Home

OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF

1. 100 150 4 200 250 4 300 4 350 400 capacity well compensated voltage time ms Figure 20 Tuning of the coarse capacity compensation Time course of the signal at ELECTRODE POTENTIAL OUTPUT is shown holding current 1 nA switching frequency 2 kHz A cell model was connected electrode resistance 100 MQ version 1 8 page 35 SEC 03M User Manual 120 4 capacity overcompensated 100 VETE EES 80 60 4 40 S E 3 T potential 8 SEE current T T gt 100 150 200 250 300 60 J WMRBETIN A ee E A A 80 time ms 120 5 466 capacity undercompensated 80 4 S 60 4 7 y potential Ed jmnna win LI LIII MITEI E IL memm NON naa rd current 40 4 gt 20 0 T T T yang 0 50 100 150 200 250 300 20 420 time ms capacity well compensated 100 gt SSA MR Am 0 eae 80 gt E 60 a potential ee a iBeseat current o 404 gt 20 0 A A A 0 50 100 150 200 250 300 20 time ms Figure 21 Capacity compensation of the electrode in the bath electrode resistance 100 MQ Current stimulus 1 nA switching frequency 2 kHz Current stimulus and electrode potential are shown version 1 8 SEC 03M User Manual 600 capacity overcompensated gt E A S current 8 voltage gt time ms
2. 22 REL push button mu Push button for activating the resistance measurement of the microelectrode When pushed the microelectrode resistance is measured and shown in POTENTIAL RESISTANCE display 2 Important An accurate measurement of Ret requires that the input capacity is well compensated see also 24 and chapter 8 6 23 GAIN potentiometer 10 turn potentiometer to set amplification factor GAIN of the VC error signal To keep the VC error as small as possible it is necessary to use high GAIN settings but the system becomes unstable and begins to oscillate if the GAIN is set too high Thus the care should be taken when setting this control Using the INTEGRATOR 20 provides a virtually infinite GAIN for slow signal e g holding potential 24 C COMP potentiometer Control for the capacity compensation of the microelectrode ten turn potentiometer clockwise Caution This circuit is based on a positive feedback circuit Overcompensation leads to oscillations that may damage the cell 5 Headstages 5 1 Standard and low noise SEC HSP headstages The SEC 03M comes with the standard headstage range 120 nA for connecting glass electrodes with high resistances or suction electrodes for whole cell patch clamp recordings with lower resistances via an electrode holder A low noise current headstage for measurement of small currents a headstage with differential input and a headstage for extracellular
3. D Electronic E Instruments for the Life Sciences ease ade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF THE SEC 03M SINGLE ELECTRODE CLAMP AMPLIFIER MODULE FOR EPMS SYSTEMS sSEC 03M or CC p vc ext C COMP E 6 API 10 2 40 A 7 pa INTEGR SW FREQ L A e ms khz Pf MODE SELECT VERSION 1 8 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 SEC 03M User Manual Table of Contents Aboutthis Manual 9 a Rb baee uo E Ur datar e iSi aides 4 1 Safety AAA A P 5 2 BPMS 07 Modular Plug In System iecore AA 6 2 1 General System Description Operation uie s rehenes picadas 6 2 2 EPMS OT PIOUSIDE iS reed uuo dotes uude ores Se a 6 2 3 BPMS E 07 Housing irc ket e t ERREUR EAD E EXATAA SETA ORE EUR S I FUNER Sd teases 6 2 4 PASOS S caecos tanto stipe t TM esten TES 6 A O 7 RET 7 EPMS E 07 5 tabtocsieliristi i sott vesti eu btc e i Sar cis ter bia ened ea ghes tad asensi 7 2 0 Techiical Dita A AA ecu asus Aa dut od E dE Satta 7 EPNIS U S eau e d aN e os 7 EPMS E 0T e ness pi tum due a 7 3 MT TU eE ENEE etl ESE EE aE EE 8 Sl Why a Single Electrode Clamp uie den e a aa 8 2 25 Principle of Operation A E R a adea e sees 10 Major Advantages of the npi SEC System sess 12 3 3 Advantages of the Modular SEC 0
4. 12 2 Speed of Response of SEC Single Electrode Clamps The maximum speed of response of any clamp system to a voltage command step is determined by the cell capacity the resistance of the current injecting electrode and the maximum output voltage of the VC amplifier Polder and Houamed 1994 dU m dt max Umax Cm Ra 1 1V s 10 3 mV ys la The standard headstages of the SEC amplifiers are equipped with a current source output with a calibration of 10 nA V Therefore with a voltage of 12 V linear range of the current source a maximum current of 120 nA can be injected into a load of maximum 100 MQ In the switched CC or VC modes the maximum current has to be multiplied with the duty cycle 1 8 1 4 or 1 2 The maximum current is 15 nA 30 nA or 60 nA Remember The duty cycle of the modular SEC 03M is fixed to 1 4 With the maximum current determined electronically by the current source for Re 100 MQ the maximum speed of response can be calculated as dUm dt max ima Cm 2 For a given command step Ucon the shortest time t to reach this level can be calculated as tr Ucom dUm dt max 3a The maximum voltage change AUmax which can be achieved in a given period of time At is AUmax dUm dt max At 3b Examples Cm 300 pF Rm 50 100 MO a Ra 5 MQ b Ra 100 MO 0 05mV us duty cycle 1 8 Equation 2 dUn dt max 0 1 mV us duty cycle 1 4 0 2 mV us duty cycle 1 2 ms duty cycle 1 8 Equa
5. Thus both current and potential output are based on discontinuous signals that are stored during each cycle in the sample and hold amplifiers SH1 and SH2 The signals will be optimal smooth at maximal switching frequencies Major Advantages of the npi SEC System npi electronic s SEC amplifiers are the only systems that use a PI controller to avoid artificial recordings known to occur in other single electrode clamp systems clamping of the electrode The PI controller design increases gain to as much as 100 uA V in frequencies less than one fourth the switching frequency The result is very sensitive control of the membrane potential with a steady state error of less than 1 and a fast response of the clamp to command steps or conductance changes The use of discontinuous current and voltage clamp in combination with high switching frequencies yields five major advantages 1 The large recording bandwidth allows recordings of even fast signals accurately 2 High clamp gains up to 100 uA V can be used in voltage clamp mode 3 Very small cells with relatively short membrane time constants can be voltage clamped 4 Series resistance effect are completely eliminated for a correct membrane potential control even with high resistance microelectrodes 5 The true membrane potential is recorded also in the voltage clamp mode whereas continuous feedback VC amplifiers only reflect the command potential 3 3 Advantages of the Modula
6. 12 3 overcompensated compensated undercompensated 5mVv 20 ms 1 nA 20 ms Figure 25 Adjustment of the bridge balance after penetrating a cell version 1 8 page 42 SEC 03M User Manual A uncompensated potential mV current nA 100 4 3 0 80 4 25 60 4 r20 40 20 0 20 40 B time ms Cstray compensated potential mV current nA 80 4 r 3 0 70 4 725 60 4 50 4 r 20 40 4 15 304 20 4 1 0 10 4 r 0 0 10 0 0 C 0 20 40 60 80 100 120 140 160 time ms Cstray and VreEL compensated potential mV current nA 60 4 3 0 we Tom 50 4 125 40 4 2 0 t 304 Cm 2415 P d 20 4 1 0 10 4 05 0 l 0 0 0 20 40 60 80 100 120 140 160 time ms potential current Figure 26 Artifacts caused by the recording electrode The measurements were done in BR mode using a cell model with 100 MQ membrane resistance 100 pF membrane capacitance and 100 MQ electrode resistance A Costay and Vang not compensated bridge not balanced B Cstray compensated and VREL not compensated C Cstray and VREL compensated bridge balanced Cm membrane capacitance Cstray electrode stray capacitance Ret electrode resistance Rm membrane resistance Tcm time constant of the cell membrane VreL potential drop at Rex see also Figure 5 version 1 8 page 43 SEC 03M User Manual 9 2 Sample Experiment using a Suction Electrode If su
7. 2005 Sharing receptive fields with your neighbors tuning the vertical system cells to wide field motion Journal of Neuroscience 25 3985 3993 L Gabbiani F Krapp H G Koch C amp Laurent G 2002 Multiplicative computation in a visual neuron sensitive to looming Nature 420 320 324 L Gabriel J P Scharstein H Schmidt J amp Buschges A 2003 Control of flexor motoneuron activity during single leg walking of the stick insect on an electronically controlled treadwheel J Neurobiol 56 237 251 L Gingl E amp French A S 2003 Active signal conduction through the sensory dendrite of a spider mechanoreceptor neuron J Neurosci 23 6096 6101 L Gingl E French A S Panek L Meisner S amp Torkkeli P H 2004 Dendritic excitability and localization of GABA mediated inhibition in spider mechanoreceptor neurons European Journal of Neuroscience 20 59 65 LI Grass D Pawlowski P G Hirrlinger J Papadopoulos N Richter D W Kirchhoff F amp Hulsmann S 2004 Diversity of functional astroglial properties in the respiratory network J Neurosci 24 1358 1365 L Hadjilambreva G Mix E Rolfs A Muller J amp Strauss U 2005 Neuromodulation by a Cytokine Interferon beta Differentially Augments Neocortical Neuronal Activity and Excitability Journal of Neurophysiology 93 843 852 LI Hepp S Gerich F J amp Mueller M 2005 Sulfhydryl Oxidation Reduces Hip
8. Dodt H U and Zieglg nsberger W 1994 Infrared videomicroscopy a new look at neuronal structure and function Trends in Neurosciences 19 11 453 458 L Haag J Denk W amp Borst A 2004 Fly motion vision is based on Reichardt detectors regardless of the signal to noise ratio Proc Natl Acad Sci U S A 101 16333 16338 1 Jacob S N Choe C U Uhlen P DeGray B Yeckel M F amp Ehrlich B E 2005 Signaling microdomains regulate inositol 1 4 5 trisphosphate mediated intracellular calcium transients in cultured neurons Journal of Neuroscience 25 2853 2864 LI Kapur A M Yeckel and Johnston D 2001 Hippocampal mossy fiber activity evokes Ca2 release in CA3 pyramidal neurons via a metabotropic glutamate receptor pathway Neuroscience 107 1 59 69 1 Single S and Borst A 1998 Dendritic Integration and Its Role in Computing Image Velocity Science 281 1848 50 1 Single S and Borst A 2002 Different Mechanisms of Calcium Entry Within Different Dendritic Compartments J Neurophysiol 87 1616 1624 L Schierloh A Eder M Zieglgansberger W amp Dodt H U 2004 Effects of sensory deprivation on columnar organization of neuronal circuits in the rat barrel cortex Eur J Neurosci 20 1118 1124 Recordings from cardiac cells LI Bollensdorff C Knopp A Biskup C Zimmer T amp Benndorf K 2004 Na current through KATP channels consequences for Na and K fluxes
9. L Lapish C C Wightman R M Phillips P E amp Seamans J K 2005 Mesocortical dopamine neurons operate in distinct temporal domains using multimodal signaling J Neurosci 25 5013 5023 LI Leger J F Stern E A Aertsen A amp Heck D 2004 Synaptic Integration in Rat Frontal Cortex Shaped by Network Activity Journal of Neurophysiology 93 281 293 L Seiffert E Dreier J P Ivens S Bechmann I Tomkins O Heinemann U amp Friedman A 2004 Lasting blood brain barrier disruption induces epileptic focus in the rat somatosensory cortex J Neurosci 24 7829 7836 L Sillaber I Rammes G Zimmermann S Mahal B Zieglg nsberger W Wurst W Holsboer F amp Spanagel R 2002 Enhanced and Delayed Stress Induced Alcohol Drinking in Mice Lacking Functional CRH1 Receptors Science 296 931 933 L Strauss U Kole M H Brauer A U Pahnke J Bajorat R Rolfs A Nitsch R amp Deisz R A 2004 An impaired neocortical I is associated with enhanced excitability and absence epilepsy Eur J Neurosci 19 3048 3058 L Weiss T Veh R W amp Heinemann U 2003 Dopamine depresses cholinergic oscillatory network activity in rat hippocampus Eur J Neurosci 18 2573 2580 Perforated Patch L Hanganu I L Kilb W amp Luhmann H J 2002 Functional synaptic projections onto subplate neurons in neonatal rat somatosensory cortex J Neurosci 22 7165 7176 L1
10. Microdomains in Hippocampal Neurons Journal of Neuroscience 25 558 565 LI Naro F De A V Coletti D Molinaro M Zani B Vassanelli S Reggiani C Teti A amp Adamo S 2003 Increase in cytosolic Ca2 induced by elevation of extracellular Ca2 in skeletal myogenic cells Am J Physiol Cell Physiol 284 C969 C976 Li Nasif F J Sidiropoulou K Hu X T amp White F J 2005 Repeated cocaine administration increases membrane excitability of pyramidal neurons in the rat medial prefrontal cortex J Pharmacol Exp Ther 312 1305 1313 LI Okabe A Kilb W Shimizu Okabe C Hanganu I L Fukuda A amp Luhmann H J 2004 Homogenous glycine receptor expression in cortical plate neurons and cajal retzius cells of neonatal rat cerebral cortex Neuroscience 123 715 724 L Panek I French A S Seyfarth E A Sekizawa S I amp Torkkeli P H 2002 Peripheral GABAergic inhibition of spider mechanosensory afferents Eur J Neurosci 16 96 104 LI Pangrsic T Stusek P Belusic G amp Zupancic G 2005 Light dependence of oxygen consumption by blowfly eyes recorded with a magnetic diver balance J Comp Physiol A Neuroethol Sens Neural Behav Physiol 191 75 84 L Pascual O Traiffort E Baker D P Galdes A Ruat M amp Champagnat J 2005 Sonic hedgehog signalling in neurons of adult ventrolateral nucleus tractus solitarius Eur J Neurosci 22 389 396 Li Pomper J K
11. Recordings under such conditions and possible applications have been presented in several papers e g Richter et al 1996 Criteria for the selection of the switching frequency Which are the most important criteria for the selection of the switching frequency This question was analyzed in detail by M Weckstrom and colleagues Juusola 1994 Weckstrom et al 1992 They presented a formula that describes the conditions for obtaining reliable results during a switching single electrode clamp version 1 8 page 31 SEC 03M User Manual fe 3fsw fsw gt 2fs fs gt 2f gt fm fe upper cutoff frequency of the microelectrode fsw switching frequency of the dSEVC fs sampling frequency of the data acquisition system fr upper cutoff frequency of the lowpass filter for current recording fm upper cutoff frequency of the membrane Example Muller et al 1999 With the time constant of 1 3 us recorded for the electrodes used in this study fe is 80 160 kHz the selected switching frequency of the dSEVC was 30 50 kHz calculated range is 25 53 kHz data were sampled at 10 kHz and the current signals have been filtered at 5 kHz These settings are currently used for recordings in many labs The principle of operation in switched mode is shown below SwF npi fast compensation 10 kHz P PME S NX a da u E 0 18 V phe compensation t 40 kHz d VVUVVVIN il Uu U 0 18V i U sampling ty lus 40 kHz 2 5 1 u
