be activated to compensate for the electrode artifact BRIDGE mode only in CC mode With the same switch the ELECTRODE RESISTANCE test mode is activated The electrode resistance is measured directly in MQ and displayed on the POTENTIAL display 4 3 2 Input configuration The amplifier has two inputs for VC and CC mode The signal applied to the analog input BNCS is converted either into a voltage command signal x1 or x10 scaling for the VC or VCx10 voltage clamp modes 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 GATE TTL input BNC This control can be also used as HOLDING potentiometer if the switch in the GATE BNC is turned off 4 3 3 Computer control of the mode of operation In the EXT position of the MODE OF OPERATION switch the amplifier can be controlled by a TTL signal connected to the MODE SELECT BNC LO CC HI VC 4 3 4 Output configuration The ELC 01MX amplifier has two output BNC connectors for POTENTIAL and one output BNC for the CURRENT signal The POTENTIAL OUTPUT 1 is a pure DC output that monitors the electrode potential The signal at the FILTERED OUTPUT can be high pass filtered 6 corner frequencies amplified x10 x100 x1000 and filtered by a 3 kHz low pass filter The current output signal is amplified x1 x10 x100 and also passed through a 3 kHz low
39. tch Switch to select the scaling of the CURRENT OUTPUT 0 1 0 1 V nA 1 V 10 nA 1 1 V nA 1V 1 10 10 V nA 1 V 20 1 nA 4 HOLD CUR nA potentiometer Ten turn potentiometer for generating a holding current in CC or BRIDGE mode or for setting the amplitude of the GATED stimulus see also 9 range 100 nA 100 10 nA 5 HOLD CUR nA polarity switch Switch for setting the polarity of the holding current in CC or BRIDGE mode or for the GATED stimulus see also 9 In zero position the holding current gated stimulus is disabled 6 C COMP potentiometer Ten turn potentiometer for compensating the capacity of the electrode clockwise range 0 30 pF Functional in all current clamp modes CC CC BRIDGE Ret OFF 7 STIM INPUT 10 nA V connector BNC connector for an external command in CC or BRIDGE mode sensitivity 10 nA V i e 1 V 10nA The voltage signal that 15 connected here is transformed to a proportional current at the electrode in CC mode The signal form remains unchanged version 2 6 page 12 ELC 01MX User Manual 8 MODE SELECT TTL connector BNC connector for remote control of the MODE of operation The TTL signal connected here selects the mode of operation remotely HI VC LO CC functional in EXT mode only 9 GATE connector ON switch BNC connector for generating a stimulus with a TTL signal in CC VC or VCx10 mode A current or a voltage stimulus respectively is ap
40. tch electrodes The system consists of a module for the EPMS 07 modular system and a small headstage with holding bar dovetail or a mounting plate It can be used in slices or in in vivo preparations using the optional headstage with a differential input or for recordings in free moving animals using a miniature headstage It has separate capacity compensation controls for VC and CC mode all controls Bridge balance CC cap comp offset holding controls are calibrated 10 turn potentiometers The ELC 01MX is capable to record extracellularly DC AC coupled to stimulate with current or voltage and to perform non invasive juxtasomal filling of cells with dyes or DNA The amplifier can also be used to record and stimulate intracellularly in current clamp CC or voltage clamp VC It is also suitable for amperometric or voltammetric investigations with carbon fiber electrodes 4 3 1 Operation modes of the amplifier The operation modes of the amplifier are selected by a rotary switch with five positions The selected mode is indicated by LEDs above the two digital displays EXT VC or CC are selected by a TTL pulse applied to the MODE SELECT BNC CC CURRENT CLAMP MODE used to inject current signals OFF CC Mode with all input signals turned off VC VOLTAGE CLAMP mode potential commands are applied to the electrode VCx10 High range VC mode for stimulation and electroporation version 2 6 page 9 ELC 01MX User Manual In addition u
41. ted at the rear of the housing see Figure 2 right 2 6 PWR 03D The external power supply PWR 03D is capable of driving up to 3 EPMS E housings Each housing is connected by a 6 pole cable from one of three connectors on the front panel of the PWR 03D to the rear panel of the respective EPMS E housing see Figure 3 Figure 4 A POWER LED indicates that the PWR 03D is powered on see Figure 3 left Power switch voltage selector and fuse are located at the rear panel see Figure 3 right Note The chassis of the PWR 03D is connected to protective earth and it provides protective earth to the EPMS E housing if connected PWR 03D npi OUTPUT A OUTPUT B OUTPUT C ROUND 50 60 Hz SLOW ISAC 50 60 Hz i npi electronic GrnbH e D 71732 Tamm Germany www npielectronic corn oe 62724752 Figure 3 Left PWR 03D front panel view Right PWR 03D rear panel view Note This power supply is intended to be used with npi EPMS E systems only version 2 6 page 5 ELC 01MX User Manual 2 7 System Grounding 2 7 1 EPMS 07 EPMS 03 The 19 cabinet is grounded by the power cable through the ground pin of the mains connector protective earth In order to avoid ground loops the internal ground is isolated from the protective earth The internal ground is used on the BNC connectors or GROUND plugs of the modules that are inserted into the EPMS 07 housing The internal ground
42. und 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 Sakmann 2006 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 for 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 allows the application of 1 2 V stimuli to only one cell Babour amp Isope 2000 In the nineties of the last century the method of juxtacellular dye application juxtasomal 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 has now become well established in the field of slice and in vivo preparations Klausberger 2004 Duque A and L Zaborszky 2006 Pinault 2011 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 Stan et al
43. warm up period of at least 30 minutes The ELC 01MX 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 trim pot see 26 in Figure 5 The tuning procedure should be performed regularly about once a month since the bias current changes over time The tuning procedure is performed using high value resistors and or a cell model It cannot be performed with an electrode since there are always unknown potentials involved tip potential junction potentials d d Disconnect all input signals except the headstage Put the holding current switch to position 0 5 Figure 5 Set the operation mode to CC Connect the Pri connector of the headstage to ground If oscillations occur take a resistor of a few kQ and connect it 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 2 Figure 5 Remember The zero position of the OFFSET control is at 5 Remove the wire and attach the cell model or a resistor with a value of about 10 MQ across the same connection The value displayed at the POTENTIAL DISPLAY is related to the BIAS current of the headstage according to O
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