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OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION FOR

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1. von 23 4 9 Capacity COMPCNSAlON cde debut 23 d Bidee Balant ses erat lero da ye ste tuc m pev ca vidas tod tube v era Ui dann Du pin unt 25 T9 HENCE CU OCS SOIC CHOI tihi evo uo wal eae 26 7 6 Extracellular Recording Stimulation and Juxtacellular Filling 26 Intracelular Sample 28 8 1 Sample Experiment using a Sharp Electrode 28 8 2 Sample Experiment using a Suction Patch 31 8 3 Recordings with the Differential Headstage 32 D Trouble SNOOUMS a Ead EM 34 10 35 11 Me CHING EL MI 38 ERE 40 version 2 0 2 BA 03X User Manual About this Manual This manual should help to setup and use the BA 03X system correctly and to perform reliable experiments If you are not familiar with the use of instruments for intracellular recording of electrical signals please read the manual completely The experienced user should read at least chapters 1 3 3 3 5 and 7 Important Please read chapter 1 carefully It contains general information about the safety regulations and how to handle highly sensitive electronic instruments Signs and conventions In this ma
2. Apply stronger suction to the pipette or carefully use the BUZZ unit to brake the cell membrane under the pipette and establish the whole cell configuration The whole cell configuration is established if you see the voltage signal getting smaller again c Figure 15 and you read the expected membrane potential L Read the membrane potential and if necessary readjust the BRIDGE BALANCE as shown in 7 4 and Figure 13 J Start the experiment version 2 0 page 31 BA 03X User Manual close the a EMEN cell membrane 10 mV gigaseal formed c whole cell configuration established omv 25 ms Figure 15 Approaching the cell forming a gigaseal and establishing the whole cell configuration 8 3 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 BA 03X 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 1 e which both electrodes re
3. D Electronic f Instruments for the Life Sciences n QS wade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION FOR THE BA 03X INTRACELLULAR BRIDGE MODE and EXTRACELLULAR AMPLIFIER VERSION 2 0 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 BA 03X User Manual Table of Contents About thus Manual au ipee aae pe tabe dtes e ated esu 3 Regulo ees aee scu Ae au e 4 2 DU X C oOmpobellts distin 5 5 EET 5 3 2 Description Of the Front Panel eo ta e eed ertt 6 2355 J Deseripaonm Or the Rear Pane a a 16 eh iad D hee haa eaten yee 17 Als Headstape Elements taste ulead auo 17 2 SEUNG Up the D A 09 18 Gz Passive Cell eus amd man aes Gears ere eee 19 COE MOGSGL De SC tip MOI scared o 19 6 2 COMME CHO MS and ODeratTOTl acne Weal satin stan rss eins 20 T Procedures utu eris tho edes v Lem desta iu 22 7 1 Headstage Bias Current Adjustment oui rie 27 1225 CompensatlOlkenrnea iem
4. 0 1 0 2 0 5 1 2 5 or 10 V nA display XX XX nA CURRENT RANGE x1 display nA CURRENT RANGE x10 Potential BNC connector POTENTIAL OUTPUT sensitivity selectable by rotary switch x10 x20 x50 x100 x200 500 x1000 display mV or connector POTENTIAL OUTPUT FROM HEADSTAGE sensitivity V V Potential LP filter 4 pole BESSEL filter other options available attenuation 24 dB octave corner frequencies Hz 20 50 100 200 300 500 700 1k 1 3k 2k 3k 5k 8k 10k 13k 20k Potential HP filter 1 pole filter other options available attenuation 6 dB octave corner frequencies Hz DC 0 1 0 3 0 5 1 3 5 10 30 50 100 300 500 800 Ik 3k Telegraph potential LP filter 8 7 V IV step Telegraph potential HP filter 8 7 V I V step Telegraph potential output sensitivity 1 7 V 1 V step Telegraph current output sensitivity 1 7 V 1 V step Inputs Current stimulus input via BNC connectors sensitivity dependent on preset CURRENT RANGE CURRENT RANGE x1 I nA orO nA V CURRENT RANGE x10 10nA Vor1lnA V Step gate input via BNC connector gated stimulus with digital control of current step size Resolution x1 10 pA Resolution x10 100 pA Polarity selectable with toggle switch Holding current range 1 10 nA Holding current range x10 100 nA Adjustable with ten turn control Dimensions 19 rackmount cabinet 19 483 mm 10 250 mm
5. 18 Figure 1 at the BA 03X E 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 the gated stimulus unit L 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 2 Figure 1 and the CURRENT OUTPUT SENSITIVITY switch 6 Figure 1 Before using the BA 03X always make the basic settings to avoid oscillations Basic settings Turn all controls to low values less than 1 and the OFFSET in the range of 5 zero position see chapter 3 2 version 2 0 page 18 BA 03X User Manual L Set the CURRENT RANGE switch 9 Figure 1 to x1 _J Turn POWER switch on Now the BA 03X 1s ready for an initial check with the cell model 6 Passive Cell Model The BA 03X 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 damaged during transport or 2 to train personnel using the instrument or 3 in case of trouble see also chapter 9 to check which part of
6. 3 5 88 mm Power requirements 115 230 V AC 60 50 Hz fuse 0 4 0 2 A slow 25 W version 2 0 page 39 BA 03X User Manual index abbreviations 3 accessories 5 AUDIO potentiometer 15 basic settings 18 bias current 34 bias current adjustment 22 BIAS current potentiometer 10 bridge balance 25 27 29 BRIDGE BALANCE unit 12 CAPACITY COMP potentiometer 12 cell model 19 connections and operation 20 description 19 components 5 CURRENT INPUT unit 13 CURRENT OUTPUT unit 9 CURRENT RANGE unit 10 Differential Headstage optional 32 electrical connections 18 electrode 23 artifacts 30 capacity compensation 23 offset compensation 23 selection 26 ELECTRODE RESISTANCE TEST button 15 front panel view 7 version 2 0 gated stimulus unit 14 general description 5 GROUND connector 15 headstage 17 elements 17 HEADSTAGE connector 12 HOLDING CURRENT unit 10 literature 35 model circuit sharp electrode 28 model circuit suction patch electrode 31 OFFSET potentiometer 11 OSCILLATION SHUT OFF unit 15 PENETRATION unit 11 POTENTIAL OUTPUT unit 8 rear panel 16 safety regulations 4 sample experiments 28 sharp electrode 28 suction patch electrode 31 seal formation 32 sharp electrodes 28 suction patch electrodes 31 technical data 38 testing 22 trouble shooting 34 tuning 22 page 40
7. E AL OUTPUT GAIN HE 9 9 F OO OOO PONOC STEP SUE OSCILLATION SHUT OFF CABLED T RESET POTENTIAL ELECTRODE RESISTANCE TES 8 e E 3 3 _ 2 a F ET zu n 2 o E I ive OWPASS Hz POTENTIAL TPUT rri Figure 1 BA 03X front panel view the numbers are related to those in the text below version 2 0 page 7 BA 03X User Manual 1 POWER pressure switch Switch to turn POWER on switch pushed or off switch released POTENTIAL OUTPUT unit The POTENTIAL OUTPUT unit consists of 2 POTENTIAL OUTPUT GAIN 34 POTENTIAL OUTPUT FILTER HIGHPASS Hz 35 POTENTIAL OUTPUT FROM HEADSTAGE V 36 POTENTIAL OUTPUT mV and 37 POTENTIAL OUTPUT FILTER LOWPASS Hz 2 POTENTIAL OUTPUT GAIN switch 7 position switch for setting the amplification factor of the recorded potential x10 to x 1k The amplified voltage signal is available at the POTENTIAL OUTPUT BNC connector 36 34 POTENTIAL OUTPUT FILTER HIGHPASS Hz switch 16 position switch for setting the corner frequency of the HIGHPASS 1 pole BESSEL FILTER for the POTENTIAL signal 35 POTENTIAL OUTPUT FROM HEADSTAGE V connector BNC connector providing the raw potential at the electrode 36 POTENTIAL OUTPUT mV connector BNC connector providing the amplified and filtered potential at the electrode 37 POTENTIAL OUTP