12. fme CUMING sce oer couse ca aaa secu acto ue Mh rial 38 8 7a resting Operation Modes Jio etd s ea ean ee OS 39 Current Clamp in BR or discontinuous CC mode sees 39 Voltage Cia aaa a a ue ut a bei Durs tu o ARENA 39 9 Sample Expermells od el Bcc nA INA Uta uidet Debet dE E R 41 9 1 Sample Experiment using a Sharp Microelectrode sene 41 9 2 Sample Experiment using a Suction Electrode eene 44 10 Tuning VC Performance cid sese oreet ense ea Me ENS PNE UNTEN ERR NN ex PU esa o YU FR Pe Rd ONU HOUR 46 General Consideratioks eei e a es i is 46 LC A A hha fade ete eae le Sena a Oe aa ech toda lees clyde ice 47 11 Trouble iD setas 48 12 PS e a a a a a N 49 version 1 8 page 2 SEC 03M User Manual I2 T EOD OP Operation dci 49 12 2 Speed of Response of SEC Single Electrode Clamps sees 50 12 3 Tuning Procedures for VC Controllers iere tete tete eh ierat ciegas 50 Practical Implicalonsc aa 51 13 Literature about npi single electrode clamp amplifiers sess 53 13 1 Paper in Journals ied nest rer a dais eagegeaatacuaeaaeounees 33 13 2 Books rx e m 64 14 SEC 03M Specifications Technical Data io 65 15 GER qe M NAT 68 version 1 8 page 3 SEC 03M User Manual About this Manual This manual should help to setup and use SEC systems correctly and to perfor
13. undercom pensated potential mV 6 a O 50 100 150 200 250 300 350 400 time ms overcom pensated potential mV 6 4 time ms compensated potential mV 3 2 1 O eres A AI I ES EE TEER 14 24 0 50 100 150 200 250 300 350 400 time ms potential Figure 16 Tuning of the BRIDGE BALANCE using 100 MQ resistor version 1 8 page 30 SEC 03M User Manual 8 5 Switching Frequency and Capacitance Compensation in switched modes For accurate measurements in switched mode it is essential that the capacity of the electrode is fully compensated Important Wrong compensation of electrode capacity leads to errors in measurements done in switched mode of the amplifier see Figure 18 Microelectrode selection As depicted in chapter 8 2 electrodes must be tested before use For details see also Richter et al 1996 Switching frequency is a key parameter of discontinuous single electrode clamp dSEVC systems The switching frequency determines the accuracy speed of response and signal to noise ratio of the dSEVC system Richter et al 1996 Muller et al 1999 Since its launch in 1984 one of the outstanding features of the SEC series of single electrode voltage current clamp systems has been the ability to record routinely with high switching frequencies in the range of tens of kilohertz regardless of the microelectrode resistance Polder et al 1984 P
14. x10 mV output impedance 250 2 potential display XXX mV CURRENT CLAMP Input 1 nA V input resistance 2100 KQ HOLD X XX nA ten turn digital control with 0 switch max 10 nA BRIDGE balance XXX MQ with ten turn digital control Noise BRIDGE MODE 400 uVpp pApp with 100 MQ resistance at 10 KHz bandwidth VOLTAGE CLAMP Input 10 mV input resistance 2100 KQ HOLD XXX mV ten turn digital control with 0 switch max 1000 mV GAIN 100 nA V 10 n A V ten turn linear control Noise potential output 400 uV pp current output 400 pApp version 1 8 page 66 SEC 03M User Manual SPEED of RESPONSE VC Mode 1 settling time lt 80 us for 10 mV step and lt 800 us for 50 mV step applied to a cell model Ret 100 MQ Rm 50 MQ Cm 470 pF duty cycle 25 switching frequency 30 kHz standard headstage DIMENSIONS Front panel 24 HP 121 5 mm x 3U 128 5 mm Housing 7 175 mm deep EPMS 07 system POWER REQUIREMENTS 115 230 V AC 60 50 Hz fuse 2 A 1 A slow 45 60 W depending on the modules plugged in DIMENSIONS 19 rackmount cabinet 3U high 1U 1 3 4 44 45 mm version 1 8 page 67 SEC 03M User Manual 15 Index abbreviations 4 Absolute value optimum 51 accessories 13 AVO method 51 basic installation 22 basic settings 22 bias current adjustment 27 BIAS current potentiometer 15 bridge balance 29 30 42 BRIDGE BALANCE potentiometer 15 C COMP potentiomet
15. D 2004 Neurogranin RC3 enhances long term potentiation and learning by promoting calcium mediated signaling J Neurosci 24 10660 10669 LI Marsicano G Wotjak C T Azad S C Bisognok T Rammes G Casciok M C Hermann H Tang J Hofmann C Zieglg nsberger W Di Marzok V amp Lutz B 2002 The endogenous cannabinoid system controls extinction of aversive memories Nature 418 530 533 LJ Nakazawa K Quirk M C Chitwood R A Watanabe M Yeckel M F Sun L D Kato A Carr C A Johnston D Wilson M A amp Tonegawa M A 2002 Requirement for Hippocampal CA3 NMDA Receptors in Associative Memory Recall Science 297 211 218 Li Rammes G Palmer M Eder M Dodt H U Zieglgansberger W amp Collingridge G L 2003 Activation of mGlu receptors induces LTD without affecting postsynaptic sensitivity of CA1 neurons in rat hippocampal slices J Physiol 546 455 460 Li Rammes G Steckler T Kresse A Schutz G Zieglgansberger W and Lutz B 2000 Synaptic plasticity in the basolateral amygdala in transgenic mice expressing dominant negative cAMP response element binding protein CREB in forebrain Eur J Neurosci 12 2534 2546 L Seeger T Fedorova I Zheng F Miyakawa T Koustova E Gomeza J Basile A S Alzheimer C amp Wess J 2004 M2 muscarinic acetylcholine receptor knock out mice show deficits in behavioral flexibility working memory an
16. Electrode connectors two gold plated SUBCLIC SMB with driven shields Driven shield output 2 3 mm connector range 15 V impedance 250 Q Ground 2 3 mm connector or headstage enclosure Holding bar diameter 8 mm length 100 mm Size 100x40x25 mm Headstage enclosure is connected to ground version 1 8 page 65 SEC 03M User Manual ELECTRODE PARAMETER CONTROLS Offset ten turn control 200 mV Capacity compensation range 0 30 pF adapts compensation circuit to electrode parameters coarse control at headstage fine control at front panel ten turn potentiometer BANDWIDTH and SPEED OF RESPONSE Full power bandwidth Rex 0 2100 kHz Rise time 10 90 Ret 100 MQ lt 30 us Rise time 10 90 Ret 5 MQ lt 8 us Electrode artifact decay switched modes 10 nA signal lt l us Ret 5 MQ 1 5 us Rer 100 MQ CAPACITY COMPENSATION tuned with no overshoot ELECTRODE RESISTANCE TEST obtained by application of square current pulses 1 nA 10 mV MQ display XXX MO SWITCHED MODES PARAMETERS Switching frequency linear control 2 40 kHz duty cycle fixed to 25 current injection CURRENT RANGE in SWITCHED MODE Standard headstage 30 nA SEC HSP headstage 3 nA SWITCHED MODE OUTPUTS Electrode potential max 12 V output impedance 250 Q Switching frequency TTL output impedance 250 Q CURRENT OUTPUT 10 nA V output impedance 250 Q current display X XX nA POTENTIAL OUPUT Sensitivity
17. Haack S Petzold G C Buchheim K Gabriel S Hoffmann U amp Heinemann U 2005 Repetitive Spreading Depression Like Events Result in Cell Damage in Juvenile Hippocampal Slice Cultures Maintained in Normoxia Journal of Neurophysiology Li Ranft A Kurz J Deuringer M Haseneder R Dodt H U Zieglgansberger W Kochs E Eder M amp Hapfelmeier G 2004 Isoflurane modulates glutamatergic and GABAergic neurotransmission in the amygdala Eur J Neurosci 20 1276 1280 LJ Rastan A J Walther T Kostelka M Garbade J Schubert A Stein A Dhein S amp Mohr F W 2005 Morphological electrophysiological and coupling characteristics of version 1 8 page 62 SEC 03M User Manual bone marrow derived mononuclear cells an in vitro model European Journal of Cardio Thoracic Surgery 27 104 110 L Reiprich P Kilb W amp Luhmann H J 2005 Neonatal NMDA Receptor Blockade Disturbs Neuronal Migration in Rat Somatosensory Cortex In Vivo Cerebral Cortex 15 349 358 LI Ren J Lee S Pagliardini S Gerard M Stewart C L Greer J J amp Wevrick R 2003 Absence of Ndn encoding the Prader Willi syndrome deleted gene necdin results in congenital deficiency of central respiratory drive in neonatal mice J Neurosci 23 1569 1573 L Ren J amp Greer J J 2006 Modulation of respiratory rhythmogenesis by chloride mediated conductances during the perinatal pe
18. M amp Richter A 2004 Increased excitability in cortico striatal synaptic pathway in a model of paroxysmal dystonia Neurobiol Dis 16 236 245 Li Ludwar B C Westmark S Buschges A amp Schmidt J 2005 Modulation of membrane potential in mesothoracic moto and interneurons during stick insect front leg walking Journal of Neurophysiology 94 2772 2784 1 Leger J F Stern E A Aertsen A amp Heck D 2005 Synaptic integration in rat frontal cortex shaped by network activity Journal of Neurophysiology 93 281 293 L Marsicano G Goodenough S Monory K Hermann H Eder M Cannich A Azad S C Cascio M G Gutierrez S O van der S M Lopez Rodriguez M L Casanova E Schutz G Zieglgansberger W Di M V Behl C amp Lutz B 2003 CB1 cannabinoid receptors and on demand defense against excitotoxicity Science 302 84 88 J Manzke T Guenther U Ponimaskin E G Haller M Dutschmann M Schwarzacher S amp Richter D W 2003 5 HT4 a receptors avert opioid induced breathing depression without loss of analgesia Science 301 226 229 L Mentel T Krause A Pabst M El Manira A amp Buschges A 2006 Activity of fin muscles and fin motoneurons during swimming motor pattern in the lamprey Eur J Neurosci 23 2012 2026 L Muller A Kukley M Stausberg P Beck H Muller W amp Dietrich D 2005 Endogenous Ca2 Buffer Concentration and Ca2
19. OPERATION switch 1 Set the membrane resistance of the cell model to 100 MQ see chapter 7 Set the holding current to 0 5 nA using the HOLD potentiometer 9 setting 50 reading 0 50 nA and the HOLD current polarity switch 9 to Make sure that the ELECTRODE RESISTANCE test is not active The POTENTIAL display should read 50 mV according to Ohm s law The voltage at POTENTIAL OUTPUT BNC 17 should be 500 mV DO DD DL Remember The voltage at POTENTIAL OUTPUT is the membrane potential multiplied by 10 _ Apply a test pulse of 1 nA to the cell model by giving a voltage step of 0 5 V to CUR STIM INPUT 12 The length of the test pulse should be at least 30 ms _ You should see a potential step of 500 mV amplitude at POTENTIAL OUTPUT BNC 17 Due to the membrane capacity the step is smoothed Note If you expect the POTENTIAL display to show the value of the potential step in this case 50 mV amplitude from a resting potential of 50 mV i e 0 mV remember that the display is rather sluggish and may not display the right value depending on the length of the step The same is true for the CURRENT display Voltage Clamp In voltage clamp mode the membrane potential is forced by a controller to maintain a certain value or to follow an external command That allows measurement of ion fluxes across the cell membrane This is the most complex mode of operation with the SEC 03M Special precaution
20. OUTPUT see Figure 16 L Make the basic settings at the amplifier see chapter 6 L Connect a cell model or immerse the electrode into the bath as deep as necessary during the experiment _ Apply current pulses to the electrode either using an external stimulator via the CUR STIM INPUT connector 12 Figure 8 L Watch the POTENTIAL OUTPUT at the oscilloscope and adjust the BRIDGE BALANCE as shown in Figure 16 using the BR BAL potentiometer 6 Figure 8 After adjustment you should see a straight voltage trace without artifacts caused by the potential drop at Ret Figure 16 illustrates the BRIDGE BALANCE procedure using a 100 MQ resistor that represents the electrode The current stimulus 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 must be tuned several times during an experiment since most parameters change during a recording session see Figure 15 OFFSET deviations can be detected by comparing the readout on the potential display before and after an experiment with the electrode in the tissue but not in a cell overcompensated cornpensated undercompensated 5mv 20 ms Figure 15 Adjustment of the bridge balance after cell penetration in BR mode version 1 8 page 29 SEC 03M User Manual
21. SO uses also the PI controller and has the best performance compensating intrinsic disturbance signals The response to a command step shows a very steep rise phase followed by a considerable overshoot maximum 43 The response to a disturbance is fast and the amplitude of the deviation is in the same range as with the AVO method The overshoot can be reduced by adequate shaping of the command pulse by a delay unit Froehr 1985 Polder and Swandulla 1990 Polder and Swandulla 2001 This method is preferred for slowly activating currents such as those evoked by agonist application The upper speed limit for all optimization methods is determined by the maximum amount of current which the clamp system can force through a given electrode see chapter 12 2 Practical Implications In the following some practical implications of the theory discussed earlier in this chapter are outlined It is assumed that the system is in VC mode with integrator turned OFF Although most of the parameters of the control chain are not known during an experiment it is possible to tune the clamp controller by optimizing the response to a test pulse applied to the VC COMM INPUT The main criterion of tuning is the overshoot seen at the potential output Since the SO method provides the tightest control it will be most sensitive to parameter settings and requires most experience Note The transitions between the optimization methods are blurred and the tuning p
22. half of the voltage command In order to achieve a voltage error of less than 1 Ra must be more than 100 times smaller than Rm This condition is not always easily to accomplish especially if recordings a performed from small cells If sharp intracellular microelectrodes are used it is virtually impossible If Ra is not negligible precise determination of the membrane potential can be achieved only if no current flows across Ra during potential measurement This is the strategy employed in npi electronic s SEC amplifier systems The SEC amplifiers inject current and record the potential in an alternating mode switched mode Therefore this technique is called discontinuous SEVC This ensures that no current passes through Ra during potential measurement and completely eliminates access resistance artefacts After each injection of current the potential gradient at the electrode tip decays much faster than the potential added at the cell membrane during the same injection The membrane potential is measured after the potential difference across Ra has completely dropped see chapter 3 2 The discontinuous current and voltage signal are then smoothed and read at the CURRENT OUTPUT and POTENTIAL OUTPUT connectors 3 2 Principle of Operation current output record potential output el SwF Figure 6 Model circuit of SEC systems version 1 8 page 10 SEC 03M User Manual current inject current free sample
