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SEC-05X manual ver 2_0 - NPI Electronic Instruments

1. 120 nA Operation The linear mode is set through the Linear Mode switch at the front panel When the switch is set to the middle position the amplifier is in switched VC or CC or in BRIDGE mode version 2 0 page 43 SEC 05X User Manual CC Setting the switch to x1 or x10 lets the amplifier work in linear mode either without or with x10 amplification J Linear Mode x1 x10 switch 39 xl The amplifier operates in linear unswitched mode see below current and or voltage are not enhanced LINEAR MODE LED 38 lights green x10 The amplifier operates in linear electroporation mode Command voltage in VC or current stimulus in CC or BRIDGE mode are multiplied by the factor of ten This allows to apply stimuli of max 120 nA In this operation mode the lights red and the voltage output at POTENTIAL OUTPUT x10mV BNC 43 connector is set to xlmV middle In the middle position of this switch the amplifier works in switched or BRIDGE mode The LINEAR MODE LED 38 does not light Important In LINEAR MODE x10 the voltage output POTENTIAL OUTPUT x10 mV BNC connector is set to x1 mV i e 1 V is 1 V and not 100 mV as in LIN mode x1 Important The linear mode must be used with low resistance patch pipettes only Ringing can be avoided by setting the GAIN in VC mode not higher than 1 and by setting the capacity compensation of the electrode to very low values best close to zero Note Be always aware that the l
2. 2002 Dopamine activates noradrenergic receptors in the preoptic area J Neurosci 22 9320 9330 I 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 version 2 0 page 66 SEC 05X User Manual I 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 J Neurosci 25 936 940 dJ Farrow K Haag J amp Borst A 2003 Input organization of multifunctional motion sensitive neurons in the blowfly J Neurosci 23 9805 9811 I Farrow K Borst A amp Haag J 2005 Sharing receptive fields with your neighbors tuning the vertical system cells to wide field motion J Neurosci 25 3985 3993 I Gabbiani F Krapp H G Koch C amp Laurent G 2002 Multiplicative computation in a visual neuron sensitive to looming Nature 420 320 324 I 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 Ld Gingl E amp French A S 2003 Active signal conduction through the sensory dendrite of a spider mechanoreceptor neuron J Neurosci 23 609
3. Polarity is set by switch to the right of the control 0 is off position version 2 0 page 13 SEC 05X User Manual 7 POTENTIAL FILTER switch 16 position switch to set the corner frequency of the Bessel filter The setting is monitored by 42 The MODE OF OPERATION switch has 6 positions The active mode of operation is indicated by a red LED next to the operation mode name VCcCC Voltage Clamp controlled Current Clamp optional VC Voltage Clamp CC Current Clamp BR Bridge Mode EXT External Mode DHC Dynamic Hybrid Clamp optional VCcCC mode optional see chapter 8 3 Voltage Clamp controlled Current Clamp mode This mode allows accurate current clamp experiments at controlled resting potentials The time constant is set by the VCcCC TIME CONST sec switch 51 on the left of the front panel In the BR amp bridge mode the electrode resistance is compensated with the BRIDGE BALANCE control 23 The range can be set to 10 MQ 100 10 MQ max resistance 99 MQ for low resistance patch microelectrodes or to the range of 100 MQ 100 100 MQ max resistance 999 MQ for sharp microelectrodes using a toggle switch 21 External mode see also Figure 6 In external mode CC or VC mode can be selected by a TTL signal applied to the MODE SELECT TTL DHC TTL connector 41 below the MODE OF OPERATION switch TTL low CC mode TTL high VC mode DHC mode optional see chapter 8 1 Dynamic Hy
4. _I Haag J amp Borst A 2001 Recurrent Network Interactions Underlying Flow Field Selectivity of Visual Interneurons J Neurosci 21 15 5685 5692 I Haag J amp Borst A 2002 Dendro Dendritic Interactions between Motion Sensitive Large Field Neurons in the Fly J Neurosci 22 8 3227 3233 I Haag J amp Borst A 2004 Neural mechanism underlying complex receptive field properties of motion sensitive interneurons Nat Neurosci 7 628 634 L Smarandache Wellmann C Gr tsch S 2014 Mechanisms of coordination in distributed neural circuits encoding coordinating information J Neurosci 34 5627 39 I Smarandache Wellmann C Weller C Mulloney B 2014 Mechanisms of coordination in distributed neural circuits decoding and integration of coordinating information J Neurosci 34 793 803 Simultaneous intracellular recordings during voltammetric measurements I Kudernatsch M amp Sutor B 1994 Cholinergic modulation of dopamine overflow in the rat neostriatum a fast cyclic voltammetric study in vitro Neurosci Letters 181 107 112 I Schl sser B Kudernatsch M B Sutor B amp ten Bruggencate G 1995 d m and k opioid receptor agonists inhibit dopamine overflow in rat neostriatal slices Neurosci Letters 191 126 130 Intra and extracellular drug application during single electrode clamping _I Biggs JE Boakye PA Ganesan N Stemkowski PL Lantero A Ballanyi K Smith PA 2014