8. potential mV 12000 8000 4000 4000 8000 0 MO Q start stop 12000 time Figure 2 buzz function of the BA 03X 16 OFFSET potentiometer Control to compensate for the electrode OFFSET ten turn potentiometer symmetrical i e 0 mV 5 on the dial range 200 mV see chapter 7 2 version 2 0 page 11 BA 03X User Manual Control for the compensation of the input capacitance ten turn potentiometer clockwise range 0 30 pF see chapter 7 3 Caution This circuit is based on a positive feedback circuit Overcompensation leads to oscillations which may damage the cell Important The capacity compensation for the electrode does NOT work if the oscillation shut off circuit 1s activated This may lead to an incorrect reading of the electrode resistance see also 33 especially when electrodes with high resistances are used 18 HEADSTAGE connector The
9. Connect the MICROELECTRODE connector of the headstage to ground If parasitic oscillations occur use a 10 resistor for grounding If you use a cell model set 3 in Figure 7 to GROUND Note 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 I Tune the OFFSET to zero using the OFFSET control 16 Figure 1 see also chapter 7 2 L After tuning the OFFSET connect the cell model via the 50 connector sharp electrode If you do not use a cell model remove the wire and attach a resistor with a value of about 5 to 10 across the same connection The value displayed at the DISPLAY 3 Figure 1 is related to the BIAS current of the headstage according to Ohm s Law Cancel this voltage by tuning the headstage BIAS current potentiometer 12 Figure 1 L Accuracy Now both displays potential current the should read 000 Due to the limited resolution of the display unbalanced offsets and thermal drifts an offset of 001 to 002 version 2 0 page 22 BA 03X User Manual can occur on the display This small deviation can be trimmed internally but this procedure 15 necessary only if very small currents in the pA range must be applied exactly 7 2 Offset Compensation If an electrode is immersed into the bath solution an offset voltage will appear even if no current is passed This offset
10. HEADSTAGE 1 connected via a flexible cable and a 8 pole connector to the mainframe see also chapter 3 3 Caution Please always adhere to the appropriate safety regulations see chapter 1 Please turn power off when connecting or disconnecting the headstage from the HEADSTAGE connector BRIDGE BALANCE unit The BRIDGE BALANCE unit consists of 20 100 10 RANGE switch and 21 BRIDGE BALANCE potentiometer 20 100 MO 10 MO RANGE switch Switch to set the RANGE of the BRIDGE BALANCE potentiometer 100 MQ position RANGE 0 MQ to 1000 MQ 10 MQ position RANGE 0 MQ to 100 MQ 21 BRIDGE BALANCE M potentiometer If current is passed through the recording electrode the potential deflection caused at the electrode resistance is compensated with this control ten turn potentiometer clockwise calibrated in RANGE set by switch 20 see also chapter 7 4 version 2 0 page 12 BA 03X User Manual The CURRENT INPUT unit consists of 22 25 ON OFF switches 23 STIMULUS INPUT 1 nA V connector and 24 STIMULUS INPUT 0 1 nA V connector 22 ON OFF switch Switch to enable ON or disable OFF STIMULUS INPUT via 1 nA V connector To avoid interferences always switch to OFF position if the INPUT 1s not used 23 STIMULUS INPUT 1 nA V connector Analog input BNC connector for applying signals from an external stimulus source The voltage signal that is connected here is transformed to a proport
11. cerebellum evidence that intrapontine communication is mediated by a reciprocal loop with the cerebellar nuclei J Neurophysiol 95 3414 3425 Neher E 1974 Elektrische Me technik in der Physiologie Springer Verlag Berlin L Nimmrich V Maier N Schmitz D amp Draguhn A 2005 Induced sharp wave ripple complexes in the absence of synaptic inhibition in mouse hippocampal slices Journal of Physiology 563 663 670 version 2 0 page 35 BA 03X User Manual 1 Ogden DC 1994 Microelectrode Techniques The Plymouth Workshop Handbook Second Edition The Company of Biologists Limited Cambridge J Prinz A A and P Fromherz 2000 Electrical synapses by guided growth of cultured neurons from the snail Lymnaea stagnalis Biol Cybern 82 LI L5 _J Prinz A A amp Fromherz P 2003 Effect of neuritic cables on conductance estimates for remote electrical synapses J Neurophysiol 89 2215 2224 1 Purves 1981 Microelectrode Methods for Intracellular Recording and Ionophoresis London Academic Press Rokem A Watzl S Gollisch T Stemmler M Herz A V amp Samengo I 2006 Spike timing precision underlies the coding efficiency of auditory receptor neurons J Neurophysiol 95 2541 2552 I Schaette R Gollisch T amp Herz V M 2005 Spike Train Variability of Auditory Neurons in vivo Dynamic Responses Follow Predictions from Constant Stimuli Journal of Neurophysiology 93 32
12. described in chapter 7 1 Problem 3 The system oscillates Possible reason 1 The capacitance of the electrode is overcompensated Solution 1 Turn the CAPACITY COMP potentiometer 17 Figure 1 to the most left position and compensate the input capacitance again Problem 4 With the cell model connected the display does not show the correct value within a tolerance of 2 Possible reasons 1 The capacitance of the electrode is not compensated using the 100 MQ electrode 2 The headstage has an error Solution 1 Turn the CAPACITY COMP potentiometer 17 Figure 1 to the most left position and compensate the input capacitance again 2 Contact version 2 0 page 34 BA 03X User Manual Problem 5 The amplifier does not provide any current Possible reason 1 The OSCILLATION SHUT OFF circuit is on LED 30 Figure 1 1 red Solution 1 Turn the CAPACITY COMP potentiometer 17 Figure 1 to the most left position and compensate the input capacitance again 2 RESET the OSCILLATION SHUT OFF circuit using switch 32 Figure 1 LED 30 Figure must become green 3 Compensate the input capacitance again 10 Literature Bridge amplifier recording I Behrend O Branoner F Zhivkov Z amp U 2006 Neural responses to water surface waves in the midbrain of the aquatic predator Xenopus laevis laevis Eur J Neurosci 23 729 744 Burrell B D amp Sahley L 2004 M