23. is split into two controls the coarse control at the headstage and a the fine control at the front panel of the amplifier The aim of the first part of the tuning procedure is to set the coarse capacity compensation at the headstage so that an optimal wide range of C COMP at the amplifier is achieved 1 Insert the electrode into the electrode holder and connect it to the amplifier 4 Immerse the electrode as deep as it will be during the experiment into the bath solution Q Set the C COMP control at the amplifier potentiometer 24 at the front panel to a value around 2 and turn COARSE CAPACITY COMPENSATION at the headstage to the leftmost position Q Connect the BNC connector ELECT POTENTIAL OUTPUT 16 at the front panel to an oscilloscope and trigger with the signal at BNC connector SWITCH FREQUENCY SYNC OUT 14 at the front panel The oscilloscope should be in external trigger mode The time base of the oscilloscope should be in the range of 250 us 4 Set the amplifier in CC mode and select the lowest switching frequency 1 to 2 kHz 4 Apply positive or negative current to the electrode using the HOLD CUR control potentiometer 9 at the front panel 4 You should see a signal at the oscilloscope similar to those in Figure 19 Turn the COARSE CAPACITY COMPENSATION carefully clockwise until the signal becomes as square as possible lower diagram in Figure 19 Important If you use a model cell e g to train yoursel
24. potential offset see chapter 8 3 compensate the input capacitance see chapter 8 6 and measure the electrode resistance using switch 22 Figure 8 L1 Apply current steps to the CUR STIM INPUT and adjust the BRIDGE BALANCE to suppress all artifacts on the POTENTIAL OUTPUT see chapter 8 4 version 1 8 page 41 SEC 03M User Manual or Now the system is preadjusted for measurements in BR mode 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 Apply a BUZZ to the electrode If you are lucky the tip of the electrode is now inside the cell If necessary readjust BRIDGE BALANCE and or C COMP as shown in Figure 25 and Figure 26 using current stimuli that do not activate ion channels or transporters 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 in BR mode Switch to discontinuous CC mode The shape of voltage and current traces should not change considerably If you intend to work in discontinuous VC mode tune the system in CC mode see above then switch to VC mode and adjust the clamp as described in chapters 10 and
25. resolution of tail currents following single action potentials J Neurosci Meth 116 55 63 Voltage clamp controlled current clamp L Schubert D Kotter R Luhmann H J amp Staiger J F 2006 Morphology electrophysiology and functional input connectivity of pyramidal neurons characterizes a genuine layer va in the primary somatosensory cortex Cereb Cortex 16 223 236 version 1 8 page 53 SEC 03M User Manual Comparison of recording methods sharp electrode whole cell perforated patch L Jarolimek W and Miseld U 1993 4 Aminopyridine induced synaptic GABA B currents in granule cells of the guinea pig hippocampus Pfliigers Arch 425 491 498 L Kapur A Yeckel M F Gray R and Johnston D 1998 L Type calcium channels are required for one form of hippocampal mossy fiber LTP J Neurophysiol 79 2181 2190 L Magistretti J Mantegazza M Guatteo E and Wanke E 1996 Action potentials recorded with patch clamp amplifiers are they genuine Trends Neurosci 19 530 534 Recordings of fast Na channels LI Inceoglu A B Hayashida Y Lango J Ishida A T amp Hammock B D 2002 A single charged surface residue modifies the activity of ikitoxin a beta type Na channel toxin from Parabuthus transvaalicus Eur J Biochem 269 5369 5376 L Hayashida Y Partida G J amp Ishida A T 2004 Dissociation of retinal ganglion cells without enzymes J Neurosci Methods 137
26. the center of the module 16 ELECT POTENTIAL connector BNC connector providing the switched signal directly from the electrode This signal is used for tuning the capacity compensation see also SEC 05 manual ELECT POTENTIAL M version 1 8 page 17 SEC 03M User Manual 17 POTENTIAL OUTPUT x10 mV connector BNC connector monitoring the recorded membrane potential with a gain of ten BNC connector for an external COMMAND in VC mode sensitivity 10 i e 0 1 V V The voltage signal that is connected here is transformed to a proportional COMMAND voltage in VC mode The signal form remains unchanged Two examples are given in Figure 9 The amplitude of the output voltage signal voltage stimulus is determined by the amplitude of the input voltage signal input voltage signal command voltage signal Figure 10 input output relation using VC COMM INPUT in VC mode 19 MODE SELECT connector MODE EN BNC connector for remote control of the MODE of operation A TTL signal is mu O connected here to select the mode of operation remotely HI VC LO CC 20 INTEGR ms potentiometer Potentiometer for setting the INTEGRATOR time constant in VC mode range 0 to 10 ms 10 turn digital control that presets a continuous command signal HOLD potential for VC Polarity is set by switch to the right of the control 0 is off position HOLD POT mV version 1 8 page 18 SEC 03M User Manual
27. timing vm es A CCS cut I sed Nil pe V IAT ee ee cm V cell 1 Vau o oo Figure 7 principle of SEVC operation Figure 6 and Figure 7 illustrate the basic circuitry and operation of npi SEC voltage clamp amplifiers A single microelectrode penetrates the cell or is connected to the cell interior in the whole cell configuration of the patch clamp technique The recorded voltage is buffered by a xl operational amplifier A1 in Figure 6 At this point the potential V A1 in Figure 7 is the sum of the cell s membrane potential and the voltage gradient which develops when current is injected at the access resistance Due to npi s unique compensation circuitry the voltage at the tip of the electrode decays extremely fast after each injection of current and therefore allows for a correct measurement of Vm after a few microseconds At the end of the current free interval when the electrode potential has dropped to zero the sample and hold circuit SH1 in Figure 6 samples Vm and holds the value for the remainder of the cycle Vsui in Figure 7 The differential amplifier A2 in Figure 6 compares the sampled potential with the command potential Vcom in Figure 6 The output of this amplifier becomes the input of a controlled current source CCS in Figure 6 if the switch S1 Figure 6 is in the current passing position The gain of this current source increases as much as 100 n A V due to a PI proportional integral c
28. 25 35 L Hayashida Y amp Ishida A T 2004 Dopamine receptor activation can reduce voltage gated Na current by modulating both entry into and recovery from inactivation Journal of Neurophysiology 92 3134 3141 Coating of sharp microelectrodes for VC recordings LJ Juusola M Seyfarth E A and French A S 1997 Fast coating of glass capillary microelectrodes for single electrode voltage clamp J Neurosci Meth 71 199 204 Recordings with high resistance 150 220 MQ sharp microelectrodes LI Highstein S M Rabbitt R D Holstein G R amp Boyle R D 2005 Determinants of spatial and temporal coding by semicircular canal afferents J Neurophysiol 93 2359 2370 LI Niven J E Vahasoyrinki M Kauranen M Hardie R C Juusola M amp Weckstrom M 2003 The contribution of Shaker K channels to the information capacity of Drosophila photoreceptors Nature 421 630 634 L Rabbitt R D Boyle R Holstein G R amp Highstein S M 2005 Hair cell versus afferent adaptation in the semicircular canals Journal of Neurophysiology 93 424 436 L Wolfram V amp Juusola M 2004 The Impact of Rearing Conditions and Short Term Light Exposure on Signaling Performance in Drosophila Photoreceptors Journal of Neurophysiology 92 1918 1927 Capacitive transients in VC recordings L Sutor B Hablitz J J 1989 Excitatory postsynaptic potentials in rat neocortical neurons in
29. 3M System sess 12 A gabe sedens esu tpa seta d ce ca utu uns Lo DIS ate 13 4 1 SECOM CODDODGDIS 5 sedentes Gaii n Gas id cedit Gu dads etre dire RRi 13 4 2 Description of the Front Panel sese ei ii ii ad Duda 14 A I REI E VER Re E E A NEQU Ed e E a Taas 19 5 1 Standard and low noise SEC HSP headstages sese 19 5 2 Liowenotsedeadstage SEC HSP ns ete beide ani tenia aee uide 21 6 Setting up the SEC 03M System inicios irene 22 de Passive Cell Model ctor cti ai 23 TAI Cel Model Description uon eM tend 23 12 CONNECTIONS and OperatlOli s tese ds SUN PRU Last aan pA sut ae Ola ia pee Utd Lei SUE 24 T3 Connections and OpOral OD sao esses ores OU EAS E ENNNAE Te uu S ses e ERN ARA 25 8 Test and Tuning Procedures eterna aesa ei i aapi e as 27 8 1 Headstage Bias Current Adjustment ics oet io 27 8 25 Electrode Selections riiin A E EEE AT EA R EE E a 28 8 3 Offset Compensa Ofiss evt essi Ia n UIN NEUE EAEE EESE E REEERE EAs 28 8 4 Bridge Balance m BR mode merino tet etae uino ete eres rep TR UE nane imple ee dup o ques a adan 29 8 5 Switching Frequency and Capacitance Compensation in switched modes 31 Criteria for the selection of the switching frequency cocoocccnoocccnnocnconancnonncconancconnncnnnns 31 8 6 Capacity Compensation Tuning Procedure sese 33 First part basic setting iso ere S DUREE RU TAN CY SUR Ea sededatacagedervansdenvs senses 33 Second patt
30. 800 700 4 capacity undercompensated gt A aa current voltage gt 45 50 55 60 100 time ms 800 4 700 4 600 4 capacity well compensated S E PCM LZ LLL 4 nh 1l1 li ll QNEM current 8 voltage gt 40 45 50 55 60 100 time ms Figure 22 Capacity compensation of the electrode using a cell model electrode resistance 100 MQ current 1 nA cell membrane 100 MQ 100 pF switching frequency 2 kHz Current stimulus and membrane potential are shown version 1 8 page 37 SEC 03M User Manual Second part fine tuning Now the basic setting of the CAPACITY COMPENSATION is achieved Since the electrode parameters change during the experiment especially after impaling a cell it is necessary to fine tune the CAPACITY COMPENSATION during the experiment using the C COMP control on the amplifier To get familiar with this connect a cell model and go through the following steps the procedure is the identical with a real cell 4 Connect POTENTIAL OUTPUT and CURRENT OUTPUT front panel to another oscilloscope O Set SWITCHING FREQUENCY to the desired value gt 15 kHz O Set the HOLDING CURRENT to zero With the amplifier in CC mode apply square pulses of a few nA or a few tens of pA for patch recordings to the cell Negative current pulses are recommended If you apply positive current pulses be sure only to elicit ohmic responses of the cell membrane i e pulses shou
31. C MOD SEAL g 160 Figure 13 SEC MOD passive cell model 1 3 connectors for the headstage 1 electrode resistance 100 MQ 3 electrode resistance 5MO 2 GND ground connector to be connected to GND jack of the headstage 4 CELL switch for cell membrane representing a membrane of either 100 MQ and 100 pF CELL 1 or 500 MQ and 22 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 GO resistor simulating the formation of a GIGASEAL with a patch electrode version 1 8 page 23 SEC 03M User Manual Whole cell Patch Sharp Microelectrode Driven Shield BNC connector o NANA O SMB connector Re 5 MO Re 100 MO SMB BNC adapter CELL 2 CELL 1 2 Rerouno seat Rm L Sm L m Rm 1kQ 160 500 MO 22 pF 100pF 100MO GND Figure 14 Schematic diagram of the passive cell model 7 2 Connections and Operation Connections L Turn POWER switch of the amplifier off a For simulation of an experiment using a suction electrode _ Connect the BNC jack 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 SMB connector of the cell model to the BNC connector Per at the headstage For headstages with BNC connector use the supplied SMB to BNC adapter For a and b L Connect GND of the cell model to
32. GND of the headstage L Do not connect DRIVEN SHIELD version 1 8 page 24 SEC 03M User Manual Simulation of electrode in the bath L Set switch 4 Figure 13 to the lower position L Set switch 5 Figure 13 to GROUND position The 1 KQ resistor simulates the resistance of the bath solution This can be used to train cancellation of offsets using the bridge balance and using the capacity compensation Simulation of SEAL formation L Set switch 4 Figure 13 to the lower position L Set switch 5 Figure 13 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 100 MQ electrode connector 1 Figure 13 for an experiment with sharp electrodes or the 5 MQ electrode connector 3 Figure 13 for simulating an experiment with patch electrodes 3 Switch the CELL membrane switch see 4 Figure 13 to the desired position CELL 1 or CELL 2 L Turn all controls at the amplifier to low values less than 1 and the OFFSET in the range of 5 zero position and the OSCILLATION SHUTOFF in the DISABLED position L Turn POWER switch of the amplifier on Now you can adjust the amplifier see below and apply test pulses to the cell model The upper position the CELL membrane switch CELL 1 simulates a cell with a res
33. Hayashida Y Partida G J amp Ishida A T 2004 Dissociation of retinal ganglion cells without enzymes J Neurosci Methods 137 25 35 L Hayashida Y amp Ishida A T 2004 Dopamine receptor activation can reduce voltage gated Na current by modulating both entry into and recovery from inactivation Journal of Neurophysiology 92 3134 3141 LI Inceoglu A B Hayashida Y Lango J Ishida A T amp Hammock B D 2002 A single charged surface residue modifies the activity of ikitoxin a beta type Na channel toxin from Parabuthus transvaalicus Eur J Biochem 269 5369 5376 L Yanovsky Y Zhang W amp Misgeld U 2005 Two pathways for the activation of small conductance potassium channels in neurons of substantia nigra pars reticulata Neuroscience 136 1027 1036 version 1 8 page 59 SEC 03M User Manual Recordings from Crustacea L DiCaprio R A 2003 Nonspiking and Spiking Proprioceptors in the Crab Nonlinear Analysis of Nonspiking TCMRO Afferents J Neurophysiol 89 1826 1836 L DiCaprio R A 2004 Information Transfer Rate of Nonspiking Afferent Neurons in the Crab Journal of Neurophysiology 92 302 310 _ Gamble E R amp DiCaprio R A 2003 Nonspiking and Spiking Proprioceptors in the Crab White Noise Analysis of Spiking CB Chordotonal Organ Afferents J Neurophysiol 89 1815 1825 LI Stein W Eberle C C amp Hedrich U B S 2005 Motor pattern selection
