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ELC-01MX User Manual

version 2.6

page 24

7.3 Extracellular Stimulation and Electroporation

Cells can be stimulated using current or voltage signals.

7.3.1

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 COMM.
INP. / 10 mV (#14, Figure 5).

❏

Set the MODE OF OPERATION switch (#23, Figure 5) to CC.

❏

Set the holding current to zero using switch #5, Figure 5).

❏

For stimulation:

Apply the stimulus signal 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

❏

Approach the cell in VC mode and apply square voltage pulses to the electrode.

❏

Contact the cell and establish the loose-patch.

❏

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).

❏

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 enabling electroporation with up to 12 V with
a 100 M

Ω

electrode.

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Содержание ELC-01MX

Страница 1: ...AMPLIFIER MODULE for EXTRA INTRACELLULAR RECORDING SINGLE CELL STIMULATION and ELECTROPORATION 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 ...

Страница 2: ...Input configuration 10 4 3 3 Computer control of the mode of operation 10 4 3 4 Output configuration 10 4 3 5 Digital displays 10 4 4 Description of the Front Panel 11 5 Headstage 16 5 1 Headstage Elements 17 5 2 Headstage Bias Current Adjustment 18 6 Passive Cell Model 19 6 1 Cell Model Description 19 6 2 Connections and Operation 20 7 Introduction into Experiments 22 7 1 Recordings with the Diff...

Страница 3: ... 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 3 STATIC ELECTRICITY Electronic equipment is sensitive to static dis...

Страница 4: ...items are shipped with the EPMS 07 housing EPMS 07 cabinet with built in power supply Mains cord Fuse 2 A 1 A slow inserted Front covers Figure 1 Left front view of empty EPMS 07 housing In order to avoid induction of electromagnetic noise the power supply unit the power switch and the fuse are located at the rear of the housing see Figure 2 right 2 3 EPMS H 07 Housing In addition to the standard ...

Страница 5: ...power supply unit the power switch and the fuse are located 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 ...

Страница 6: ...he 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 Import...

Страница 7: ... boutons are accessible to loose patch recordings Auger 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 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 base...

Страница 8: ...ion 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...

Страница 9: ...age 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 or 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 stimul...

Страница 10: ... 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 POTENTIA...

Страница 11: ... 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 electr...

Страница 12: ...uid 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 switch Switch to select the scaling ...

Страница 13: ...wer position the built in low pass filter is disabled i e 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 en...

Страница 14: ... 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 τ 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 capa...

Страница 15: ...ngs are indicated by LEDs 26 The BRIDGE balance circuit compensates for the electrode artifact and allows accurate intracellular measurements of the membrane 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...

Страница 16: ...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 ch...

Страница 17: ...rolyte 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 ACl 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 ...

Страница 18: ...connect 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 PEL connector of the headstage to ground If oscillations occur take a resistor of a few kΩ 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 head...

Страница 19: ...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 MΩ and 22 pF CELL 1 represents an astrocyte like cell or a cell with 200 MΩ membrane resistance and 100 pF memb...

Страница 20: ...ELL 2 5 In GROUND lower position the electrodes are connected to ground via a 1 kΩ resistor In SEAL upper position are connected to a 1 GΩ resistor simulating the formation of a GIGASEAL with a patch electrode Figure 9 Schematic diagram of the passive cell model 6 2 Connections and Operation Checking the configuration Turn POWER switch of the EPMS housing off a For simulation of an experiment usin...

Страница 21: ...o SEAL position The 1 GΩ 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 MΩ electrode connector 1 Figure 8 for an experiment with sharp electrodes or the 10 MΩ electrode connector 3 Figure 8 for simulating an experiment ...

Страница 22: ...e 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 grounded enclosure or electrode holders PEL is connected to the measurin...

Страница 23: ... 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 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 loose patch Set the MODE OF OPERATION switch 23 Figure 5 to OFF Se...

Страница 24: ...e 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 Approach the cell in VC mode and apply square voltage pulses to the electrode Contact the cell and establish the loose patch For stimulation apply a voltage signal of the required amplitude and duration to the COMM INP 10 mV connector 14 Figure 5...

Страница 25: ...timuli 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 Approach the cell in VC mode and apply square voltage pulses to the electrode Contact the cell set a holding potential using the HOLD POT mV potentiometer 18 Figure 5 and establish the whole cell patch clamp configuration If necess...

Страница 26: ... 526 1 3 11 Barbour B Isope P 2000 Combining loose cell attached stimulation and recording J Neurosci Methods 103 199 208 Bureau I Shepherd G M G Svoboda K 2004 Precise Development of Functional and Anatomical Columns in the Neocortex Neuron 42 789 801 Duque A and L Zaborszky 2006 Juxtacellular Labeling of Individual Neurons In Vivo From Electrophysiology to Synaptology in Zaborszky L F G Wouterlo...

Страница 27: ...yi P 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 Fuentealba P Tomioka R Dalezios Y Marton L F Studer M Rockland K Klausberger T Somogyi P 2008 Rhythmically active enkephalin expressing GABAergic cells in the CA1 area of the hippocampus project to the subiculum and preferen...

Страница 28: ...an R Koch M Bückers J Kastrup L Roux I Petit C Hell SW Brose N Rhee JS Kügler S Brigande JV Moser T 2011 Probing the Functional Equivalence of Otoferlin and Synaptotagmin 1 in Exocytosis J Neurosci 31 4886 4895 Strenzke N Chanda S Kopp Scheinpflug C Khimich D Reim K Bulankina A V Neef A Wolf F Brose N Xu Friedman M A Moser T 2009 Complexin I is required for high fidelity transmission at the endbul...

Страница 29: ...09 120 April 14 2011 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 Tang Q Burgalossi A Ebbesen CL Ray S Naumann R Schmidt H Spicher D Brecht M 2014 Pyramidal and stellate cell specificity of grid and border representations in...

Страница 30: ...ast trim pots min response time 30 µs BIAS control CC only range 100 pA current adjustable with trim potentiometer Bridge balance 0 100 MΩ 0 1000 MΩ possible Adjustable with ten turn control Electrode resistance test Sensitivity 1 mV MΩ application of square current pulses 1 nA Display 3 digit XXX MΩ activated by push button same as POTENTIAL display Bandwidth and speed response Full power bandwid...

Страница 31: ...Input range 12 V Input impedance digital TTL 10 kΩ Input TTL range 0 5 V LO 0 8 V HI 1 V Current stimulus input via BNC connectors sensitivity 10 nA V Step gate input via BNC connector TTL Gated stimulus 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...

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