13. is recommended first to connect a cell model to the amplifier in order to perform some basic adjustments and to get familiar with these procedures It is assumed that all connections are built as described in chapter 5 Important Except for Headstage Bias Current adjustment see 7 1 all adjustments described below should be carried out every time before starting an experiment or after changing the electrode 7 1 Bias Current Adjustment Caution It is very important that this tuning procedure is performed ONLY after a warm up period of at least 30 minutes since the bias current changes significantly during warm up The BA 03X system 1 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 control 12 Figure 1 chapter 3 2 The tuning procedure must be performed regularly about once a month since the bias current changes over time If very small currents are used in the 10 pA range the procedure must be repeated in shorter intervals The tuning procedure 1 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 etc L Disconnected all input signals except the headstage Put the holding current switch to position 0 0 switch 11 Figure 1 L
14. potential is the sum of various effects at the tip of the electrode filled with electrolyte tip potential junction potential etc This offset voltage must be compensated i e set to zero carefully with the OFFSET control 16 Figure 1 before recording from a cell When adjusting the OFFSET make sure that no current flows through the electrode Thus it is recommended to disconnect CURRENT INPUT and to disable GATE TTL STEP SIZE and the HOLDING CURRENT unit see chapter 3 2 If a cell model 15 connected the OFFSET control should read a value around 5 otherwise it 1 likely that the headstage or the amplifier is damaged 7 3 Capacity Compensation High resistances of electrodes and stray capacitances Cstray form a low pass filter that deteriorates the shape of recorded intracellular signals see also Figure 11 The frequency response of the amplifier is improved considerably by using the capacitance compensation function This function is based on positive feedback negative capacitance circuit The tuning of the capacitance compensation control is performed using pulses applied to the CURRENT INPUT or pulses provided by the electrode resistance test circuit With the cell model connected or the electrode in the bath the CAPACITY COMP control is turned clockwise until there is no capacitance artifact on the POTENTIAL OUTPUT see Figure 9 I Make the basic settings at the amplifier see chapter 5 Connect a cell mode
15. 12 1622 1627 version 2 0 page 37 BA 03X User Manual 11 Technical Data Headstage Input voltage range 1000 mV Operating voltage 15 V Enclosure Size 23 x 70 x 26 mm grounded Holding bar Size length 150 mm 8 mm Electrode connector BNC with driven shield Ground connector 2 4 mm connector REF connector optional SMB connector Input resistance 10P Q internally adjustable Current range 1 12 nA into 1 GO Current range x10 120 nA into 100 MQ Electrode parameter controls BIAS range 200 pA current adjustable with trim potentiometer OFFSET range 200 mV ten turn control CAPACITY COMPENSATION range 0 30 pF ten turn control Cell penetration overcompensation of capacitance compensation BUZZ duration 2 5 100 ms dependent on the setting of capacitance compensation timer controlled with linear control accessible with remote switch Electrode clear circuit Application of max DC currents Imax or Imax Bridge balance Adjustable with ten turn control and RANGE switch 0 100 MQ or 0 1000 MQ Electrode resistance test mV MQ obtained by application of square current pulses 1 nA display XXXX MQ Bandwidth and speed response Full power bandwidth Ret 0 gt 30 kHz rise time 1096 90 10 us Ret 100 MQ 5 us Ret 5 MQ version 2 0 page 38 BA 03X User Manual Outputs Resistance 50 9 Current BNC connector sensitivity selectable by rotary switch
16. 30 minutes 1 sufficient HANDLING Please protect the device from moisture heat radiation and corrosive chemicals version 2 0 page 4 BA 03X User Manual 2 BA 03X Components The following items are shipped with the BA 03X system BA 03X amplifier Headstage Ground connector for headstage 2 4 mm Power cord User manual SS NS SN Optional accessories L Sharp electrode holder Suction patch electrode holder gt Electrode holder adapter with BNC connectors L electrode holder Remote switch for penetration unit Active cell model Passive cell model see Figure 7 Low noise low bias current headstage with a reduced current range 10 headstage 1 e maximal current 1s 1 2 nA Headstage with differential input 3 BA 03X System This manual is related to the standard configuration of the BA 03X system consisting a standard headstage and standard calibrations of bridge balance electrode resistance display range etc Other configurations are available e g if the BA 03X system is used only for whole cell patch clamp recordings with suction electrodes the BA 03X system can be delivered with adapted calibrations and a low noise low bias current headstage see Optional accessories in chapter 2 For details contact 3 1 System Description The npi BA 03X recording systems are precise current clamp instruments with a bridge circuit to compensate for the resistance of the recording intracellul
17. 4 A MEE 4 ad 3 T T T T T T T 1 0 50 100 150 200 250 300 350 400 time ms potential Figure 10 Tuning of the BRIDGE BALANCE using 100 MQ resistor version 2 0 page 27 BA 03X User Manual 8 Intracellular Sample Experiments In the following the basics of a simple intracellular experiment are described either using a sharp or a suction electrode It is assumed that all connections are built as described in chapter 5 Before starting remove the cell model 8 1 Sample Experiment using a Sharp Electrode Rer to amplifier electrode DA cell qe ee C stray amp 7 A ground qol en jm ground Figure 11 Model circuit for intracellular recording using a sharp electrode Cm membrane capacitance Cstray electrode stray capacitance Rex electrode resistance Rm membrane resistance I Connect the electrode cable holder to the BNC connector and the Ag AgCl pellet or the agar bridge for grounding the bath with GND at the headstage Make the basic settings see chapter 5 Again It is of major importance that the BA 03X systems are used only in warmed up condition 1 e 20 to 30 minutes after turning power on Adjust BIAS CURRENT to zero if necessary see chapter 7 1 Reconnect the STIMULUS INPUT and or the GATE TTL STEP SIZE and put an electrode into the electrode holder L Immerse the electrode into the bath not in a cell as deep as necessary during t
18. 70 3281 1 Schoen I amp Fromherz P 2007 The Mechanism of Extracellular Stimulation of Nerve Cells on an Electrolyte Oxide Semiconductor Capacitor Biophys J 92 1096 1111 L Vogel A Hennig M amp Ronacher 2005 Increase of neuronal response variability at higher processing levels as revealed by simultaneous recordings Journal of Neurophysiology 93 3548 3559 1 Zeck and P Fromherz 2001 Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip PNAS Vol 98 no 18 10457 10462 Juxtasomal Filling Loose Patch Techniques General Auger C amp Marty A 2000 Topical Review Quantal currents at singlesite central synapses J Physiol 526 1 3 11 Barbour B amp Isope 2000 Combining loose cell attached stimulation and recording J Neurosci Methods 103 199 208 I Bureau I Shepherd G M G amp Svoboda 2004 Precise Development of Functional and Anatomical Columns in the Neocortex Neuron 42 789 801 1 Joshi S amp Hawken M J 2006 Loose patch juxtacellular recording in vivo A method for functional characterization and labeling of neurons in macaque Vl J Neurosci Methods 156 37 49 1 Khaliq Z M amp Raman I 2005 Axonal Propagation of Simple and Complex Spikes in Cerebellar Purkinje Neurons J Neurosci 25 454 463 I Klausberger T Marton L F Baude A Roberts J D Ma