34. M F Johnston D amp Zucker R S 2004 Photolysis of postsynaptic caged Ca2 can potentiate and depress mossy fiber synaptic responses in rat hippocampal CA3 pyramidal neurons Journal of Neurophysiology 91 1596 1607 L Wolfram V amp Juusola M 2004 The Impact of Rearing Conditions and Short Term Light Exposure on Signaling Performance in Drosophila Photoreceptors Journal of Neurophysiology 92 1918 1927 version 1 8 page 63 SEC 03M User Manual 13 2 Books L Boulton A A Baker G B and Vanderwolf C H eds 1990 Neurophysiological techniques Basic methods and concepts Humana Press Clifton New Jersey L Cole K S 1968 Membranes ions and impulses University of California Press Berkely CA L Ferreira H G and Marshall M W 1985 The biophysical basis of excitability Cambridge University Press Cambridge LJ Fr hr F 1985 Electronic control engineering made easy An introduction for beginners Siemens AG Berlin and Munich L Horowitz P and Hill W 1989 The art of electronics Cambridge University Press NY L Jack J J B Noble D and Tsien R W 1975 Electric current flow in excitable cells Claredon Press Oxford L Kettenmann H and Grantyn R eds 1992 Practical electrophysiological methods Wiley Liss New York _ Neher E 1974 Elektrische Me technik in der Physiologie Springer Verlag Berlin L Numberger M and Draguhn A eds 1996 Patch Cla
35. M J amp Smith P A 2006 Sciatic Chronic Constriction Injury Produces Cell type Specific Changes in the Electrophysiological Properties of Rat Substantia Gelatinosa Neurons J Neurophysiol L Bickmeyer U Heine M Manzke T amp Richter D W 2002 Differential modulation of Ih by 5 HT receptors in mouse CAI hippocampal neurons Eur J Neurosci 16 209 218 L Bucher D Akay T DiCaprio R A amp Buschges A 2003 Interjoint coordination in the stick insect leg control system the role of positional signaling J Neurophysiol 89 1245 1255 Li Cornil C A Balthazart J Motte P Massotte L amp Seutin V 2002 Dopamine activates noradrenergic receptors in the preoptic area J Neurosci 22 9320 9330 version 1 8 page 60 SEC 03M User Manual L Daw M I Bannister N V amp Isaac J T 2006 Rapid activity dependent plasticity in timing precision in neonatal barrel cortex J Neurosci 26 4178 4187 1 Dong Y Nasif F J Tsui J J Ju W Y Cooper D C Hu X T Malenka R C amp White F J 2005 Cocaine induced plasticity of intrinsic membrane properties in prefrontal cortex pyramidal neurons adaptations in potassium currents Journal of Neuroscience 25 936 940 L Farrow K Haag J amp Borst A 2003 Input organization of multifunctional motion sensitive neurons in the blowfly J Neurosci 23 9805 9811 LI Farrow K Borst A amp Haag J
36. SEC 03M User Manual 3 Introduction npi electronic s SEC Single Electrode Clamp systems are based on the newest developments in the field of modern electronics and control theory see also chapter 11 These versatile current voltage clamp amplifiers permit extremely rapid switching between current injection and current free recording of true intracellular potentials The use of modern high voltage operational amplifiers and a new improved method of capacity compensation makes it possible to inject very short current pulses through high resistance microelectrodes up to 200 MQ and more and to record membrane potentials accurately i e without series resistance error within the same cycle Although the system has been designed primarily to overcome the limitations related to the use of high resistance microelectrodes in intracellular recordings it can also be used to do conventional whole cell patch clamp recordings with suction electrodes or perforated patch recordings The whole cell configuration allows to investigate even small dissociated or cultured cells as well as cells in slice preparations in both current and voltage clamp mode while the intracellular medium is being controlled by the pipette solution 3 1 Why a Single Electrode Clamp Voltage clamp techniques permit the analysis of ionic currents flowing through biological membranes at preset membrane potentials Under ideal conditions the recorded current is directly rel
37. ated to the conductance changes in the membrane and thus gives an accurate measure of the activity of ion channels and electrogenic pumps The membrane potential is generally kept at a preselected value command or holding potential Ionic currents are then activated by sudden changes in potential e g voltage gated ion channels by transmitter release at synapses e g electrical stimulation of fiber tracts in brain slices or by external application of an appropriate agonist Sudden command potential changes used to activate voltage gated currents are especially challenging because the membrane will adopt the new potential value only after it s capacitance Cm in Figure 4and Figure 5 has been charged Therefore the initial transient current following the voltage step should be as large as possible to achieve rapid membrane charging In conventional patch clamp amplifiers this requires a minimal resistance between the amplifier and the cell interior a simple consequence of Ohm s law AU R D i e for a given voltage difference AU the current I is inversely proportional to the resistance R In this context R is the access or series resistance Ra in Figure 4 and Figure 5 between the electrode and the cell interior The time constant for charging a cell is x ReL Cm Ra is largely determined by certain electrode properties mainly electrode resistance and the connection between the electrode and the cell Typical Ra values are be
38. aus W 1999 Switched single electrode amplifiers allow precise measurement of gap junction conductance Amer J Physiol Cell 276 4 C980 C988 J Polontchouk L Ebelt B Jackels M amp Dhein S 2002 Chronic effects of endothelin 1 and angiotensin II on gap junctions and intercellular communication in cardiac cells FASEB J 16 87 89 Li Weng S Lauven M Schaefer T Polontchouk L Grover R amp Dhein S 2002 Pharmacological modification of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation FASEB J 16 1114 1116 LI Xing D Kjolbye A L Nielsen M S Petersen J S Harlow K W Holstein Rathlou N H amp Martins J B 2003 ZP123 increases gap junctional conductance and prevents reentrant ventricular tachycardia during myocardial ischemia in open chest dogs J Cardiovasc Electrophysiol 14 510 520 Simultaneous recordings with two SEC amplifiers L Haag J and Borst A 1996 Amplification of high frequency synaptic inputs by active dendritic membrane processes Nature 379 639 641 LI Haag J and Borst A 2001 Recurrent Network Interactions Underlying Flow Field Selectivity of Visual Interneurons J Neurosci 21 15 5685 5692 L Haag J and Borst A 2002 Dendro Dendritic Interactions between Motion Sensitive Large Field Neurons in the Fly J Neurosci 22 8 3227 3233 L Haag J amp Borst A 2004 Neural mechanism underlying complex rec
39. by nitric oxide in the stomatogastric nervous system of the crab European Journal of Neuroscience 21 2767 2781 Recordings from plant cells L Raschke K 2003 Alternation of the slow with the quick anion conductance in whole guard cells effected by external malate Planta 217 651 657 L Raschke K Shabahang M amp Wolf R 2003 The slow and the quick anion conductance in whole guard cells their voltage dependent alternation and the modulation of their activities Planta 217 639 650 SEC 03 recordings L Martin Pena A Acebes A Rodriguez J R Sorribes A de Polavieja G G Fernandez Funez P amp Ferrus A 2006 Age independent synaptogenesis by phosphoinositide 3 kinase J Neurosci 26 10199 10208 Extracellular recordings SEC EXT LI Beckers U Egelhaaf M amp Kurtz R 2007 Synapses in the fly motion vision pathway evidence for a broad range of signal amplitudes and dynamics J Neurophysiol 97 2032 2041 Other L Akay T Haehn S Schmitz J amp Buschges A 2004 Signals From Load Sensors Underlie Interjoint Coordination During Stepping Movements of the Stick Insect Leg Journal of Neurophysiology 92 42 51 L Albrecht J Hanganu I L Heck N amp Luhmann H J 2005 Oxygen and glucose deprivation induces major dysfunction in the somatosensory cortex of the newborn rat Eur J Neurosci 22 2295 2305 L Balasubramanyan S Stemkowski P L Stebbing
40. cell membrane with 20 MQ and 500 pF is simulated The middle position simulates the electrode immersed into the bath and can be used to train cancellation of offsets and using the capacity compensation version 1 8 page 26 SEC 03M User Manual 8 Test and Tuning Procedures Important The SEC 03M should be used only in warmed up condition i e 20 to 30 minutes after turning power on The following test and tuning procedures are necessary for optimal recordings It is recommended to first connect a cell model to the amplifier to perform some basic adjustments and to get familiar with these procedures It is assumed that all connections are built as described in chapter 6 Many of the tuning procedure can be performed analogue to those described in the manual for the SEC 05L X Important Except for Headstage bias current adjustment see 8 1 all adjustments described below should be carried out every time before starting an experiment or after changing the electrode 8 1 Headstage Bias Current Adjustment Caution It is important that this tuning procedure is performed ONLY after a warm up period of at least 30 minutes This tuning procedure is very important since it determines the accuracy of the SEC system Therefore it must be done routinely with great care SEC systems are equipped with a current source that is connected to the current injecting electrode and performs the current injection This current source has a high imp
41. chter D W Pierrefiche O Lalley P M amp Polder H R 1996 Voltage clamp analysis of neurons within deep layers of the brain J Neurosci Meth 67 121 131 L Schubert D Staiger J F Cho N Koetter R Zilles K and Luhmann H J 2001 Layer Specific Intracolumnar and Transcolumnar Functional Connectivity of Layer V Pyramidal Cells in Rat Barrel Cortex J Neurosci 21 10 3580 3592 L Schubert D Kotter R Zilles K Luhmann H J amp Staiger J F 2003 Cell Type Specific Circuits of Cortical Layer IV Spiny Neurons J Neurosci 23 2961 2970 L Schubert D Kotter R Luhmann H J amp Staiger J F 2005 Morphology Electrophysiology and Functional Input Connectivity of Pyramidal Neurons Characterizes a Genuine Layer Va in the Primary Somatosensory Cortex Cerebral Cortex bhil00 L Weiss T Veh R W amp Heinemann U 2003 Dopamine depresses cholinergic oscillatory network activity in rat hippocampus Eur J Neurosci 18 2573 2580 version 1 8 page 56 SEC 03M User Manual Tracer injection and intracellular recording L Poulet J F amp Hedwig B 2006 The cellular basis of a corollary discharge Science 311 518 522 L R hrig G Klausa G amp Sutor B 1996 Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurons J Physiol 490 1 31 49 Visualization imaging and infrared video microscopy L
42. ction electrodes are used for whole cell recordings they are usually called pipettes Thus in this subchapter pipette means suction electrode to amplifier electrode _ cell ground Figure 27 Model circuit for whole cell patch clamp recording using a suction electrode Cm membrane capacitance Cstray electrode stray capacitance Re electrode resistance Rm membrane resistance L Prepare the setup and proceed as described in the previous subchapter 9 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 _ Apply test pulses to the pipette about 10 pA The resulting voltage signals at the pipette are very small 50 uV with a 5 MQ pipette L Approach the cell until the voltage signal changes a Figure 28 Often you can observe a slight dent in the cell membrane L Release pressure from the pipette Now forming of the seal is indicated by the voltage deflections getting much larger L If the seal does not form apply gentle suction to the pipette until a gigaseal is established b Figure 28 _ Apply stronger suction to the pipette or use the BUZZ unit to brake the cell membrane under the pipette opening and establish the whole cell configuration The whole cell configuration is established if you see the voltage signal getting smaller again c Figure 28 and you read the expected membrane p
43. d hippocampal plasticity J Neurosci 24 10117 10127 L Wang J Yeckel M F Johnston D amp Zucker R S 2004 Photolysis of Postsynaptic Caged Ca2 Can Potentiate and Depress Mossy Fiber Synaptic Responses in Rat Hippocampal CA3 Pyramidal Neurons Journal of Neurophysiology 91 1596 1607 LI Yeckel M F Kapur A amp Johnston D 1999 Multiple forms of LTP in hippocampal CA3 neurons use a common postsynaptic mechanism Nat Neurosci 2 625 633 version 1 8 page 58 SEC 03M User Manual Performance test with active cell model L Draguhn A Pfeiffer M Heinemann U and Polder H R 1997 A simple hardware model for the direct observation of voltage clamp performance under realistic conditions J Neurosci Meth 78 105 113 Intra and extracellular low noise recording LI DeBock F Kurz J Azad S C Parsons C G Hapfelmeier G Zieglg nsberger W amp Rammes G 2003 a2 Adrenoreceptor activation inhibits LTP and LTD in the basolateral amygdala involvement of Gio protein mediated modulation of Ca channels and inwardly rectifying K channels in LTD Eur J Neurosci 17 1411 1424 Li Kukley M Stausberg P Adelmann G Chessell I P amp Dietrich D 2004 Ecto nucleotidases and nucleoside transporters mediate activation of adenosine receptors on hippocampal mossy fibers by P2X7 receptor agonist 2 3 O 4 benzoylbenzoyl ATP J Neurosci 24 7128 7139 LI Lavin A Nogueira
44. during early myocardial ischemia Am J Physiol Heart Circ Physiol 286 H283 H295 3 Linz K and Meyer R 1997 Modulation of L type calcium current by internal potassium in guinea pig ventricular myocytes Cardiovascular Research 33 110 122 LI Lu J Dalton IV J F Stokes D R and Calabrese R L 1997 Functional role of Ca2 currents in graded and spike synaptic transmission between leech heart interneurons J Europhysiol 77 1779 1794 L M ller A et al 1997 Increase in gap junction conductance by an antiarrhythmic peptide Europ J Pharmacol 327 65 72 L M ller A et al 1997 Actions of the antiarrhythmic peptide AAPIO on intracellular coupling Naunyn Schmiedeberg s Arch Pharmacol 356 76 82 L Pillekamp F Reppel M Dinkelacker V Duan Y Jazmati N Bloch W Brockmeier K Hescheler J Fleischmann B K amp Koehling R 2005 Establishment and characterization of a mouse embryonic heart slice preparation Cell Physiol Biochem 16 127 132 LI R cke H F et al 1994 Fosinoprilate prolongs the action potential reduction of Ik and enhancement of L type calcium current in guinea pig ventricular myocytes Cardiovascular Research 28 201 208 version 1 8 page 57 SEC 03M User Manual LTP LDP LTD Investigations Li Azad S C Monory K Marsicano G Cravatt B F Lutz B Zieglgansberger W amp Rammes G 2004 Circuitry for associative plasticity