5. signal and consequently reduce the overshoot according to the requirements of the experiment see also Figure 25 version 2 0 page 57 SEC 05X User Manual 13 Literature 13 1 Papers in Journals and Book Chapters about npi Single electrode Clamp Amplifiers Recording methods and voltage clamp technique I Dietzel I D Bruns D Polder H R amp Lux H D 1992 Voltage Clamp Recording in Kettenmann H and R Grantyn eds Practical Electrophysiological Methods Wiley Liss NY J Lalley P M Moschovakis A K amp 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 I Misgeld U M ller W amp Polder H R 1989 Potentiation and Supression by Eserine of Muscarinic Synaptic Transmission in the Guinea Pig Hipocampal Slice J Physiol 409 191 206 I Polder H R amp Houamed K 1994 A new ultra high voltage oocyte voltage current clamp amplifier In G ttingen Neurbiology Report eds Elsner N amp H Breer Thieme Verlag Stuttgart I Polder H R amp 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 I Richter D W Pierrefiche O Lalley P M amp Polder H R 1996 Voltage clamp ana
6. 8 V 7 V connector BNC output connector monitoring the setting of POTENTIAL FILTER Hz switch 7 Resolution 1 V STEP i e 5 V indicate a filter frequency of 10 kHz 43 POTENTIAL OUTPUT x 10 mV connector version 2 0 page 18 SEC 05X User Manual BNC connector monitoring the POTENTIAL at the tip of the electrode sensitivity x10 mV Important In LINEAR MODE x10 the voltage output POTENTIAL OUTPUT x10 mV BNC connector is set to x1 mV i e 1 V is 1 V and not 100 mV as in LIN mode x1 VC COMMAND INPUT unit 44 Toggle switch to activate INPUT 45 45 47 BNC connectors for an external COMMAND INPUT in VC mode Sensitivity 10 mV 45 or 40 mV 47 48 Toggle switch to activate INPUT 47 Sometimes it 1s necessary to limit the rise time of a voltage clamp pulse especially in connection with PlI controllers to avoid overshooting of the potential 49 GROUND connector 50 AUDIO potentiometer Banana jack providing the internal GROUND not connected to PROTECTIVE EARTH Volume control for the AUDIO MONITOR The potential at the electrode is monitored by a sound The pitch of sound is related to the value of the potential 51 VCcCC TIME CONST rotary switch see 8 version 2 0 page 19 SEC 05X User Manual 3 3 Description of the Rear Panel N 4 N f f N a y N f NS A NA NJ 9 o LS Q T Fin T L fas FR J0 9 P es l 5 4 3 Figure
7. Calabrese R L 1997 Functional role of Ca2 currents in graded and spike synaptic transmission between leech heart interneurons J Neurophysiol 77 1779 1794 I M ller A et al 1997 Increase in gap junction conductance by an antiarrhythmic peptide Europ J Pharmacol 327 65 72 I M ller A et al 1997 Actions of the antiarrhythmic peptide AAP10 on intracellular coupling Naunyn Schmiedeberg s Arch Pharmacol 356 76 82 I 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 I Racke 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 Cardiovasc Res 28 201 208 LTP LDP LTD Investigations I Azad S C Monory K Marsicano G Cravatt B F Lutz B Zieglgansberger W amp Rammes G 2004 Circuitry for associative plasticity in the amygdala involves endocannabinoid signaling J Neurosci 24 9953 9961 I Blank T Nijholt I Eckart K amp 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 I Blank T Nijholt I Gramma
8. D Electronic amp Instruments for the Life Sciences pease ade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF THE SEC 05X SINGLE ELECTRODE CLAMP AMPLIFIER i i _ SEC 05X npl lina i CLAMP me CURRENT LAMP mx lt z i 5 x oe r VERSION 2 0 npi 2015 npi electronic GmbH Bauhofring 16 D 71732 Tamm Germany Phone 49 0 7141 9730230 Fax 49 0 7141 9730240 support npielectronic com http www npielectronic com SEC 05X User Manual Table of Contents ADOLFIN Manu AN 5 ores Fehcttene aie a ecciot awe decteaca ES 4 Saer ING aO aa a a eds earn enda hay aa Seal ansaid antag 5 2 MING OGU CL OMiceiacsstioussua seen a S a E A cee 6 2k Why a SINS le Electrode Clamp recensa n R 6 22 PRNCIPle OFODCI Oaa n vir niasaeseesteteaaasargenn ad ian gee eteaeeeant 8 Major advantages of the npi SEC System airnn a E E 10 Js ee De VS Ue MM ress hats tetera A coca tena ennee ce eces as 10 Jle DEC 05 X Components esses cewere sisi bee manevaiee T aereact nase 10 5 22 DESC APUN OLNE Pron Panel epena EE ERE a e ESSR EERENS 12 5 52 Description or the Rear Pane leeu octlishdetaieuecesas asennad oeceniehdetacde 20 A STICAGSLAGCS aertansisyrscaidedcueesdatapansdeei oust eveiecicileetaassduswctudadahaledave mesaauniadiddnsVecestadahere ta vactaaisineds 21 dl Standard TICACUSCAG ES i usssuscac setts A mlend ass sealeviendaoak senna ieasels ete 21 4 2 oweanoise Headstace SECHS BP osctitcsccious cis tilanebutd
9. Dhein S amp Mohr F W 2005 Morphological electrophysiological and coupling characteristics of version 2 0 page 68 SEC 05X User Manual bone marrow derived mononuclear cells an in vitro model Eur J Cardio Thoracic Surgery 27 104 110 I Reiprich P Kilb W amp Luhmann H J 2005 Neonatal NMDA Receptor Blockade Disturbs Neuronal Migration in Rat Somatosensory Cortex In Vivo Cerebr Cortex 15 349 358 d 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 LJ Ren J amp Greer J J 2006 Modulation of respiratory rhythmogenesis by chloride mediated conductances during the perinatal period J Neurosci 26 3721 3730 I 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 J Stalbovskiy AO Briant LJ Paton JF Pickering AE 2014 Mapping the cellular electrophysiology of rat sympathetic preganglionic neurones to their roles in cardiorespiratory reflex integration a whole cell recording study in situ J Physiol 592 2215 2236 I Stett A Bucher V Burkhardt C Weber U amp Nisch W 2003 Patch clamping of primary cardiac cells with