19. ION 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 system to mains Always use a three wire line cord and a mains power plug with a protection contact connected to ground protective earth Before opening the cabinet unplug the instrument Unplug the instrument when replacing the fuse or changing line voltage Replace fuse only with an appropriate specified type STATIC ELECTRICITY Electronic equipment is sensitive to static discharges Some devices such as sensor inputs are equipped with very sensitive FET amplifiers which can be damaged by electrostatic charge and must therefore be handled with care Electrostatic discharge can be avoided by touching a grounded metal surface when changing or adjusting sensors Always turn power off when adding or removing modules connecting or disconnecting sensors headstages or other components from the instrument or 19 cabinet TEMPERATURE DRIFT WARM UP TIME All analog electronic systems are sensitive to temperature changes Therefore all electronic instruments containing analog circuits should be used only in a warmed up condition 1 after internal temperature has reached a steady state value In most cases a warm up period of 20
20. UT FILTER LOWPASS Hz switch 16 position switch for setting the corner frequency of the LOWPASS 4 pole BESSEL FILTER for the POTENTIAL signal 3 POTENTIAL RESISTANCE display LC Display for the recorded potential in mV XXXX mV or the electrode resistance in MQ XXXX i e 100 correspond to 100 MQ version 2 0 page 8 BA 03X User Manual 4 mV LED 5 MQ LED ie LEDs indicating the unit of the reading of the DISPLAY 3 The resistance is measured accurately regardless of the correct setting of other front panel elements such as OFFSET BRIDGE BALANCE etc Caution When the ELECTRODE RESISTANCE button is pressed the BA 03X automatically applies current pulses of 1 nA to the electrode Therefore it should not be used during recordings from cells since this current may stimulate or damage the cell Note With high resistance electrodes Ret gt 20 the displayed value is dependent on the accurate setting of the capacity compensation 17 CURRENT OUTPUT unit The CURRENT OUTPUT unit consists of 6 CURRENT OUTPUT SENSITIVITY V nA switch and 29 CURRENT OUTPUT connector 6 CURRENT OUTPUT SENSITIVITY V nA switch 7 position switch for setting the amplification factor of the recorded current 0 1 V nA to 10 V nA 29 CURRENT OUTPUT connector BNC connector monitoring the stimulating current passed through the electrode sensitivity set by switch 6 Note The maximum voltage an this con
21. UT via 0 1 nA V connector Hint To avoid interferences always switch to OFF position if the INPUT is not used gated stimulus unit The gated stimulus unit consists of 26 0 switch STEP SIZE switch 27 GATE TTL connector and 28 STEP SIZE digital potentiometer 26 switch STEP SIZE switch 0 toggle switch to disable the gated stimulus set by STEP SIZE 28 and gated by GATE TTL 27 or to select the polarity of the gated stimulus gated stimulus positive 0 gated stimulus disabled gated stimulus negative 27 GATE TTL connector With this input a current step gated stimulus can be generated set by the digital potentiometer STEP SIZE 28 and the polarity switch 26 This current step is gated by a positive TTL pulse 3 5 V applied to the BNC connector Two examples are given in Figure 4 The duration of the current step is set by the duration of the gating signal The amplitude of the current step is set by STEP SIZE 28 28 STEP SIZE digital potentiometer Control to set the amplitude of the gated stimulus digital potentiometer X XX nA range 9 99 nA resolution 10 pA if switch 9 is set to x1 or XX X nA range 99 9 nA resolution 100 pA if switch 9 is set to x10 input voltage signal gt 3 V output current signal J U L 4 U L nana UUU Figure 4 input output relation using gated stimuli version 2 0 page 14 BA 03X User Manual OSCILLATION SHUT OFF u
22. al 1 pA 5 on the Py dial range 200 pA CURRENT RANGE x1 see chapter 7 1 version 2 0 page 10 BA 03X User Manual PENETRATION unit The PENETRATION unit consists of 13 ELECTR CLEAR switch 14 BUZZ push button 15 DURATION potentiometer and 19 BUZZ REMOTE connector 13 ELECTR CLEAR switch The ELECTR CLEAR switch is used to activate the ELECTRODE CLEAR circuit that can be used to clean the tip of the electrode by passing large amounts of positive or negative current The circuit is operated by pushing this switch to Imax maximum positive current or Imax maximum negative current The maximum current is dependent on the setting of the current range see also 9 14 BUZZ push button Push button for activating the BUZZ or ELECTR CLEAR circuit duration set by 15 To facilitate the penetration of the cell membrane the BUZZ circuit is provided which is based on oscillations caused by overcompensating the capacity compensation system The overcompensation of capacity compensation yields to very powerful high frequency oscillations see Figure 2 15 DURATION potentiometer Control to set the duration of the BUZZ potentiometer clockwise range 1 ms to 100 ms The duration is dependent on the setting of CAPACITY COMP 17 It is effective in REMOTE control and when pushing the BUZZ button 14 19 BUZZ REMOTE connector BNC connector to attach a remote switch to the PENETRATION unit
23. ar electrode For current injection and potential recording a very high impedance voltage to current converter with a special input capacitance compensation circuit is used The BA 03X is suited for extracellular recording as well because of the built in potential gain up to 1000 and potential high and lowpass filters For methodical reviews see Lalley et al 1999 Ogden 1994 and Boulton et al 1990 version 2 0 page 5 BA 03X User Manual The system consists of standard desktop cabinet and a headstage which should be placed close to the recording site The recording electrode is connected to the headstage via an electrode holder see also Figure 6 In some setups there is restricted space for placing the headstage very close to the recording site In that case the electrode holder can be connected to the headstage via an electrode adapter see Optional accessories in chapter 2 All electrode connectors use a driven shield approach for details of this approach see Ogden 1994 to minimize the effect of the connecting cables In addition all headstages are equipped with a ground connector providing system ground The standard system is equipped with a headstage capable of injecting a maximal current of approximately 12 nA 120 nA into a resistance of 100 10 dependent on the preset current range With this headstage the system can be used either with high resistance electrodes for intracellular recordings or with low resis