45. e design and the optimal tuning of feedback systems Froehr 1985 Most voltage clamp systems are composed only of delay elements i e elements which react with a retardation to a change This type of closed loop systems can be optimized easily by adequate shaping of the frequency characteristic magnitude F jw of the associated transfer function F s output to input ratio in the frequency domain LAPLACE transform of the differential equation of the system Polder and Swandulla 2001 Using controllers with a proportional integral characteristic PI controllers it is possible to force the magnitude of the frequency characteristic to be as close as possible to one over a wide frequency range modulus hugging see Froehr 1985 Polder and Swandulla 1990 Polder 1993 Polder and Houamed 1994 Polder and Swandulla 2001 For voltage clamps this means that the controlled membrane potential rapidly reaches the desired command value The PI controller yields an instantaneous fast response to changes proportional gain while the integral part increases the accuracy by raising the gain below the corner frequency of the integrator i e for slow signals to very high values theoretically to infinite for DC signals i e an error of 096 without affecting the noise level and stability Since the integrator induces a O in the transfer function the clamp system will tend to overshoot if a step command is used Therefore the tuning of the contr
46. e overshoot of the selected optimization method 4 with the AVO method and 43 with the SO method With the AVO method the response to a command step is very fast with 4 overshoot potential output The response to a disturbance e g an activating channel is slow and has a slight deviation With the SO method the response to an unsmoothed command step is fast with 43 overshoot potential output The response to a disturbance e g an activating channel is very fast and has a slight deviation Now the steady state error must disappear Note If the SO is used an external command input filter can be used to smooth the command signal and consequently reduce the overshoot according to the requirements of the experiment see also Figure 29 version 1 8 page 52 SEC 03M User Manual 13 Literature about npi single electrode clamp amplifiers 13 1 Paper in Journals Recording Methods and Voltage Clamp Technique L Dietzel I D Bruns D Polder H R and Lux H D 1992 Voltage Clamp Recording in Kettenmann H and R Grantyn eds Practical Electrophysiological Methods Wiley Liss NY LJ Lalley P M Moschovakis A K 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 LI Misgeld U M ller W and Polder H R 1989 Potentiation and Supre
47. e value optimum AVO that provides the fastest response to a command step with very little overshoot maximum 4 or 3 the symmetrical optimum SO has the best performance compensating intrinsic disturbance signals but shows a considerable overshoot maximum 43 to a step command version 1 8 page 46 Response to a command variable step Response to a disturbance variable step Linear optimum LO aperiodic response P Controller slow response no overshoot Pus slow response large deviation Absolute value optimum AVO PI Controller fastest response 4 overshoot ee slow response slight deviation Symmetrical optimum SO Unsmoothed com mand variable PI Controller Smoothed com mand variable 1 fast response 43 overshoot slow response 8 overshoot Mr very fast response slight deviation LO Only a P Controller is used The response to a command step is slow and has no overshoot potential output The response to a disturbance e g an activating channel is slow and has a large deviation AVO A PlI Controller is used The response to a command step is very fast with 4 overshoot potential output The response to a disturbance e g an activating channel is slow and has a slight deviation SO A PlI Controller is used The response to an unsmoothed command step is fast with 43 overshoot potential output The response t
48. edance floating output Therefore the zero position the zero of the bias current i e with no input signal there is no output current of this device has to be defined Since the highly sensitive FET amplifiers in the headstage become warm from the internal heat dissipation and their characteristics are strongly temperature dependent the calibration procedure has to be done periodically by the user The tuning procedure is done in BR MODE using the BIAS control 8 Figure 8 range approx 500 pA and a resistance of a few ten MQ or a cell model It is based on Ohm s Law The voltage deflection caused by the bias current generated by the headstage on a test resistor is displayed on the digital meter The output current that is proportional to the monitored voltage deflection is tuned to zero with the HEADSTAGE BIAS CURRENT control This tuning procedure cannot be performed with an electrode since there always are unknown offset voltages involved tip potential junction potentials etc Therefore a test resistor of 10 100 MQ must be used The procedure is described step by step version 1 8 page 27 SEC 03M User Manual L1 First the headstage electrode connector must be grounded as if an electrode with a very low resistance were attached To avoid damage of the headstage amplifiers please use a 10 kQ resistor which is small enough compared to a 10 100 MQ resistor Now the offset potential of the POTENTIAL output can be tuned to zer
49. eptive field properties of motion sensitive interneurons Nat Neurosci 7 628 634 Simultaneous intracellular recordings during voltammetric measurements L Kudernatsch M Sutor B 1994 Cholinergic modulation of dopamine overflow in the rat neostriatum a fast cyclic voltammetric study in vitro Neurosci Letters 181 107 112 L Schl sser B Kudernatsch M B Sutor B and ten Bruggencate G 1995 d m and k opioid receptor agonists inhibit dopamine overflow in rat neostriatal slices Neurosci Letters 191 126 130 version 1 8 page 55 SEC 03M User Manual Intra and extracellular drug application during single electrode clamping LI Scuvee Moreau J Liegeois J F Massotte L amp Seutin V 2002 Methyl laudanosine a new pharmacological tool to investigate the function of small conductance Ca 2 activated K channels J Pharmacol Exp Ther 302 1176 1183 L Dutschmann M Bischoff M Busselberg D amp Richter W 2003 Histaminergic modulation of the intact respiratory network of adult mice Pflugers Arch 445 570 576 L Eder M Becker K Rammes G Schierloh A Azad S C Zieglgansberger W amp Dodt H U 2003 Distribution and Properties of Functional Postsynaptic Kainate Receptors on Neocortical Layer V Pyramidal Neurons J Neurosci 23 6660 6670 L Hanganu I L Kilb W and Luhmann H J 2001 Spontaneous synaptic activity of subplate neurons in neona
50. er 19 cell model 23 connections and operation 24 25 description 23 clamp performance 50 closed loop system 49 control theory 49 CUR STIM INPUT connector 16 CURRENT display 15 CURRENT OUTPUT connector 17 ELECT POTENTIAL connector 17 electrical connections 22 electrode 28 artifacts 43 capacity compensation 31 offset compensation 28 selection 28 GAIN potentiometer 19 HEADSTAGE connector 17 Headstages 19 HOLD POT mV potentiometer 18 HOLD CUR nA potentiometer 16 INTEGR ms potentiometer 18 Linear optimum 51 LO method 51 MODE OF OPERATION LEDs 15 MODE OF OPERATION switch 15 MODE SELECT connector 18 model circuit sharp electrode 9 41 model circuit suction patch electrode 9 modulus hugging 49 OFFSET potentiometer 15 operation modes testing 39 PI controllers 49 POTENTIAL RESISTANCE display 15 POTENTIAL OUTPUT connector 18 REL push button 19 sample experiments 41 sharp electrode 41 suction patch electrode 44 sealing 45 Selection of the switching frequency 31 sharp electrode 41 sharp electrodes 9 SO method 51 Speed of Response 50 suction electrodes 44 SW FREQ kHz potentiometer 16 SWITCH FREQUENCY SYNC OUT connector 17 Symmetrical optimum 51 SYNC INTERN switch 16 testing 27 Trouble Shooting 48 tuning 27 VC optimization methods 46 VOLTAGE COMMAND INPUT connector 18 version 1 8 page 68
51. f in adjusting the capacity compensation the capacity of the model cell is always present Thus you will get an approximately square shaped signal with a slight slope as shown in Figure 20 lower panel Q Increase the switching frequency to at least 15 kHz The amplitude and shape of the signal should not change considerably version 1 8 page 33 SEC 03M User Manual 200 5 capacity overcompensated 100 100 200 voltage voltage mV 300 4 400 4 500 600 T T T T T T T 1 3 3 5 4 45 5 5 5 6 6 5 7 time ms capacity undercompensated 50 100 150 4 voltage voltage mV 200 250 4 300 T T T T T T T 1 time ms 50 capacity well compensated 50 100 4 150 4 200 4 voltage voltage mV 250 4 300 4 350 400 4 450 T T T T T T T 1 3 3 5 4 4 5 5 5 5 6 6 5 7 time ms Figure 19 Tuning of the coarse capacity compensation with an electrode resistance 100 MQ in the bath Time course of the signal at ELECTRODE POTENTIAL OUTPUT is shown holding current 1 nA switching frequency 2 kHz version 1 8 page 34 SEC 03M User Manual voltage mV voltage mV voltage mV 200 4 200 4 400 600 4 800 1000 capacity overcompensated 100 150 4 200 4 250 4 300 time ms capacity undercompensated voltage
52. gh 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 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 POWER w amp EPMS E 07 A i The 19 cabinet is connected to the CHASSIS connector at the rear panel The CHASSIS is linked to protective earth as soon as the PWR 03D is connected It can be connected also to the SYSTEM GROUND SIGNAL GROUND on the rear panel of the instrument see Figure 3 S Important Always adhere to the appropriate safety measures Gnouwo _ CHASSIS Figure 3 Rear panel connectors of the EPMS E 07 2 6 Technical Data 19 rackmount cabinet for up to 7 plug in units Dimensions 3U high 1U 1 3 4 44 45 mm 254 mm deep EPMS 07 Power supply 115 230 V AC 60 50 Hz fuse 2 A 1 A slow 45 60 W EPMS E 07 External power supply for EPMS E 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 version 1 8 page 7
53. gure 11 standard headstage electrode holder optional and electrode holder adapter optional of the SEC 03M The standard headstage consists of the following elements see Figure 11 Headstage cable to amplifier Coarse capacity compensation potentiometer Holding bar GROUND Ground connector ELECTRODE SMB connector for microelectrode DRIVEN SHIELD connector cO UR WN version 1 8 page 20 SEC 03M User Manual 5 2 Low noise headstage SEC HSP The low noise low bias headstage range 12 nA see also Optional accessories in chapter 4 1 has an external capacity compensation and a BNC electrode holder connector Electrode holder External capacity compensation Headstage with mounting plate Figure 12 low noise headstage with electrode holder optional The headstage is mounted to a non conducting mounting plate 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 1 8 page 21 SEC 03M User Manual 6 Setting up the SEC 03M System The following steps should help you set up the SEC 03M correctly Always adhere to the appropriate safety measures see chapter 1 Usually the SEC 03M is shipped mounted in an EPMS 07 hous
54. ight value depending on the length of the step The same is true for the CURRENT display version 1 8 page 40 SEC 03M User Manual 9 Sample Experiments In the following the basics of a simple experiment are described either using a sharp or a suction patch electrode It is assumed that all connections are built as described in chapter 6 Before starting remove the cell model 9 1 Sample Experiment using a Sharp Microelectrode to amplifier electrode cell m d C stray X a ground C m m ground Figure 24 Model circuit for intracellular recording using a sharp electrode Cm membrane capacitance Cstray electrode stray capacitance Re electrode resistance Rm membrane resistance _ Connect the electrode cable holder to the SMB connector and the Ag AgCl pellet or the agar bridge for grounding the bath with GND at the headstage L Make the basic settings see chapter 6 Again It is of major importance that SEC 03M systems are used only in warmed up condition i e 20 to 30 minutes after turning power on L Adjust BIAS CURRENT to zero if necessary see chapter 8 1 L Reconnect the CUR STIM INPUT and or the VC COMMAND INPUT put an electrode into the electrode holder and attach it to the headstage L Immerse the electrode into the bath not in a cell as deep as necessary during the experiment Test the capability of the electrode to carry current see chapter 8 2 compensate the
55. in the amygdala involves endocannabinoid signaling J Neurosci 24 9953 9961 LJ Blank T Nijholt I Eckart K and Spiess J 2002 Priming of long term potentiation in mouse hippocampus by corticotropin releasing factor and acute stress implications for hippocampus dependent learning J Neurosci 22 3788 94 Blank T Nijholt I Grammatopoulos D K Randeva H S Hillhouse E W amp Spiess J 2003 Corticotropin releasing factor receptors couple to multiple G proteins to activate diverse intracellular signaling pathways in mouse hippocampus role in neuronal excitability and associative learning J Neurosci 23 700 707 LI DeBock F Kurz J Azad S C Parsons C G Hapfelmeier G Zieglg nsberger W amp Rammes G 2003 a2 Adrenoreceptor activation inhibits LTP and LTD in the basolateral amygdala involvement of Gio protein mediated modulation of Ca channels and inwardly rectifying K channels in LTD Eur J Neurosci 17 1411 1424 L Dodt H Eder M Frick A and Zieglgansberger W 1999 Precisely localized LTD in the neocortex revealed by infrared guided laser stimulation Science 286 110 113 L Eder M Zieglgansberger W amp Dodt H U 2002 Neocortical long term potentiation and long term depression site of expression investigated by infrared guided laser stimulation J Neurosci 22 7558 7568 L Huang K P Huang F L Jager T Li J Reymann K G amp Balschun
56. ing If a single SEC 03M module is delivered the user has to mount the module into the EPMS 07 housing This is done by performing the basic installation steps Basic installation L Turn off the EPMS 07 system L Remove two front covers from the EPMS 07 housing 1 Plug in the SEC 03M and fasten the amplifier module with four screws The screws are important not only for mechanical stability but also for proper electrical connection to the EPMS 07 housing After installation the SEC 03M is attached to the setup by assembling the electrical connections It is assumed that a cell model will be attached Q Electrical connections L Connect the headstage to the HEADSTAGE connector 15 Figure 8 at the SEC 03M _ Connect a cell model see chapter 7 if you want to test the system with a cell model _ Connect a digital analog timing unit or a stimulation device to CUR STIM INPUT for CC experiments and or to VC COMM INPUT for VC experiments _ Connect a store oscilloscope or a data recording device i e a computer with data acquisition card to the POTENTIAL OUTPUT and to the CURRENT OUTPUT triggered from the stimulation device Before using the SEC 03M always start with the basic settings to avoid oscillations Basic settings L Turn all controls to low values less than 1 and the OFFSET in the range of 5 zero position see chapter 8 3 L Set MODE OF OPERATION to BR bridge mode L Turn POWER switch