10. INTEGRATOR TIME CONST 5 Figure 5 The controller is now in PI mode proportional integral Tune the GAIN again see above Ld Watch the potential output and tune the time constant using INTEGRATOR TIME CONST 5 Figure 5 until the overshoot of the desired tuning method appears see also Figure 25 version 2 0 page 52 SEC 05X User Manual 11 Trouble Shooting In the following section some common problems possible causes and their solutions are described Important Please note that the suggestions for solving the problems are only hints and may not work In a complex setup it is impossible to analyze problems without knowing details In case of trouble always contact an experienced electrophysiologist in your laboratory if possible and connect a cell model to see whether the problem occurring with electrodes and real cells persists 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 seal
11. 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 I Beckers U Egelhaaf M Kurtz R 2009 Precise timing in fly motion vision is mediated by fast components of combined graded and spike signals Neuroscience 160 639 650 Other I 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 J Neurophysiol 92 42 51 I 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 I Balasubramanyan S Stemkowski P L Stebbing 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 I Bickmeyer U Heine M Manzke T amp Richter D W 2002 Differential modulation of Ih by 5 HT receptors in mouse CA1 hippocampal neurons Eur J Neurosci 16 209 218 I 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 I Cornil C A Balthazart J Motte P Massotte L amp Seutin V
12. 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 set point 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 the design and the optimal tuning of feedback systems Froehr 1985 Most voltage clamp systems are composed only of delay elements 1 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
13. electrode A SITO cell ma i ns ground Co R ground Figure 2 Model circuit for intracellular recording using a sharp electrode Cm membrane capacitance Cstray electrode stray capacitance Ra access resistance Rm membrane resistance Besides slowing down 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 1 and Figure 2 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 version 2 0 page 7 SEC 05X User Manual potential If in an extreme case Ra Rm the membrane potential will follow only one 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 easy to accomplish especially if recordings are 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 mo
14. 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 2 0 page 23 SEC 05X User Manual 5 Setting up the SEC 05X System The following steps should help you set up the SEC 05X correctly Always adhere to the appropriate safety measures see chapter 1 It is assumed that a cell model will be attached Electrical connections 1 Connect the headstage to the HEADSTAGE connector 28 Figure 5 at the SEC 05X 1 Connect a cell model see chapter 6 if you want to test the system with a cell model 1 Connect a digital analog timing unit or a stimulation device to one of the CURRENT STIMULUS INPUT connectors 31 33 for CC experiments and or to one of the VC COMMAND INPUT connectors 45 47 for VC experiments 1 Connect a storage oscilloscope or a data recording device 1 e a computer with data acquisition card to the POTENTIAL OUTPUT 43 and to the CURRENT OUTPUT 35 triggered from the stimulation device Before using the SEC 05X always start with the basic settings to avoid oscillations Basic settings I Turn all controls to low values less than 1 and the
15. or 50 of each switching period Potentiometer for setting the switching frequency in VC or CC mode range circa 10 Hz to 70 kHz indicated on display 40 version 2 0 page 15 SEC 05X User Manual 16 CURRENT OUTPUT SENSITIVITY V nA switch 7 position switch to set the CURRENT OUTPUT gain The setting is monitored by 36 OSCILLATION SHUT OFF unit In SHUTOFF condition the amplifier is set into CC mode and all outputs including holding current and CAPACITY COMPENSATION are disabled The inputs and the ELECTRODE RESISTANCE test are activated 17 THRESHOLD potentiometer Control to set the activation THRESHOLD of the OSCILLATION SHUTOFF circuit potentiometer linear clockwise range 0 1200 mV 18 OSCILLATION SHUTOFF LED Indicates whether the OSCILLATION SHUTOFF circuit is in SHUTOFF condition LED red or not LED green 19 DISABLED RESET switch Switch to DISABLE the OSCILLATION SHUTOFF unit or to RESET the circuit A RESET is carried out if one wants to reset the circuit after a previous SHUTOFF condition After resetting the OSCILLATION SHUT OFF unit is active again PENETRATION ELECTRODE CLEAR unit This unit is used to clean the tip of the electrode and to facilitate the puncture of the cell membrane e 20 PENETRATION push button activates the unit 22 ELECTRODE CLEAR rotary switch o BUZZ mode overcompensation of the capacity compensation effective in all six modes of operation VCcC