24. at Ret affects the recording of membrane potential Therefore this deflection must be compensated carefully by means of the BRIDGE BALANCE control This control is calibrated in MQ and has two ranges selected by a RANGE switch 20 Figure 1 With the cell model connected or the electrode in the bath the BRIDGE BALANCE control is turned on clockwise until there is no artifact on the POTENTIAL OUTPUT see Figure 10 I Make the basic settings at the amplifier see chapter 5 I Connect a cell model or immerse the electrode into the bath as deep as necessary during the experiment _J Tune the OFFSET to zero see chapter 7 2 and compensate the input capacitance see chapter 7 3 This is very important since a badly compensated input capacitance prevents setting the BRIDGE BALANCE to correct values L Determine the electrode resistance using the ELECTRODE RESISTANCE switch and set the BRIDGE BALANCE RANGE switch 20 Figure 1 accordingly L Apply current pulses to the electrode either using an external stimulator via the CURRENT INPUT connector 23 24 Figure 1 or by using the gated stimulus unit L Watch the POTENTIAL OUTPUT at the oscilloscope and adjust the BRIDGE BALANCE as shown in Figure 10 using the BRIDGE BALANCE potentiometer 21 Figure 1 After adjustment you should see a straight voltage trace without artifacts caused by the potential drop at Rex Figure 10 illustrates the BRIDGE BALANCE procedure using a 100 MQ resi
25. be incorrect if the oscillation shut off circuit is activated 38 GROUND connector 39 AUDIO monitor potentiometer Banana jack that 1 linked to the internal system ground which has no connection to the 19 cabinet and the mains ground to avoid ground loops Potentiometer for setting the volume of the internal speaker The AUDIO function of the BA 03X converts the potential output into a tone with a frequency equivalent to the amplitude of the potential Note Very small signals of only one or a few mV are probably not well monitored by the AUDIO monitor especially in extracellular recordings This function is intended to monitor version 2 0 page 15 BA 03X User Manual acoustically the transition of extracellular to intracellular when penetrating a cell which is accompanied by a large voltage drop of several tens of mV 3 3 Description of the Rear Panel X 20000 69000 Figure 5 BA 03X rear panel view the numbers are related to those in the text below 1 Mains connector Plug for connecting the BA 03X to mains Caution Always switch to the appropriate voltage before connecting the BA 03X to power 2 Voltage SELECTOR Switch for selecting the mains voltage 110 V 120 V 220 V 240 V Caution Always switch to the appropriate voltage before connecting the BA 03X to power 3 FUSE holder Holder for the line fuse For changing the fuse rotate the holder counter clockwise using a screw drive
26. cord e g noise For differential measurements both inputs of the headstage REF and are connected to microelectrodes using cables with 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 16 If differential measurement is not required single ended measurement configuration see Figure 16 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 0 page 32 BA 03X User Manual Experimental chamber Ag AgCl pellet REF electrode Electrode holder BNC jack EL npi electronic GND REF Ground SUBCLICK jack connector REF Cable to amplifier differential measurement configuration Experimental chamber Slice Ag AgCl pellet Electrode holder BNC jack Q C REF Ground SUBCLICK jack connector REF Cable to amplifier single ended measurement configuration Figure 16 headstage connections A differential measurement B single ended measurement Differential measurements can also be done in intracellular recordings The electrode connected to is i
27. gill P J amp Somogyi 2004 Spike timing of dendrite targeting bistratified cells during hippocampal network oscillations in vivo Nature Neuroscience 7 41 47 I Nunemaker S DeFazio A amp Moenter S 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 version 2 0 page 36 BA 03X User Manual I Rathenberg J Nevian T amp Witzemann 2003 High efficiency transfection of individual neurons using modified electrophysiology techniques J Neurosci Methods 126 91 98 L 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 LJ Strickholm 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 1 Bruno R M amp Sakmann B 2006 Cortex is driven by weak but synchronously active thalamocortical synapses Science 3
28. he experiment Test the capability of the electrode to carry current see chapter 7 4 compensate the potential offset see chapter 7 2 measure the electrode resistance see 33 chapter 3 2 and compensate the input capacitance see chapter 7 3 and Figure 9 version 2 0 page 28 BA 03X User Manual L Enable the OSCILLATION SHUT OFF unit 32 Figure 1 and set the THRESHOLD 31 Figure 1 in a way that the OSCILLATION SHUT OFF unit activates if the system begins to oscillate Test this by overcompensating the electrode capacitance in several positions of the THRESHOLD potentiometer L Now the system is preadjusted for measurements Find a cell Approach the desired cell There are several indications that the electrode is very close to the cell membrane the electrode resistance increases the bridge balance appears undercompensated extracellular action potentials APs are recorded the acoustic monitor signal changes L Set the BUZZ DURATION potentiometer to one fourth and apply a BUZZ to the electrode If you are lucky the tip of the electrode is now inside the cell If necessary readjust the BRIDGE BALANCE as shown in Figure 12 L You read the membrane potential and can apply current pulses to the cell After penetration the voltage responses of the cell to the test pulses should reflect the cell membrane resistance and time constant Start the experiment overcompensated compensated undercompensa
29. iding a voltage monitoring the position of the CURRENT OUTPUT SENSITIVITY switch 1 V to 7 IV STEP 4 Headstage The BA 03X comes with the standard headstage range 12 nA voltage range xl or 120 nA voltage range x10 for connecting glass electrodes with high resistances or suction electrodes for whole cell patch clamp recordings with lower resistances via an electrode holder see Figure 6 low noise low bias current headstage range 1 2 nA voltage range 1 or 12 nA voltage range x10 see also Optional accessories in chapter 2 for very small currents is also available For details contact npi Figure 6 electrode holder optional and headstage of the BA 03X 4 1 M Headstage Elements 1 BNC connector for the optional electrode holder 2 REF connector for the reference electrode differential headstage only 3 GND Ground connector version 2 0 page 17 BA 03X User Manual 4 headstage cable holding bar The electrode filled with electrolyte is inserted into an electrode holder optional see Figure 6 that fits into the BNC connector of the headstage or into an electrode holder adapter optional see also Optional accessories in chapter 2 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 p