57. ing 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 tip 8 3 Offset Compensation If an electrode is immersed into the bath solution an offset voltage will appear even if no current is passed This offset 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 7 Figure 8 before recording from a cell When adjusting the OFFSET make sure that no current flows through the electrode Thus it is recommended to disconnect all inputs If a cell model is connected the OFFSET control should read a value around 5 otherwise it is likely that the headstage or the amplifier is damaged version 1 8 page 28 SEC 03M User Manual 8 4 Bridge Balance in BR mode If current is passed through an electrode the occurring voltage deflection potential drop at Re affects the recording of membrane potential in BRIDGE mode Therefore this deflection must be compensated carefully by means of the BR BAL control This control is calibrated in MO With the cell model connected or the electrode in the bath the BR BAL control is turned on clockwise until there is no artifact on the POTENTIAL
58. ipment 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 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 sen
59. 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 Each control element has a label and frequently a calibration e g CURRENT OUTPUT 10nA V version 1 8 page 14 SEC 03M User Manual 1 MODE OF OPERATION switch Voltage Clamp voltage and current clamp OFF In this position the amplifier does not apply any voltage or current to the cell The potential at the electrode tip is measured and displayed The BUZZ function is active Current Clamp Bridge Mode EXT EXTernal control if this position is selected the mode of operation VC CC can be set by a TTL pulse applied to the MODE SELECT BNC 19 TTL high VC TTL low CC MODE OF OPERATION 2 POTENTIAL RESISTANCE display LC Display for the POTENTIAL at the electrode tip in mV or the electrode RESISTANCE in MQ POTENTIAL RESISTANCE 3 MODE OF OPERATION LEDs VC OFF CC BR EXT DUAL ri LEDs indicating the active mode of operation see also 1 i If operated together with the HVC 03M module the DUAL LED indicates that the SEC 03M works in two electrode voltage clamp mode 4 MQ mV LEDs AENEA LEDs indicating that RESISTANCE MQ or POTENTIAL mV is revealed in DISPLAY 2 5 CURRENT display LC Display for the CURRENT passed through the CURRENT electrode in nA X XX nA CURRENT nA 6 BR BAL pote
60. istance of 100 MQ and a capacitance of 100 pF In the lower position CELL 2 a cell membrane with 500 MX and 22 pF is simulated 7 3 Connections and Operation Checking the configuration L Turn POWER switch of the amplifier off a For simulation of an experiment using a suction electrode _ Connect the BNC jack 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 SUBCLICK connector of the cell model to the BNC connector Pex at the headstage For headstages with BNC connector use the supplied SMB to BNC adapter For a and b L Connect GND of the cell model to GND of the headstage version 1 8 page 25 SEC 03M User Manual Leave REF untouched L Switch the CELL membrane switch see Figure 13 to the desired position L Turn all controls at the amplifier to low values less than 1 and the OFFSET in the range of 5 and the OSCILLATION SHUTOFF in the DISABLED position L Turn POWER switch of the amplifier on Now you can adjust the amplifier see below and apply test pulses to the cell model Connection to the BNC jack gives access to the cell via an electrode with 5 MQ resistance Connection to SUBCLICK adapter simulates access to the cell via an electrode with 100 MQ resistance The upper position the CELL membrane switch simulates a small cell with a resistance of 100 MQ and a capacitance of 100 pF In the lower position a
61. ld not elicit openings of voltage gated channels Q The POTENTIAL OUTPUT should show the ohmic response of the cell membrane without an artifact as illustrated in Figure 22 and Figure 23 undercompensated compensated overcompensated Ss IM 5m Figure 23 Capacity compensation of the electrode inside a cell in CC mode Current stimulus and membrane potential are shown Hint The results of this procedure look very similar to tuning of the bridge balance If the BRIDGE is balanced accurately no differences in the potential outputs should occur when switching between CC and BR mode Important Always monitor the OUTPUT from ELECT POTENTIAL using a second oscilloscope The signals must be always square If not CAPACITY COMPENSATION has to be readjusted or the switching frequency must be lowered version 1 8 page 38 SEC 03M User Manual 8 7 Testing Operation Modes Current Clamp in BR or discontinuous CC mode The cell s response to current injections is measured in the current clamp CC mode Current injection is performed by means of a current source connected to the microelectrode Basically the test procedure in BR and CC mode is the same In the following it is assumed that the basic settings and the tuning procedures are carried out as described in chapters 8 1 to 8 6 All numbers refer to Figure 8 Connect the cell model see Set the amplifier to CC or BR mode respectively using the MODE OF
62. m 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 4 8 and 10 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 manual all elements of the front panel are written in capital letters as they appear on the front panel System components that are shipped in the standard configuration are marked with Y optional components with In some chapters the user is guided step by step through a certain procedure These steps are marked with L1 Important information and special precautions are highlighted in gray Abbreviations Cm cell membrane capacitance Cstray electrode stray capacitance GND ground Imax maximal current Ra access resistance Rm cell membrane resistance Rex electrode resistance SwF switching frequency Tcm time constant of the cell membrane VreL potential drop at REL version 1 8 page 4 SEC 03M 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 Equ
63. measurements is also available see chapter 5 2 The electrode filled with electrolyte is inserted into an electrode holder optional see Figure 11 that fits into the electrode holder adapter optional see also Optional accessories in chapter 4 1 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 For optimal chlorinating of sliver wires an automated chlorinating apparatus ACL 01 is available 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 1 Figure 11 and a 12 pole connector The headstage is mounted to a holding bar that fits to most micromanipulators version 1 8 page 19 SEC 03M User Manual Note The shield of the SMC 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 0 Please turn power off when connecting or disconnecting the headstage from the HEADSTAGE connector Headstage Fi
64. mp Technik Spektrum Akad Verl Heidelberg Berlin Oxford L Ogden D C ed 1994 Microelectrode techniques The Plymouth Workshop Handbook 2nd edition The Company of Biologists Limited Cambridge L Polder H R 1984 Entwurf und Aufbau eines Ger tes zur Untersuchung der Membranleitf higkeit von Nervenzellen und deren Nichtlinearit it nach der potentiostatischen Methode Voltage Clamp Methode mittels einer Mikroelektrode Diplomarbeit Technische Universitat M nchen L Rudy B and Iverson L E eds 1992 Ion channels In Methods in enzymology Vol 207 Academic Press San Diego CA USA L Sahm M W H and Smith M W eds 1984 Optoelectronics manual 3rd edition General Electric Company Auburn NY USA L Sakmann B and Neher E eds 1995 Single channel recording 2nd Edition Plenum NY L Smith T G Jr Lecar H Redmann S J and Gage P W eds 1985 Voltage and patch clamping with microelectrodes American Physiological Society Bethesda The Williams amp Wilkins Company Baltimore L Windhorst U and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin Heidelberg New York 1999 version 1 8 page 64 SEC 03M User Manual 14 SEC 03M Specifications Technical Data MODES OF OPERATION VC Voltage Clamp mode discontinuous CC Current Clamp mode discontinuous OFF Current and Voltage Clamp disabled BR Bridge Mode conti
65. nector for application of 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 1 nA V i e an input voltage of 5 V is transformed to an output current of 5 nA The signal form remains unchanged The amplitude of the output current signal current stimulus is determined by the amplitude of the CUR STIM INPUT Two examples are given in Figure 9 In A the amplitude of the CUR STIM INPUT is 1 V that gives a current stimulus of 1 nA in B the CUR STIM INPUT amplitude is 2 V that is transformed into a current stimulus of 2 nA version 1 8 page 16 SEC 03M User Manual CURRENT STIMULUS INPUT current stimulus A I Figure 9 Input output relation using CUR STIM INPUT 1V 1 nA gt Important The current injected through the electrode is always the sum of the input signal at CUR STIM INPUT 12 and the holding current set by HOLD CUR 9 and polarity switch 13 CURRENT OUTPUT 10 nA V connector 9 BNC connector providing the CURRENT OUTPUT signal scaling 10 nA V i e 1V corresponds to 10 nA CURRENT OUTPUT 1OnAV 14 SWITCH FREQUENCY SYNC OUT connector BNC connector providing the switching frequency for synchronization of an oscilloscope triggering for tuning the capacity compensation 15 HEADSTAGE connector The HEADSTAGE cable is connected to the unit at this 12 pin connector in
66. ntiometer 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 MQ range 0 1000 MQ Control to set the output of the electrode preamplifier to zero ten turn potentiometer symmetrical i e O mV 5 on the dial range 200 mV Note Position 5 on the OFFSET control corresponds to 0 mV offset OFFSET 8 BIAS bias current potentiometer version 1 8 page 15 SEC 03M User Manual With this trim potentiometer the output current of the headstage headstage BIAS current can be tuned to zero 9 HOLD CUR nA potentiometer and polarity switch 10 turn digital control that presets a continuous command signal for CC mode HOLD current Polarity is set by switch to the left of the control 0 is off position Potentiometer for setting the switching frequency in VC or CC mode range 2 kHz to 40 kHz 11 SYNC INTERN switch a WEA TR Switch for setting the synchronization mode of the switching frequency SYNC Switching frequency is synchronized with the Master amplifier for double cell recordings SW FREQ potentiometer 10 is disabled and the switching frequency is set by the Master amplifier INTERN Switching frequency is set by SW FREQ potentiometer 10 for single cell recordings 12 CUR STIM INPUT 1 nA V connector Analog input BNC con
67. nuous CC EXT External control mode the mode of operation can be set by a TTL pulse applied to the MODE SELECT BNC MODE selection toggle switch LED indicators remote selection by TTL pulse HEADSTAGES Standard headstage Operation voltage 15 V Input resistance lt 10 Q internally adjustable Current range continuous mode 120 nA into 100 MQ CC control Coarse control for input capacity compensation Electrode connector gold plated SUBCLIC SMB with driven shield Driven shield output 2 3 mm connector range 15 V impedance 250 Q Ground 2 3 mm connector or headstage enclosure Holding bar diameter 8 mm length 100 mm Size 100x40x25 mm Headstage enclosure is connected to ground Low noise whole cell headstage SEC HSP Operation voltage 15 V Input resistance 10P Q internally adjustable Current range continuous mode 12 nA into 100 MQ external CC control Coarse control for input capacity compensation Electrode connector BNC connector with driven shield Driven shield output 1 mm connector range 15 V impedance 250 Q Ground 1 mm connector or headstage enclosure Mounting plate 60x50 mm with four 6 mm holes Headstage enclosure is connected to ground Differential input headstage SEC HSD Operation voltage 15 V Input resistance lt 10 Q internally adjustable CMR gt 90 dB Current range continuous mode 120 nA into 100 MQ CC control Coarse control for input capacity compensation
68. o Watch the left digital display and set the POTENTIAL output to zero with the OFFSET control L Next a resistance of 10 100 MQ is connected from the headstage output to ground as if an electrode with a high resistance were attached L The left digital display and the POTENTIAL OUTPUT BNC connector x10mV now show a voltage deflection which is proportional to the flowing output current bias current L This bias current can be tuned to zero with the BIAS control The current is zero when the voltage deflection is zero i e the meter shows zero L As a rule the current output CURRENT OUTPUT BNC and the CURRENT DISPLAY lower digital display should also read zero Important All headstages are equipped with very sensitive FET amplifiers which can be damaged with electrostatic charge and must therefore be handled with care This can be avoided by touching a grounded metal surface when changing or adjusting the electrodes If a headstage is not used the input should always be connected to ground either using an appropriate connector or with aluminum foil wrapped around the headstage Always turn power off when connecting or disconnecting headstages from the 19 cabinet 8 2 Electrode Selection Electrodes must be tested before use This is done by applying positive and negative current pulses Electrodes which show significant changes in resistance rectification cannot be used for intracellular recordings By increas
69. o a disturbance e g an activating channel is very fast and has a slight deviation Figure 29 tuning VC according LO AVO or SO The potential output is shown Tuning Procedure Important First use a cell model for the tuning procedure You will get familiar with the different settings and the consequences for the system without any damage to cells or electrodes LI Before you switch to VC mode tune all parameters related to the recording electrode offset capacity compensation etc in CC mode set GAIN to a low save level and turn INTEGR 20 Figure 8 to OFF L Switch to VC mode and apply identical test pulses to the cell model _ The controller is now in P mode proportional only Watch the potential output and increase the GAIN so that no overshoot appears _ Turn the integrator on INTEGR 20 Figure 8 The controller is now in PI mode proportional integral Tune the GAIN again see above L Watch the potential output and tune the time constant using INTEGR 20 Figure 8 until the overshoot of the desired tuning method appears see also Figure 29 11 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 elec