16. the MODE OF OPERATION rotary switch 8 and a rotary switch to set the time constants 10 100 1000 5000 and 10000 sec for the low pass filter 51 To start using the VCcCC mode the amplifier must be tuned accurately in the fast VC mode The holding potential control must be set to the desired value or a holding potential signal must be provided from an external device e g a computer This holding potential will be the preset membrane potential for the VCcCC mode Under these conditions PSPs or other changes of the membrane potential will be voltage clamped VCcCC mode is activated by switching the MODE OF OPERATION 8 rotary switch to VCcCC A red LED indicates its function Depending on the preset time constant fast changes of the membrane potential will not be voltage clamped any more This is a condition that corresponds to an accurate current clamp Fast changes of the membrane potential are monitored on the potential output slow changes are compensated by the VCcCC circuit The time constant should be selected so that the signals under investigation are not altered by the VCcCC please compare with current clamp recordings Important The average membrane potential can be changed only through the VOLTAGE COMMAND INPUT If changes are required please select a short time constant 1 or 10 s Note Please don t use DHC and VCcCC mode simultaneously Current Clamp Input The current clamp input CURRENT STIMULUS BNC connect
17. 16338 J 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 J Neurosci 25 2853 2864 _I Kapur A M Yeckel amp 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 I Single S amp Borst A 1998 Dendritic Integration and Its Role in Computing Image Velocity Science 281 1848 50 I Single S amp Borst A 2002 Different Mechanisms of Calcium Entry Within Different Dendritic Compartments J Neurophysiol 87 1616 1624 version 2 0 page 62 SEC 05X User Manual I 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 I Bollensdorff C Knopp A Biskup C Zimmer T amp Benndorf K 2004 Na current through KATP channels consequences for Na and K fluxes during early myocardial ischemia Am J Physiol Heart Circ Physiol 286 H283 H295 I Linz K amp Meyer R 1997 Modulation of L type calcium current by internal potassium in guinea pig ventricular myocytes Cardiovasc Res 33 110 122 _J Lu J Dalton IV J F Stokes D R amp
18. 17 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 2 0 page 38 SEC 05X User Manual 800 700 600 capacity overcompensated gt oT ype ieee current F voltage gt time ms 800 700 capacity undercompensated gt w A aa current voltage gt 4 60 100 time ms 800 700 4 600 capacity well compensated gt me GC current F voltage gt 40 45 50 59 60 100 time ms Figure 18 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 2 0 page 39 SEC 05X 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 4 Set SWITCHIN
19. 6 SEC 05X rear panel view the numbers are related to those in the text below 1 FUSE holder Holder for the line fuse and line voltage selector For changing the fuse or selecting line voltage open the flap using a screw driver The fuse is located below the voltage selector Pull out the holder indicated by an arrow in order to change the fuse For selecting the line voltage rotate the selector drum until the proper voltage appears in the front 2 Mains connector Plug socket for the mains power plug Important Check line voltage before connecting the TEC amplifier to power Always use a three wire line cord and a mains power plug with a protection contact connected to ground Disconnect mains power plug when replacing the fuse or changing line voltage Replace fuse only by appropriate specified type Before opening the cabinet unplug the instrument 3 PROTECTIVE EARTH connector Banana plug providing mains ground see below 4 INTERNAL GROUND connector Banana plug providing internal ground see below 5 8 MODE OF OPERATION TTL IN connectors BNC connectors for external control of MODE OF OPERATION see 8 front panel 9 ELECTRODE POTENTIAL V connector BNC connector monitoring the electrode potential i e the response of the electrode to the discontinuous current injection 10 SWITCHING FREQUENCY TTL connector BNC connector monitoring the selected switching frequency 5 V pulses used to trigger t
20. 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 I 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 I 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 I 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 1 Pomper J K 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 J Neurophysiol I 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 d Rastan A J Walther T Kostelka M Garbade J Schubert A Stein A
21. Analysis of the long term actions of gabapentin and pregabalin in dorsal root ganglia and substantia gelatinosa J Neurophysiol 112 2398 2412 d Briant LJ Stalbovskiy AO Nolan MF Champneys AR Pickering AE 2014 Increased intrinsic excitability of muscle vasoconstrictor preganglionic neurons may contribute to the elevated sympathetic activity in hypertensive rats J Neurophysiol 112 2756 78 J 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 I 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 d Hanganu I L Kilb W and Luhmann H J 2001 Spontaneous synaptic activity of subplate neurons in neonatal rat somatosensoric cortex Cerebral Cortex 11 5 400 410 I Hanganu I L amp Luhmann H J 2004 Functional nicotinic acetylcholine receptors on subplate neurons in neonatal rat somatosensory cortex J Neurophysiol 92 189 198 I 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 I Lalley P M 1999 Microiontophoresis and Pressure Ejection in U Windhorst