30. ional current at the electrode with a sensitivity of 1 nA V 1 an input voltage of 5 V is transformed to an output current of 5 nA if switch 9 is set to x1 The signal form remains unchanged Two examples are given in Figure 3 The amplitude of the output current signal CURRENT STIMULUS is determined by the amplitude of the input voltage signal input voltage signal output current signal LN J UOU Loe Jo LJ L Figure 3 input output relation using STIMULUS INPUT 24 STIMULUS INPUT 0 1 nA V connector Analog input BNC connector for applying signals from an external stimulus source The voltage signal that is connected here is transformed to a proportional current at the electrode with a sensitivity of 0 1 nA V i e an input voltage of 5 V is transformed to an output current of 0 5 nA if switch 9 is set to x1 The signal form remains unchanged see also 23 and Figure 3 Very Important If switch 9 is set to x10 all current input signals are multiplied by the factor of 10 1 e STIMULUS INPUT 1 nA V is then 10 nA V and 0 1 nA V is 1 nA V respectively Important The current injected through the electrode is always the sum of the input signals at STIMULUS INPUT 23 or 24 the holding current set by HOLDING CURRENT 10 and switch 11 and the gated stimulus set by STEP SIZE 28 and switch 26 version 2 0 page 13 BA 03X User Manual 25 ON OFF switch Switch to enable ON or disable OFF STIMULUS INP
31. ip or by using the BUZZ or ELECTRODE CLEAR facilities of the amplifier 7 6 Extracellular Recording Stimulation and Juxtacellular Filling The BA 03X can also be used for extracellular recordings with glass or metal electrodes 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 The recorded signal is amplified with the factor set by switch 2 Figure 1 and filtered using the LOWPASS filter and or HIGHPASS filter 34 Figure 1 Since the BA 03X is capable of current injection the amplifier can be used for extracellular stimulation or electroporation as well Normally a current of few nA are used for extracellular stimulation Electroporation can be done using the extracellular stimulation procedure but usually the applied current is much higher and the stimulus duration is shorter Therefore most electroporation experiments are done in CURRENT RANGE x10 mode 9 Figure 1 This enables the BA 03X to perform juxtacellular filling of individual cells with dyes or nucleic acids version 2 0 page 26 BA 03X User Manual undercom pensated potential mV 6 0 50 100 150 200 250 300 350 400 time ms overcom pensated potential mV 6 time ms compensated potential mV 3 a 4 1
32. l or immerse the electrode into the bath as deep as necessary during the experiment L Tune the OFFSET to zero see chapter 7 2 L Press the ELECTRODE RESISTANCE button 33 Figure 1 or apply rectangular pulses to the CURRENT STIMULUS INPUT and watch the POTENTIAL OUTPUT Compensate the input capacitance as shown in Figure 9 using the CAPACITY COMP potentiometer 17 Figure 1 Figure 9 illustrates the capacitance compensation procedure using a 100 MQ resistor that represents the electrode The pulses were generated using the automated electrode resistance test circuit of the 03 The upper diagram shows an undercompensated capacitance In the diagram in the middle the capacitance is slightly overcompensated and in the lower diagram it 1s well compensated version 2 0 page 23 BA 03X User Manual undercompensated potential mV 250 200 150 100 50 0 10 20 30 40 50 time ms overcompensated potential mV 600 400 200 o 200 4 400 600 10 20 30 40 o0 time ms compensated potential mV 290 4 200 150 100 90 0 im zx Lp m 0 10 20 30 40 50 time ms potential Figure 9 Tuning of the capacitance compensation using a 100 MQ resistor version 2 0 page 24 BA 03X User Manual 7 4 Bridge Balance If current is passed through an electrode the occurring voltage deflection potential drop
33. lance and using the capacity compensation Simulation of SEAL formation L Set switch 4 Figure 7 to the upper position L Set switch 5 Figure 7 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 7 for an experiment with sharp electrodes or the 10 electrode connector 3 Figure 7 for simulating an experiment with patch electrodes I Switch the CELL membrane switch see 4 Figure 7 to the desired position CELL 1 or CELL 2 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 _J 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 0 page 21 BA 03X User Manual 7 Test and Tuning Procedures Important The BA 03X should be used only in warmed up condition i e 30 minutes after turning power on The following test and tuning procedures are necessary for optimal recordings It
34. nector is 12 V This has to be considered if large currents are applied 7 CURRENT nA display IZ LC display showing the stimulating current passed through the electrode version 2 0 page 9 BA 03X User Manual CURRENT RANGE unit Sms The CURRENT OUTPUT unit consists of 8 CURRENT RANGE LED and 9 CURRENT RANGE switch x 10 x 8 CURRENT RANGE LED The CURRENT RANGE LED indicates the setting x10 120 nA of switch 9 LED ON 9 CURRENT RANGE switch Switch for setting the CURRENT RANGE of the amplifier x1 12 nA into 1 GQ x10 max 120 nA into 100 MQ HOLDING CURRENT unit The HOLDING CURRENT unit consists of 10 HOLDING CURRENT nA potentiometer and 11 0 switch holding current switch 10 HOLDING CURRENT nA potentiometer With this control a constant current holding current can be generated ten turn potentiometer clockwise calibrated in nA Imax 10 nA or 100 nA dependent on setting of CURRENT RANGE switch 9 The polarity of the holding current is set by toggle switch 11 11 0 switch holding current switch Switch to disable holding current generation or to set the polarity of the holding current current positive 0 holding current disabled current negative 12 BIAS bias current potentiometer 10 turn potentiometer for setting the output current of the headstage headstage fr BIAS current to zero ten turn potentiometer symmetric