70. older and Swandulla 2001 The main considerations are Do I expect rapid or slow responses to voltage changes How much noise can I accept Is it possible to use an electrode with low resistance General The speed and accuracy of the voltage clamp control circuit is mainly determined by the question how much current can be injected and how fast can this happen Thus the more current the system can inject within a short time the better the quality of the clamp see chapter 12 2 General Considerations The key to accurate and fast recording is a properly built setup e Make sure that the internal system ground is connected to only one point on the measuring ground and originates from the potential headstage Multiple grounding should be avoided all ground points should originate from a central point The electrode used for grounding the bath should have a low resistance and must not produce offsets Use electrodes with resistances as low as possible Keep cables short Check regularly whether cables and or connections are broken Make sure that chlorinating of silver wires for the electrodes is proper and that there are no unwanted earth bridges e g salt bridges originating from experimental solutions SEC systems can be tuned according to one of three optimization methods see also chapter 12 3 1 the linear optimum LO that provides only slow response to a command step and a maximal accuracy of 90 9796 2 the absolut
71. olerance of 2 Possible reason 1 Electrode capacity is not well compensated 2 The headstage has an error Solution 1 Turn the COARSE CAPACITY COMPENSATION at the headstage and C COMP potentiometer 24 Figure 8 to the most left positions and compensate the input capacitance again see chapter 8 6 2 Contact npi SEC 03M User Manual 12 Appendix 12 1 Theory of Operation Voltage clamp instruments are closed loop control systems with two inputs external to the control loop An electronic feedback network is used to force the membrane potential of a cell to follow a voltage command setpoint input as fast and as accurately as possible in the presence of incoming disturbances disturbance input correlated with the activities of the cell e g activation of ion channels This is achieved by injecting an adequate amount of charge into the cell The current injected by the clamp instrument is a direct measure of the ionic fluxes across the membrane Ferreira et al 1985 Jack et al 1975 Ogden 1994 Smith et al 1985 The performance evaluation and optimal tuning of these systems can be done by considering only the command input since the mathematical models setpoint transfer function and the disturbance transfer function see Froehr 1985 Polder 1984 Polder and Swandulla 1990 Polder 1993 Polder and Houamed 1994 Polder and Swandulla 2001 are closely related Modern control theory provides adequate solutions for th
72. oller is performed following optimization rules which yield a well defined system performance AVO and SO see below The various components of the clamp feedback electronics can be described as first or second order delay elements with time constants in the range of microseconds The cell capacity can be treated as an integrating element with a time constant Tm which is always in the range of hundreds of milliseconds Compared to this physiological time constant the electronic time constants of the feedback loop can be considered as small and added to an equivalent time constant Te The ratio of the small and the large time constant determines the maximum gain which can be achieved without oscillations and thus the accuracy of the clamp With the gain adjusted to this level the integrator time constant and small time constant determine the speed of response of the system version 1 8 page 49 SEC 03M User Manual The clamp performance can be increased considerably if the influence of the current injecting electrode is excluded as far as possible from the clamp loop since the electrode resistance is nonlinear This is achieved if the output of the clamp system is a current source rather than a voltage source In this case the clamp transfer function has the magnitude of a conductance A V Another advantage of this arrangement is that the clamp current can be determined by a differential amplifier with no need of virtual ground
73. on Now the SEC 03M is ready for an initial check with the cell model Important All signal outputs are coarse and unfiltered directly from the headstages Thus we recommend to use always additional filters for signal conditioning version 1 8 page 22 SEC 03M User Manual 7 Passive Cell Model The SEC 03M can be ordered with a passive SEC Single Electrode Clamp amplifier cell model as an optional accessory An active cell model is also available on request for ref see Draguhn et al 1997 The cell model is designed to be used to check the function of the SEC amplifier either 1 to train personnel in using the instrument or 2 incase of trouble to check which part of the setup does not work correctly e g to find out whether the amplifier is broken or if something is wrong with the electrodes or holders etc 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 medium sized cell with 100 MQ membrane resistance and 100 pF membrane capacitance or a small cell with 500 MO and 22 pF 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 7 1 Cell Model Description SE
74. ontroller and improved electrode capacity compensation In Figure 6 S1 is shown in the current passing position when a square current is applied to the electrode When the current passes the electrode a steep voltage gradient develops at the electrode resistance Veet Figure 7 is only slightly changed due to the slow charging of the membrane capacitance The amplitude of injected current is sampled in the sample and hold amplifier SH2 Figure 6 multiplied by the fractional time of current injection within each duty cycle 1 8 to 1 2 in SEC 05 and SEC 10 1 4 in SEC 03 systems and read out as current output IsH2 in Figure 7 S1 then switches to the voltage recording position input to CCS is zero The potential at Al decays rapidly due to the fast relaxation at the compensated electrode capacity Exact capacity compensation is essential to yield an optimally flat voltage trace at the end of the version 1 8 page 11 SEC 03M User Manual current free interval when Vceen is measured see also Figure 17 The cellular membrane potential however will drop much slower due to the large uncompensated membrane capacitance The interval between two current injections must be long enough to allow for complete lt 1 settling of the electrode potential but short enough to minimize loss of charges at the cell membrane level i e minimal relaxation of Vceen At the end of the current free period a new Vm sample is taken and a new cycle begins
75. otential 1 Read the membrane potential and if necessary readjust BRIDGE BALANCE and or CAP COMP as shown in Figure 25 and Figure 26 using current stimuli that do not activate ion channels or transporters version 1 8 page 44 SEC 03M User Manual L Start the experiment in BR mode Or L Switch to discontinuous CC mode The shape of voltage and current traces should not change considerably L If you intend to work in discontinuous VC mode tune the system in CC mode see above then switch to VC mode and adjust the clamp as described in chapters 10 and 12 3 a close to the A A AS cell membrane b 10 mV gigaseal formed SS c whole cell configuration established 5 mV 25 ms Figure 28 Approaching the cell forming a gigaseal and establishing the whole cell configuration version 1 8 page 45 SEC 03M User Manual 10 Tuning VC Performance In VC mode there is the problem that the voltage step is often not strictly angular shaped But for instance increasing the clamp speed by tuning the CAPACITY COMPENSATION of the electrode or increasing GAIN also increases noise Therefore the settings of the different parameters result always in a compromise between the stability accuracy noise and control speed In this chapter we will give some practical hints how to optimize the accuracy and speed of the clamp The theoretical background of adjustment criteria is discussed in chapter 11 see also P
76. pocampal Susceptibility To Hypoxia Induced Spreading Depression By Activating BK Channels Journal of Neurophysiology 00291 LI Hoger U Torkkeli P H amp French A S 2005 Calcium concentration changes during sensory transduction in spider mechanoreceptor neurons European Journal of Neuroscience 22 3171 3178 L Hu X T Basu S amp White F J 2004 Repeated Cocaine Administration Suppresses HVA Ca2 Potentials and Enhances Activity of K Channels in Rat Nucleus Accumbens Neurons Journal of Neurophysiology 92 1597 1607 LI Jiang Z G Nuttall A L Zhao H Dai C F Guan B C Si J Q amp Yang Y Q 2005 Electrical coupling and release of K from endothelial cells co mediate ACh induced smooth muscle hyperpolarization in inner ear artery J Physiol 564 475 487 1 Juusola M and Hardie R C 2001 Light Adaptation in Drosophila Photoreceptors I Response Dynamics and Signaling Efficiency at 25 C J Gen Physiol 117 3 25 L Juusola M and Hardie R C 2001 Light Adaptation in Drosophila Photoreceptors II Rising Temperature Increases the Bandwidth of Reliable Signaling J Gen Physiol 117 27 41 LI Juusola M Niven J E amp French A S 2003 Shaker k channels contribute early nonlinear amplification to the light response in Drosophila photoreceptors J Neurophysiol 90 2014 2021 version 1 8 page 61 SEC 03M User Manual LI Kohling R Koch U R Hamann
77. power supply unit the power switch and the fuse are located at the rear of the housing 2 3 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 Front covers KKK KK 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 4 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 the one of the three connectors on the front panel of the PWR 03D to the rear panel of the respective EPMS E housing see Figure 1 Figure 3 A POWER LED indicates that the PWR 03D is powered on see Figure 1 Power switch voltage selector and fuse are located at the rear panel see Figure 2 Note The chassis of the PWR 03D is connected to protective earth and it provides protective earth to the EPMS E housing if connected version 1 8 page 6 SEC 03M User Manual e e E i OB A cE Figure 1 PWR 03D front panel view Figure 2 PWR 03D rear panel view Note This power supply is intended to be used with npi EPMS E systems only 2 5 System Grounding EPMS 07 The 19 cabinet is grounded by the power cable throu
78. r SEC 03M System The SEC 03M system is based on the well proven npi SEC technology and designed as a module for the EPMS 07 system Several combinations with other modules are possible Because this amplifier is small and handy it is possible to combine up to three synchronized SEC 03M in one 19 EPMS 07 housing e g for recording from coupled cells simultaneously For recording from one cell only it is recommended to add one or two filters to the SEC 03M module Such a recording system can further be enhanced by adding a stimulus isolator a iontophoretic amplifier or a controller for pressure ejection When using CellWorks the combination with the modular breakout box INT 20M facilitates building up a setup All signals from or to amplifiers or filters in an EPMS housing can be linked to each other and directly to the breakout box making additional BNC cabling unnecessary Two additional modules HVC 03M and PEN 03 can supplement an SEC 03M amplifier in order to allow one and two electrode voltage clamp experiments with enhanced cell penetration facilities Please ask npi for an optimal configuration according to your needs version 1 8 page 12 SEC 03M User Manual 4 SEC 03M System 4 1 SEC 03M Components The following items are shipped with the SEC 03M system Y Amplifier module for the EPMS 07 system Y Headstage Y GND and DRIVEN SHIELD 2 6 mm banana plug connectors Please open the box and inspect contents upon
79. receipt If any components appear damaged or missing please contact npi electronic or your local distributor immediately support npielectronic com Optional accessories gt Electrode holder set with one holder for sharp microelectrodes without port one suction patch electrode holder with one port and an electrode holder adapter SEC EH SET gt Active cell model SEC MODA Passive cell model SEC MOD see chapter 7 Low noise low bias current headstage SEC HSP with a reduced current range 10 headstage 1 e maximal current is 12 nA Headstage with differential input SEC HSD Headstage for extracellular measurements SEC EXT Filter for the EPMS system Data acquisition module Stimulus isolator module Iontophoresis module Pressure ejection module CellWorks hard and software 5 nl version 1 8 page 13 SEC 03M User Manual 4 2 Description of the Front Panel DO 5 Q EN npi orr CC BR vc EXT SW FREQ kHz VC COMM 20 Q HEADSTAGE Q ot CUR STIM 0 9 SYNC SELECT HOLD CUR nA TIL HOLD POT mV INTERN GVO VO E Hom POTENTIAL ELECT SWITCH CURRENT Ed OUTPUT POTENTIAL FREQUENCY OUTPUT O x10 mV V SYNC OUT 10nA V O O Q9 9 9 3 Figure 8 SEC 03M front panel view In the following description of the front panel elements each element has a number that is related to that in Figure 8 The number
80. rinciples of the dSEVC technique are described in chapter 3 2 and in Polder et al 1984 Polder amp Swandulla 2001 Looking back In the early eighties when the design of the SEC 1L system was started single electrode clamping began to gain importance beside the two classical intracellular methods bridge recording or whole cell patch clamp recording The great advantage compared to the whole cell recording method using a patch amplifier was the elimination of series resistance due to the time sharing protocol see also chapter 3 2 No current flow during voltage recording means no interference from the series resistance regardless of its value Thus voltage clamp recordings with sharp microelectrodes in deep cell layers became possible The historical weak point of this method was the low switching frequency due to the fact that stray capacities around the microelectrode could not be compensated sufficiently The SEC systems provides a solution for this problem With their improvements on capacity compensation electronics they can be used with switching frequencies of tens of kHz even with high resistance microelectrodes What are the technical principles that make possible such high switching frequencies In SEC systems a special protocol is used to rapidly compensate the microelectrode Figure 17 shows the compensation scheme of a sharp microelectrode immersed 3 mm into the cerebrospinal fluid Here the increase in speed can be seen clearly