22. VC mode is the insufficient steepness of voltage steps In principle this problem can be alleviated by tuning the CAPACITY COMPENSATION of the electrode or increasing GAIN to increase clamp speed However these tuning procedures may also increase 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 Polder 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 magnitude of current flow during injectionand by how fast this can 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 re
23. and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin New York version 2 0 page 61 SEC 05X User Manual I Lalley P M A K Moschovakis amp 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 I 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 I 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 Mol Pharmacol 67 1434 1443 I Richter 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 I 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 I Schubert D Kotter R Zilles K Luhmann H J amp Staiger J F 2003 Cell Type Specifi
24. electrode selection 29 offset compensation 29 patch electrode 49 sharp electrode 7 46 version 2 0 ELECTRODE connector 20 EXT mode 14 FREQ MON 8 V 7 V 18 19 FUSE holder 20 GAIN potentiometer 13 GROUND connector 19 grounding 21 headstage 22 bias current adjustment 28 HEADSTAGE BIAS CURRENT 17 HEADSTAGE connector 17 Headstage types 21 low noise headstage 23 HOLDING CURRENT nA potentiometer 18 HOLDING POTENTIAL 13 INTEGRATOR TIME CONST 13 INTERNAL GROUND connector 20 linear mode 43 LINEAR MODE 18 Linear optimum 56 LO method 56 mains connector 20 MODE OF OPERATION TTL IN connectors 20 MODE OF OPERATION switch 14 MODE SELECT TTL DHC TTL connector 19 model circuit patch electrode 7 sharp electrode 7 model circuit sharp electrode 46 model ciruit SEC systems 8 modulus hugging 54 mV MQ LEDs 15 OFFSET potentiometer 17 operation modes testing 41 OSCILLATION SHUTOFF LED 16 OSCILLATION SHUT OFF unit 16 PENETRATION ELECTRODE CLEAR unit 16 PI controllers 54 POTENTIAL RESISTANCE display 15 POTENTIAL FILTER 14 POTENTIAL OUTPUT x 10 mV 19 POWER pressure switch 13 PROTECTIVE EARTH connector 20 POTENTIAL V page 74 SEC 05X User Manual rear panel view 20 REL switch 15 REMOTE TTL connector 17 RISE TIME control 19 sample experiments 46 patch electrode 49 sharp microelectrode 46 sealing 50 SO method 56 SW FREQ kHz potentiometer 15 SWITCHING FREQUENCY _ kHz display 18 SWITCHING FREQUENCY
25. 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 1 e for slow signals to very high values theoretically to infinite for DC signals i e an error of 0 without affecting the noise level and stability Since the integrator induces a zero value in the transfer function the clamp system will tend to overshoot if a step command is used Therefore the tuning of the controller is performed following optimization rules which yield a well defined system performance A VO 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 achi
26. switch CELL 1 simulates a cell with a resistance of 100 MQ and a capacitance of 100 pF In the lower position CELL 2 a cell membrane with 500 MQ and 22 pF is simulated version 2 0 page 27 SEC 05X User Manual 7 Test and Tuning Procedures Important The SEC 05X 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 5 Many of the tuning procedures can be performed analogue to those described in the manual for the SEC O5LX Important Except for headstage bias current adjustment see 7 1 all adjustments described below should be carried out every time before starting an experiment or after changing the electrode 7 1 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 impedance floating output Therefore th
27. 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 often a calibration e g CURRENT OUTPUT 10 nA V 1 POWER pressure switch Switch to turn POWER on switch pushed or off switch released VOLTAGE CLAMP unit 2 VC OUTPUT LIMITER potentiometer Under certain experimental conditions it is necessary to limit the current in the voltage clamp mode e g in order to prevent the blocking of the electrode or to protect the preparation This is possible with an electronic limiter which sets the current range between 0 100 The VC ERROR display shows the error in the VC voltage clamp mode command minus recorded potential The desired range of operation is around zero 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 OSCILLATION SHUT OFF circuit see 17 19 should be activated when setting this control 5 INTEGRATOR TIME CONST ms switch and potentiometer Potentiometer for setting the INTEGRATOR TIME CONSTANT in VC mode S g range 0 1 to 10 ms switchable to off position 10 turn digital control that presets a continuous command signal HOLDING POTENTIAL XXX mV maximum 999mV for VC
28. 2 in Figure 4 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 2 0 page 9 SEC 05X User Manual current free interval when Vecer is measured see also Figure 13 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 Vcen At the end of the current free period a new Veen sample is taken and a new cycle begins 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 recordings artefacts known to occur in other single electrode clamp systems clamping of the electrode The PI controller design increases gain to as much as 100 wA V in frequencies less than one fourth the switching frequency The result is very sensitive contro