35. nit e The OSCILLATION SHUT OFF unit consists of 30 OSCILLATION SHUT OFF LED 31 THRESHOLD potentiometer and 32 DISABLED RESET switch 30 OSCILLATION SHUT OFF LED Indicates whether the OSCILLATION SHUT OFF circuit 1 active LED red or not EU LED green OBAM 3 3 pm 31 THRESHOLD potentiometer Control to set the activation THRESHOLD of the OSCILLATION SHUT OFF circuit potentiometer linear clockwise range 0 1200 mV 32 DISABLED RESET switch Switch to DISABLE the OSCILLATION SHUT OFF unit or RESET the circuit RESET is done if one wants to reset the circuit after previous activation After resetting the OSCILLATION SHUT OFF unit is active again Note If the OSCILLATION SHUT OFF unit is active the output of the amplifier to the headstage is disabled Potential measurement works Important The capacity compensation for the electrode does NOT work if the oscillation shut off circuit 1s activated This may lead to an incorrect reading of the electrode resistance see also 33 especially when electrodes with high resistances are used 33 ELECTRODE RESISTANCE TEST push button Push button to start the measurement of the electrode resistance When this button 1s pressed the DISPLAY 3 shows the electrode resistance in MQ see also 3 Important The reading of the electrode resistance is correct only if the electrode capacitance is accurately compensated Therefore it might
36. nserted into cell and the reference electrode connected to REF is positioned outside the cell version 2 0 page 33 BA 03X User Manual 9 Trouble Shooting In the following section some common problems possible reasons and their solutions are described Important Please note that the suggestions for solving the problems are only hints and may not work In a complex setup it is impossible to analyze problems without knowing details In case of trouble always contact an experienced electrophysiologist in your laboratory if possible and connect a cell model to see whether the problem occurring with electrodes and real cells persists Problem 1 After immersing the electrode into the bath there is an unusual high potential offset Possible reasons 1 The Ag AgCl coating of the silver wire in the electrode holder is damaged 2 The Ag AgCl pellet or Ag AgCl coating of the silver wire in the agar bridge are damaged 3 There is an unwanted GND bridge e g caused by a leaky bath 4 The headstage or the amplifier has an error Solutions 1 Chloride the silver wire again 2 Exchange the pellet or chloride the silver wire in the agar bridge 3 Try to find the GND bridge and disconnect it e g by sealing the bath 4 Contact npi Problem 2 Even if no stimulus is given a current flows through the electrode Possible reason 1 The BIAS current is not adjusted Solution 1 Adjust the BIAS current according the procedure
37. nual all elements of the front panel are written 1n capital letters as they appear on the front panel System components that are shipped in the standard configuration are marked with V optional components with In some chapters the user is guided step by step through certain procedure These steps are marked with Important information and special precautions are highlighted in gray Abbreviations Cm cell membrane capacitance Cstray electrode stray capacitance GND ground Imax maximal current Rm cell membrane resistance Ret electrode resistance Tcm time constant of the cell membrane potential drop at REL version 2 0 page 3 BA 03X User Manual 1 Safety Regulations VERY IMPORTANT Instruments and components supplied by npi electronic are NOT intended for clinical use or medical purposes e g for diagnosis or treatment of humans or for any other life supporting system npi electronic disclaims any warranties for such purpose Equipment supplied by npi electronic must be operated only by selected trained and adequately instructed personnel For details please consult the GENERAL TERMS OF DELIVERY AND CONDITIONS OF BUSINESS of npi electronic D 71732 Tamm Germany 1 2 3 4 5 GENERAL This system is designed for use in scientific laboratories and must be operated only by trained staff General safety regulations for operating electrical devices should be followed AC MAINS CONNECT
38. otentials 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 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 4 Figure 6 and 8 connector The headstage is mounted to a holding bar that fits to most micromanipulators or optionally to a mounting plate or a dovetail adapter Note The shield of the BNC connector is linked to the driven shield output and must not be connected to ground 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 5 Setting up the BA 03X The following steps should help you set up the BA 03X correctly Always adhere to the appropriate safety measures see chapter 1 After unpacking the BA 03X is attached to the setup by assembling the electrical connections It is assumed that a cell model will be attached The connection of the Ag AgCl pellet or the agar bridge for grounding the bath is described in chapter 8 D Electrical connections L Turn POWER off LJ Plug the power cord of the instrument into a grounded outlet I Connect the headstage to the HEADSTAGE connector
39. position are connected to a 1 G resistor simulating the formation of a GIGASEAL with a patch electrode Whole cell Patch sharp Microelectrode Driven Shield BNC connector BNC connector 10 MQ Re 50 MQ CELL CELL 1 GROUND 2 SEAL m Cm Cm Rm 1 1 200 100 pF 22pF 2 50MOQ GND Figure 8 Schematic diagram of the passive cell model 6 2 Connections and Operation It is assumed that all connections are built as described in chapter 5 Checking the configuration _J Turn POWER switch of the amplifier off a For simulation of an experiment using a suction electrode version 2 0 page 20 BA 03X User Manual L Connect the BNC jack labeled 10 of the cell model to the BNC connector Pet of the headstage b For simulation of an experiment using a sharp electrode L Connect the BNC jack labeled 50MQ of the cell model to the BNC connector Pet at the headstage For headstages with SMB connector use the supplied SMB to BNC adapter For a and b L 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 1n the bath L Set switch 4 Figure 7 to the upper position L Set switch 5 Figure 7 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 ba
40. r The appropriate fuse type is listed above the holder 4 CHASSIS This connector is linked to mains ground green yellow wire protective earth 5 GROUND This connector is linked to the internal system ground which has no connection to the 19 cabinet CHASSIS and the mains ground to avoid ground loops MODES of OPERATION connectors 6 Ret connector BNC connector for connecting a TTL signal that activates the electrode resistance test TTL HI Rz on TTL LOW Rez off 7 x10 MODE connector BNC connector for connecting a TTL signal that activates the x10 current mode see also 9 Figure 1 TTL HI x10 current mode TTL LOW x1 current mode version 2 0 page 16 BA 03X User Manual REMOTE connector 8 BUZZ connector BNC connector for connecting a TTL signal that activates the BUZZ function see also 14 Figure 1 TTL HI BUZZ on TTL LOW BUZZ off MONITOR OUTPUT connectors 9 POTENTIAL SENSITIVITY connector BNC connector providing a voltage monitoring the position of the POTENTIAL OUTPUT GAIN switch 1 V to 7 V 1V STEP 10 HP FILTER POTENTIAL connector BNC connector providing a voltage monitoring the position of the POTENTIAL HIGHPASS FILTER switch 8 V to 7 V 1V STEP 11 LP FILTER POTENTIAL connector BNC connector providing a voltage monitoring the position of the POTENTIAL LOWPASS FILTER switch 8 V to 7 V IV STEP 12 CURRENT SENSITIVITY connector BNC connector prov