81. riod J Neurosci 26 3721 3730 LI Sacchi O Rossi M L Canella R amp Fesce R 2006 Synaptic and somatic effects of axotomy in the intact innervated rat sympathetic neuron J Neurophysiol 95 2832 2844 1 Stett A Bucher V Burkhardt C Weber U amp Nisch W 2003 Patch clamping of primary cardiac cells with micro openings in polyimide films Med Biol Eng Comput 41 233 240 LI Salgado V L amp Saar R 2004 Desensitizing and non desensitizing subtypes of alpha bungarotoxin sensitive nicotinic acetylcholine receptors in cockroach neurons J Insect Physiol 50 867 879 L Torkkeli P H Sekizawa Ss and French A S 2001 Inactivation of voltage activated Na currents contributes to different adaptation properties of paired mechanosensory neurons J Neurophysiol 85 1595 1602 L Torkkeli P H and French A S 2001 Simulation of Different Firing Patterns in Paired Spider Mechanoreceptor Neurons The Role of Na Channel Inactivation J Neurophysiol 87 1363 1368 L Vahasoyrinki M Niven J E Hardie R C Weckstrom M amp Juusola M 2006 Robustness of neural coding in Drosophila photoreceptors in the absence of slow delayed rectifier K channels J Neurosci 26 2652 2660 J Vassanelli S and Fromherz P 1999 Transistor Probes Local Potassium Conductances in the Adhesion Region of Cultured Rat Hippocampal Neurons J Neurosci 19 16 6767 6773 LI Wang J Yeckel
82. rocedure is adapted to the experimental requirements Often the adequate tuning of a clamp system can be tested by specific test signals e g stimulus evoked signals etc version 1 8 page 51 SEC 03M User Manual Very important All parameters that influence clamp performance microelectrode offset capacity compensation etc must be optimally tuned before starting the PI controller tuning procedure The tuning procedure involves the following steps Again The main criterion of tuning is the amount of overshoot seen at the potential output Tuning of the proportional gain m d Use the command input without smoothing and apply adequate identical pulses to the cell e g small hyperpolarizing pulses The controller is in P mode proportional only Watch the potential output and rise the GAIN so that no overshoot appears LO method The response to a command step is slow and has no overshoot potential output The response to a disturbance e g synaptic input or an activating channel is slow and has a large deviation Since the integral part of the controller is disconnected a steady state error in the range of a few percents will be present Tuning the integrator d m m Reconnect the integrator to form the complete PI controller by turning the INTEGR potentiometer 20 Figure 8 on Apply adequate test pulses without filtering Adjust the integrator time constant 20 Figure 8 to achieve th
83. s sampling Figure 17 Microelectrode artifact settling Compensation of stray capacities with a SEC 05 amplifier The upper trace shows the comparison between the standard capacity compensation and the fast capacity compensation of the SEC systems After full compensation the settling time of the microelectrode is reduced to a few microseconds allowing very high switching frequencies here 40 kHz middle and lower trace The microelectrode was immersed 3 mm deep in cerebrospinal fluid Microelectrode resistance 45 MQ current 1 nA duty cycle 1 4 SwF switching frequency Original data kindly provided by Prof Diethelm W Richter Goettingen For details see Richter et al 1996 version 1 8 page 32 SEC 03M User Manual 20 mV step capacitance optimally compensated potential output from SEC during voltage step CC mode VC mode membrane potential recorded by a optimal compensation second electrode undercompensated 20 mV step capacitance undercompensated optimal compensation 2 5 mV membrane potential recorded by a second electrode 10 ms potential output from SEC during voltage step Figure 18 Errors resulting from wrong compensation of the electrode capacity Original data kindly provided by Ajay Kapur For details see Kapur et al 1998 8 6 Capacity Compensation Tuning Procedure First part basic setting In SEC systems the capacity compensation of the electrode
84. s must be taken while tuning the control circuit in order avoid stability problems _ Make sure that the amplifier works correctly with the cell model in CC mode see above Leave the membrane resistance of the cell model at 100 MQ Set the holding potential to 50 mV using the HOLD potentiometer 21 setting 050 reading 050 mV and the HOLD potential polarity switch 21 to Disable the INTEGRATOR by setting the INTEGR switch 20 to OFF Set the GAIN 23 to 0 1 Set the amplifier with the MODE OF OPERATION switch 1 to VC mode The upper display should show the holding potential of 50 mV and the lower display the holding current of 0 5 nA according to Ohm s law UCC LL version 1 8 page 39 SEC 03M User Manual Hint If the system oscillates as soon as you switch to VC mode switch back to CC mode and check the settings GAIN too high CAPACITY COMPENSATION not properly adjusted i e not overcompensated INTEGRATOR switch not to OFF 1 Apply a test pulse of 20 mV to the cell model by giving a voltage step of 0 2 V to VC COMM INPUT 18 The length of the test pulse should be at least 30 ms _ You should see a potential step of 200 mV amplitude at POTENTIAL OUTPUT 17 Note If you expect the POTENTIAL display to show the value of the potential step in this case 20 mV amplitude from a holding potential of 50 mV i e 30 mV remember that the display is rather sluggish and may not display the r
85. sors Always turn power off when adding or removing modules connecting or disconnecting sensors headstages or other components from the instrument or 19 cabinet TEMPERATURE DRIFT WARM UP TIME All analog electronic systems are sensitive to temperature changes Therefore all electronic instruments containing analog circuits should be used only in a warmed up condition i e after internal temperature has reached steady state values In most cases a warm up period of 20 30 minutes is sufficient HANDLING Please protect the device from moisture heat radiation and corrosive chemicals version 1 8 page 5 SEC 03M 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 rackmount 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 EPMS 07 Housing The following items are shipped with the EPMS 07 housing Y EPMS 07 cabinet with built in power supply X Mains cord X Fuse2A 1 A slow V Front covers In order to avoid induction of electromagnetic noise the
86. ssion by Eserine of Muscarinic Synaptic Transmission in the Guinea Pig Hipocampal Slice J Physiol 409 191 206 LI Polder H R and Swandulla D 2001 The use of control theory for the design of voltage clamp systems a simple and standardized procedure for evaluating system parameters J Neurosci Meth 109 97 109 L Richter D W Pierrefiche O Lalley P M and Polder H R 1996 Voltage clamp analysis of neurons within deep layers of the brain J Neurosci Meth 67 121 131 1 Sutor B Grimm C amp Polder H R 2003 Voltage clamp controlled current clamp recordings from neurons an electrophysiological technique enabling the detection of fast potential changes at preset holding potentials Pflugers Arch 446 133 141 Selection of switching frequency electrode time constant capacity compensation L Juusola M 1994 Measuring complex admittance and receptor current by single electrode voltage clamp J Neurosci Meth 53 1 6 L Torkkeli P H amp French A S 1994 Characterization of a transient outward current in a rapidly adapting insect mechanosensory neuron Pflugers Arch 429 72 78 L Weckstr m M Kouvaleinen E and Juusola M 1992 Measurement of cell impedance in frequency domain using discontinuous current clamp and white noise modulated current injection Pfliigers Arch 421 469 472 Dynamic Hybrid Clamp LJ Dietrich D Clusmann H and T Kral 2002 Improved hybrid clamp
87. tal rat somatosensoric cortex Cerebral Cortex 11 5 400 410 L Hanganu I L amp Luhmann H J 2004 Functional nicotinic acetylcholine receptors on subplate neurons in neonatal rat somatosensory cortex Journal of Neurophysiology 92 189 198 LI Heck N Kilb W Reiprich P Kubota H Furukawa T Fukuda A amp Luhmann H J 2006 GABA A Receptors Regulate Neocortical Neuronal Migration In Vitro and In Vivo Cereb Cortex doi 10 1093 cercor bhj135 L1 Lalley P M 1999 Microiontophoresis and Pressure Ejection in U Windhorst and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin New York Li Lalley P M A K 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 L1 Lalley P M 2003 micro Opioid receptor agonist effects on medullary respiratory neurons in the cat evidence for involvement in certain types of ventilatory disturbances Am J Physiol Regul Integr Comp Physiol 285 R1287 R1304 1 Ponimaskin E Dumuis A Gaven F Barthet G Heine M Glebov K Richter D W amp Oppermann M 2005 Palmitoylation of the 5 Hydroxytryptamine4a Receptor Regulates Receptor Phosphorylation Desensitization and beta Arrestin Mediated Endocytosis Molecular Pharmacology 67 1434 1443 L Ri
88. tion 3a t 0 5 ms duty cycle 1 4 Ucom 50 mV 0 25 ms duty cycle 1 2 12 3 Tuning Procedures for VC Controllers version 1 8 page 50 SEC 03M User Manual The initial settings using GAIN only guarantee only a stable clamp that is not very accurate and insufficiently rapid for certain types of experiments e g investigation of fast voltage activated ion channels or gating currents Thus for successful and reliable experiments it is necessary to tune the clamp loop It depends on the type of experiment to which method one should follow see below o Linear Optimum LO with this method only the proportional part GAIN of the PI controller is used The response to a command step is slow but produces no overshoot The response to a disturbance is also slow with a large deviation of the membrane potential Clamp accuracy is maximum of 90 97 Finkel and Redman 1985 Therefore this method should only be used only if it is very important to avoid overshoots of the membrane potential o Absolute Value Optimum AVO uses the PI controller and provides the fastest response to a command step with very little overshoot maximum 4 The response to a disturbance is of moderate speed and the amplitude of the deviation is only half the amplitude obtained with LO It is applied if maximum speed of response to a command step is desirable e g if large voltage activated currents are investigated o Symmetrical Optimum
89. trophysiologist in your laboratory if possible and connect a cell model to see whether the problem occurring with electrodes and real cells persists with the cell model Problem 1 After immersing an 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 current electrode Possible reason 1 The BIAS current is not adjusted Solution 1 Adjust the BIAS current according the procedure described in chapter 8 1 Problem 3 The system oscillates see also voltage clamp in chapter 8 7 Possible reason 1 The capacitance of the electrode is overcompensated Solution 1 Turn the COARSE CAPACITY COMPENSATION at the headstage and C COMP potentiometer 24 Figure 8 to the most left positions and compensate the input capacitance again see chapter 8 6 Problem 4 With the cell model connected the ReL display does not show the correct value within a t
90. tween 5 to 10 MQ which results in a time constant of 0 5 to 1 ms for a cell with a membrane capacity of 100 pF i e the membrane needs roughly a millisecond to follow the command voltage step Sharp microelectrodes usually have much larger resistances 30 to 150 MQ or even more version 1 8 page 8 SEC 03M User Manual to amplifier electrode eee C stay C i I m ground Figure 4 Model circuit for whole cell patch clamp recording using a suction electrode Cm membrane capacitance Cstray electrode stray capacitance Rer electrode resistance Rm membrane resistance to amplifier electrode C xU d stray hy ground a C 2 BE m ground Figure 5 Model circuit for intracellular recording using a sharp electrode Cm membrane capacitance Cstray electrode stray capacitance Rer electrode resistance Rm membrane resistance version 1 8 page 9 SEC 03M User Manual Besides slowing the voltage response of the cell Ra can also cause additional adverse effects such as error in potential measurement Ra together with the membrane resistance Rm forms a voltage divider see Figure 4 and Figure 5 Current flowing from the amplifier to the grounded bath of a cell preparation will cause a voltage drop at both Ra and Rm If Ra lt lt Rm the majority of the voltage drop will develop at Rm and thus reflect a true membrane potential If in an extreme case Ra Rm the membrane potential will follow only one
91. vitro I Electrophysiological evidence for two distinct EPSPs J Neurophysiol 61 607 620 Leak subtraction Li Sutor B Zieglg nsberger W 1987 A low voltage activated transient calcium current is responsible for the time dependent depolarizing inward rectification of rat neocortical neurons in vitro Pfl gers Arch 410 102 111 version 1 8 page 54 SEC 03M User Manual Double cell voltage clamp method 1 Dhein St 1998 Cardiac Gap Junction Channels Physiology Regulation Pathophysiology and Pharmacology Karger Basel Double Cell Recordings Gap Junctions L Bedner P Niessen H Odermatt B Willecke K amp Harz H 2003 A method to determine the relative CAMP permeability of connexin channels Exp Cell Res 291 25 35 L Bedner P Niessen H Odermatt B Kretz M Willecke K amp Harz H 2005 Selective permeability of different connexin channels to the second messenger cyclic AMP J Biol Chem LI Dhein S Wenig S Grover R Tudyka T Gottwald M Schaefer T amp Polontchouk L 2002 Protein kinase Calpha mediates the effect of antiarrhythmic peptide on gap junction conductance Cell Adhes Commun 8 257 264 L Dupont E Hanganu I L Kilb W Hirsch S amp Luhmann H J 2006 Rapid developmental switch in the mechanisms driving early cortical columnar networks Nature 439 79 83 LJ M ller A Lauven M Berkels R Dhein S Polder H R and Kl

OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF

Contents

Download Pdf Manuals

Related Search

Related Contents