29. 269 5369 5376 I Hayashida Y Partida G J amp Ishida A T 2004 Dissociation of retinal ganglion cells without enzymes J Neurosci Meth 137 25 35 I 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 J Neurophysiol 92 3134 3141 Coating of sharp microelectrodes for VC recordings J Juusola M Seyfarth E A amp 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 I Heimonen K Salmela I Kontiokari P amp Weckstr m M 2006 Large functional variability in cockroach photoreceptors optimization to low light levels J Neurosci 26 13454 13462 I 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 I 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 d Rabbitt R D Boyle R Holstein G R amp Highstein S M 2005 Hair cell versus afferent adaptation in the semicircular canals J Neurophysiol 93 424 436 version 2 0 pag
30. 6 6101 I Gingl E French A S Panek I Meisner S amp Torkkeli P H 2004 Dendritic excitability and localization of GABA mediated inhibition in spider mechanoreceptor neurons Eur J Neurosci 20 59 65 I 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 I 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 J Neurophysiol 93 843 852 I Hepp S Gerich F J amp Mueller M 2005 Sulfhydryl Oxidation Reduces Hippocampal Susceptibility To Hypoxia Induced Spreading Depression By Activating BK Channels J Neurophysiol 00291 I Hoger U Torkkeli P H amp French A S 2005 Calcium concentration changes during sensory transduction in spider mechanoreceptor neurons Eur J Neurosci 22 3171 3178 _I 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 J Neurophysiol 92 1597 1607 I 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
31. 81 293 version 2 0 page 64 SEC 05X User Manual _I 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 I 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 I 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 I Weiss T Veh R W amp Heinemann U 2003 Dopamine depresses cholinergic oscillatory network activity in rat hippocampus Eur J Neurosci 18 2573 2580 L Zhang Y Bonnan A Bony G Ferezou I Pietropaolo S Ginger M Sans N Rossier J Oostra B LeMasson G Frick A 2014 Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1 y mice Nat Neurosci 17 1701 1709 Perforated Patch _I 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 I Hayashida Y Partida G J amp Ishida A T 2004 Dissociation of r
32. C VC CC BR EXT DHC O Imax Imax modes Application of maximum positive or negative current to the microelectrode 100 nA standard headstage o OFF 26 DURATION potentiometer sets duration of pulse 29 REMOTE TTL connector active LOW for connection of a remote switch version 2 0 page 16 SEC 05X User Manual With this 10 turn potentiometer the output current of the headstage headstage BIAS current can be tuned to 0 see chapter 7 1 25 OFFSET potentiometer Control to compensate the electrode potential ten turn potentiometer symmetrical 1 e 0 mV 5 on the dial range 200 mV see chapter 7 3 26 DURATION potentiometer see 20 27 CAPACITY COMPENSATION potentiometer Control for the capacity compensation of the electrode ten turn potentiometer clockwise range 0 30 pF see chapter 7 6 Caution This circuit is based on a positive feedback circuit Overcompensation leads to oscillations that may damage the cell 28 HEADSTAGE connector The HEADSTAGE is connected via a flexible cable and a 12 pole connector to the mainframe see also chapter 4 Caution Please always adhere to the appropriate safety regulations see chapter 1 Please turn power off when connecting or disconnecting the potential headstage from the POTENTIAL HEADSTAGE connector 29 REMOTE TTL connector for PENETRATION unit see 20 CURRENT CLAMP unit CURRENT STIMULUS INPUT unit 30 Toggle switch to a
33. G FREQUENCY to the desired value gt 25 kHz 4 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 should not elicit openings of voltage gated channels 4 The POTENTIAL OUTPUT should show the ohmic response of the cell membrane without an artifact as illustrated in Figure 18 and Figure 19 undercompensated compensated overcompensated NE 1 n 20 ms Figure 19 Capacity compensation of the electrode inside a cell n 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 rear panel 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 2 0 page 40 SEC 05X User Manual 7 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
34. 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 Performance test with active cell model I Draguhn A Pfeiffer M Heinemann U amp 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 I DeBock F Kurz J Azad S C Parsons C G Hapfelmeier G Zieglgansberger 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 I 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 J Lavin A Nogueira 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 I Leger J F Stern E A Aertsen A amp Heck D 2004 Synaptic Integration in Rat Frontal Cortex Shaped by Network Activity J Neurophysiol 93 2
35. OFFSET 25 in the range of 5 zero position see chapter 7 3 J Set MODE OF OPERATION 8 to BR bridge mode L Turn POWER switch 1 on Now the SEC 05X is ready for an initial check with the cell model 6 Passive Cell Model The SEC 05X 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 1s wrong with the electrodes or holders etc The passive cell model consists only of passive elements 1 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 MQ 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 version 2 0 page 24 SEC 05X User Manual 6 1 Cell Model Description Figure 9 SEC MOD passive ce