41. stor that represents the electrode The current stimuli were generated using the gated stimulus unit gated by two TTL pulses The amplitude was set to 0 5 nA In the upper diagram the bridge is slightly undercompensated and in the diagram in the middle it is slightly overcompensated The lower diagram shows a well balanced bridge compensated Important BRIDGE BALANCE and CAPACITY COMP must be tuned several times during an experiment since most parameters change during a recording session Figure 13 shows artifacts caused by uncompensated stray capacitance and bridge during recording from a cell It also shows how to cancel these artifacts by tuning with CAPACITY COMP and BRIDGE BALANCE OFFSET deviations can be detected by comparing the readout on the potential display before and after an experiment with the electrode in the tissue or bath but not in a cell version 2 0 page 25 BA 03X User Manual 7 5 Electrode Selection Electrodes must be tested before use This is done by applying positive and negative current pulses and by compensating with the BRIDGE BALANCE control Electrodes which show significant changes in resistance rectification cannot be used for intracellular recordings By increasing the current amplitude the capability of the electrode to carry current can be estimated The test current must cover the full range of currents used in the experiment Sometimes the performance of electrodes can be improved by breaking the t
42. tance suction electrodes for whole cell patch clamp recordings The 03 is also suitable for extracellular measurements and stimulation with glass metal electrodes An extended CURRENT RANGE x10 allows electroporation of single cells for non invasive juxtacellular filling of cells with dyes or plasmids as well To cover all the needs of electrophysiological research all systems have a large variety of operation and control elements such as cell penetration mode BUZZ ELECTRODE CLEAR facility ten turn controls for BRIDGE BALANCE CAPACITANCE COMPENSATION OFFSET and HOLDING CURRENT an automated electrode resistance test digital displays for potential current and electrode resistance a GATE and linear CURRENT STIMULUS INPUT 3 2 Description of the Front Panel In the following description of the front panel elements each element has a number 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 Some elements are grouped in functional units e g BUZZ unit and are described as units regardless of the order of numbers version 2 0 page 6 BA 03X User Manual BUZZ PENETRATION ELECTR CLEAR CURRENT n CURRENT CURRENT ind URRENI SENSITIVITY Viri m 2 Er a x i A
43. ted 5 mv 20 ms odo ooo LL m 1 20 ms Figure 12 Adjustment of the bridge balance after penetrating a cell version 2 0 page 29 BA 03X User Manual A uncompensated potential mV current nA 100 r 3 0 80 d 2 5 60 REL a 20 40 20 ee VREL 0 20 40 T T T 0 0 0 20 40 60 80 100 120 140 160 B time ms Cstray compensated potential mV current nA 80 7 3 0 70 5 2 5 60 50 2 0 40 30 T T T F 0 0 0 20 40 60 80 100 120 140 160 time ms Cstray and Vre compensated potential mV current nA 60 7 3 0 we tom 50 125 40 4 1 20 ap Tom 15 20 4 110 10 4 0 5 0 0 0 20 40 60 80 100 120 140 160 time ms potential current Figure 13 Artifacts caused by the recording electrode The measurements were done using a cell model with 100 membrane resistance 100 pF membrane capacitance and 100 MQ electrode resistance A Cstray and VreL not compensated bridge not balanced B Cstray compensated and Vret not compensated Cstray and compensated bridge balanced Cm membrane capacitance Cstray electrode stray capacitance Rex electrode resistance Rm membrane resistance Tcm time constant of the cell membrane Vnzr potential drop at Rex see also Figure 11 version 2 0 page 30 BA 03X User Manual 8 2 Sample Experiment using a Suction Patch Electrode If s
44. the setup does not work correctly e g to find out whether the amplifier or headstage 1s 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 membrane resistance and 100 pF membrane capacitance CELL 1 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 ELC MOD GROUND Re 1kQ d o 1 GROUND SND 2 npi electronic GmbH B B D 71732 Tamm Gemnany www npielectronic com Figure 7 passive cell model version 2 0 page 19 BA 03X User Manual 1 3 connectors for the headstage 1 electrode resistance 50 MQ 3 electrode resistance 10 MQ 2 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 2 In GROUND upper position the electrodes are connected to ground via a 1 kQ resistor In SEAL lower
45. uction patch electrodes are used for whole cell recordings they are usually called pipettes Thus in this subchapter pipette means suction electrode to amplifier e n a oe Pd fer ff M uy ground Jy F 1 1 L 1 1 a ff AN ff hy o NS M p gt 7 b b p g emt ins Lu D m 4 bh Paul R ground Figure 14 Model circuit for whole cell patch clamp recording using a suction electrode Cm membrane capacitance Cstray electrode stray capacitance Rex electrode resistance Rm membrane resistance Prepare the setup and proceed as described in the previous subchapter 8 1 until you have selected a cell Before immersing the pipette into the bath apply slight positive pressure to the pipette to prevent settling of particles at the tip I Apply test pulses to the pipette about 10 pA The resulting voltage signals at the pipette are very small 50 u V with a 5 MQ electrode Approach the cell until the voltage signal changes a Figure 15 Often you can observe slight dent in the cell membrane Release pressure from the pipette Now forming of the seal is indicated by the voltage deflections getting much larger I If the seal does not form apply gentle suction to the pipette until a gigaseal is established b Figure 15
46. ultiple forms of long term potentiation and long term depression converge on a single interneuron in the leech CNS J Neurosci 24 4011 4019 _I Burrell B D amp Sahley L 2005 Serotonin Mediates Learning Induced Potentiation Of Excitability Journal of Neurophysiology 94 4002 4010 L Gollisch T Schutze H Benda J amp Herz V 2002 Energy integration describes sound intensity coding in an insect auditory system J Neurosci 22 10434 10448 L Hutzler M Lambacher A Eversmann B Jenkner M Thewes R amp Fromherz 2006 High resolution multi transistor array recording of electrical field potentials in cultured brain slices J Neurophysiol Kettenmann amp Grantyn R eds 1992 Practical Electrophysiological Methods Wiley Liss New York J Lalley P M Moschovakis and U Windhorst 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 L Maier N Nimmrich V amp Draguhn A 2003 Cellular and network mechanisms underlying spontaneous sharp wave ripple complexes in mouse hippocampal slices J Physiol 550 873 887 L1 Mayer Waarden 1975 Einf hrung in die biologische und medizinische Me technik Schattauer Verlag Stuttgart LI M ck M Butovas S amp Schwarz C 2006 Functional unity of the ponto

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