36. TTL connector 20 version 2 0 Symmetrical optimum 56 testing 28 THRESHOLD potentiometer 16 Trouble Shooting 53 tuning 28 VC COMMAND INPUT connectors 19 VC ERROR display 13 VC optimization methods 51 VC OUTPUT LIMITER 13 VCcCC VCcCC mode 14 44 VCcCC TIME CONST 19 voltage clamp 41 page 75
37. Verl Heidelberg Berlin Oxford I Ogden D C ed 1994 Microelectrode techniques The Plymouth Workshop Handbook 2nd edition The Company of Biologists Limited Cambridge I Polder H R 1984 Entwurf und Aufbau eines Ger tes zur Untersuchung der Membranleitfahigkeit von Nervenzellen und deren Nichtlinearitat nach der potentiostatischen Methode Voltage Clamp Methode mittels einer Miukroelektrode Diplomarbeit Technische Universitat Miinchen I Rudy B and Iverson L E eds 1992 Ion channels In Methods in enzymology Vol 207 Academic Press San Diego CA USA LJ Sahm IM W H and Smith M W eds 1984 Optoelectronics manual 3rd edition General Electric Company Auburn NY USA J Sakmann B and Neher E eds 1995 Single channel recording 2nd Edition Plenum NY I 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 Ld Windhorst U and H Johansson eds Modern Techniques in Neuroscience Research Springer Berlin Heidelberg New York 1999 version 2 0 page 70 SEC 05X User Manual 14 SEC 05X Specifications Technical Data MODES OF OPERATION VCcCC Voltage Clamp controlled Current Clamp discontinuous VC Voltage Clamp mode discontinuous CC Current Clamp mode discontinuous BR Bridge Mode continuous CC EXT External control m
38. a R sda uilauebued dis almaaniaiens 23 Di CULM OUP ICS EC OUSA SY SUSI aan tictie st al a Beaten rsad dhcteon tact atuaane 24 Oe BAS SEV CU IMO Decre ee Su eras tenes oleae ceed eee esa ONS 24 Od Cell MOdel DESCr pllOM iss tiie eatin oc use os radon sialiaa ties saeenee ns dicwensveint ats ducannvenee sys antes 25 6 22 CONMECHONS and Operadorea a E deeds 26 Je Testand Duin Procedures manisan a a Ra aes 28 Tels Headstage Bias Current Adjustment iste cesciccieatiatinncinatiaa testis liatiwetendimaldieeats 28 Tale NCC POGUE SCLC CHON a 29 Toe OUSset COmpens aul On crease eek cee E as 29 TA Bridge Balance Gn Bik MOde yarina tin iti iaaaanm ede 30 7 5 Switching Frequency and Capacitance Compensation in switched modes a2 Criteria for the selection of the switching frequency ccceeeeeeeeccceceeeeeeeseeeeeeeeeenaas 32 7 6 Capacity Compensation Tuning Procedure eesessssseereessssssseeeresssssssseerrsssssssees 34 First part DaSiC Sete onn a A 34 Second part Hne TUNNE sesde a a a a he taaxdaaands 40 dads Tesna Opera on ModS siea r a G 4 Current Clamp in BR or discontinuous CC mde ccceeeeesecceeeeeeeeeeeseeeeeeeeeeaas 41 OS E a E A A E eset ucma aces 4 8 gt Pectal Modes OF Cpe rato cies ssw sso sestescintw a a Naied tonal e 43 8 1 Dynamic Hybrid Clamp DHC Mode optional ee ccccccccccesssseeeeeeeeeeeeeaeeeeees 43 Ge Metal DCS Chi Oana E a E 43 RDP TAU OI arash Beare eatin at
39. amp is disabled and the command potential is provided by the sample and hold electronics e g the command potential represents the last membrane potential before switching to VC mode In practice the investigator additionally needs a spike detector and a timing unit The spike detector detects an action potential and triggers with a delay set by the timing unit the transition from CC mode to VC mode 8 2 Linear Mode optional General Description The linear mode of the SEC amplifier is an unswitched operation mode of the SEC working in voltage clamp VC and current clamp CC In contrast to standard patch clamp amplifiers the electrode voltage is nevertheless measured also in VC However due to current flow during voltage measurement this measurement is distorted by the series resistance This is the reason why the linear mode should be used only for recordings where only little current flows In the linear mode the background noise of the amplifier 1s substantially reduced Therefore the linear mode is predestined for low noise recordings in VC and CC mode The linear mode allows also loose patch or macro patch recordings and can be used to approach the cell and form a gigaseal in VC mode The LIN x10 mode can be used for iontophoresis or electroporation 1 e juxtacellular non invasive filling of cells with or single cell transfection with DNA The stimulus amplitude range in CC or BRIDGE mode is also enhanced to max
40. and potential changes used to activate voltage gated currents are especially challenging because the membrane will adopt the new potential value only after its capacitance Cm in Figure and Figure 2 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 I 1 e for a given voltage difference AU the current I is inversely proportional to the resistance R In this context R is the access resistance Ra in Figure and Figure 2 between the electrode and the cell interior Ra is largely determined by certain electrode properties mainly electrode resistance and the connection between the electrode and the cell Sharp microelectrodes usually have much larger resistances 30 to 150 MQ or even more than patch clamp electrodes This makes rapid charging of the cell membrane to attain a new voltage level more difficult than in patch clamp experiments version 2 0 page 6 SEC 05X User Manual to amplifier gt R q electrode S 7 A e C stray A ground cell ground Figure 1 Model circuit for whole cell patch clamp recording Cm membrane capacitance Cstray electrode stray capacitance Ra access resistance Rm membrane resistance to cm IS
41. anges and spikes between identified visual interneurons Eur J Neurosci 34 705 716 I 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 I 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 Pfliigers Arch 446 133 141 Comparison of recording methods sharp electrode whole cell perforated patch I Jarolimek W amp Miseld U 1993 4 Aminopyridine induced synaptic GABA B currents in granule cells of the guinea pig hippocampus Pfliigers Arch 425 491 498 I Kapur A Yeckel M F Gray R amp Johnston D 1998 L Type calcium channels are required for one form of hippocampal mossy fiber LTP J Neurophysiol 79 2181 2190 I Magistretti J Mantegazza M Guatteo E amp Wanke E 1996 Action potentials recorded with patch clamp amplifiers are they genuine Trends Neurosci 19 530 534 Recordings of fast Na channels I 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
42. apacitance and 100 MQ electrode resistance A Cotray and VreL 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 Rpr electrode resistance Rm membrane resistance Tcm time constant of the cell membrane VreL potential drop at Rex see also Figure 2 version 2 0 page 48 SEC 05X User Manual 9 2 Sample Experiment using a Patch Electrode If patch electrodes are used for whole cell recordings they are usually called pipettes Thus in this subchapter pipette means patch electrode to amplifier gt R q electrode ae P a we C stray Ss ground cell ground Figure 23 Model circuit for whole cell patch clamp recording using a patch electrode Cm membrane capacitance Cstray electrode stray capacitance Ra access resistance Rm membrane resistance J 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 1 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 I Approach the cell until the voltage signal changes a Figure 24 Often you can observe a slight dent in the cell
43. apacitance undercompensated optimal compensation 2 5 mV membrane potential recorded by a 10 mv second elecirode 10 ms l 40 ms potential output from SEC during voltage step Figure 14 Errors resulting from wrong compensation of the electrode capacity Original data kindly provided by Ajay Kapur For details see Kapur et al 1998 7 6 Capacity Compensation Tuning Procedure First part basic setting In SEC systems the capacity compensation of the electrode is split into two controls the coarse control at the headstage and 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 CAPACITY COMPENSATION 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 O Set the CAPACITY COMPENSATION control at the amplifier potentiometer 27 at the front panel to a value around 2 and turn COARSE CAPACITY COMPENSATION at the headstage to the leftmost position 44 Connect the BNC connector ELECT POTENTIAL OUTPUT at the rear panel to an oscilloscope and trigger with the signal at BNC connector SWITCHING FREQUENCY at the rear panel The oscilloscope should be in external trigger mode The time base of the oscilloscope should be in the range o
44. ather 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 O5X Special precautions must be taken while tuning the control circuit in order to avoid stability problems I 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 6 setting 050 reading 050 mV and the HOLD potential polarity switch 6 to Disable the INTEGRATOR by setting the INTEGRATOR TIME CONST switch 5 to OFF Set the GAIN 4 to 0 1 Set the amplifier with the MODE OF OPERATION switch 8 to VC mode UU O Uo version 2 0 page 41 SEC 05X User Manual 1 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 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 1 e overcompensated INTEGRATOR switch not to OFF I Apply a test pulse of 20 mV to the cell m
45. bric Clamp mode see also additional sheet In DHC mode CC or VC mode is also selected by a TTL signal applied to the MODE SELECT TTL DHC TTL connector 41 below the MODE OF OPERATION switch TTL low CC mode TTL high DHC mode version 2 0 page 14 SEC 05X User Manual 9 CURRENT nA display LED Display for the CURRENT passed through the electrode in nA 10 POTENTIAL RESISTANCE display Meant matt LED Display for the POTENTIAL at the electrode tip in mV or the electrode RESISTANCE in MQ Note When measuring electrode resistance in LINEAR x10 mode the reading at the RESISTANCE display 10 must be multiplied by 10 to obtain the correct value Example display reading 01 5 MQ means a resistance of 15 MQ 11 mV MQ LEDs LEDs indicating that POTENTIAL mV or RESISTANCE MQ is revealed in display 10 12 Ret switch Toggle switch for activating the resistance measurement of the microelectrode When pushed the microelectrode resistance is measured and shown in the POTENTIAL RESISTANCE display 10 Important An accurate measurement of Ret requires that the input capacity is well compensated see also 27 and chapter 7 6 13 CURRENT FILTER Hz switch 16 position switch to set the corner frequency of the Bessel filter The setting is monitored by 37 In the discontinuous modes VC and CC modes this switch sets the ratio between current injection and potential recording mode 12 5 25
46. 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 7 1 to 7 6 All numbers refer to Figure 5 Connect the cell model see 6 2 Set the amplifier to CC or BR mode respectively using the MODE OF OPERATION switch 8 Set the membrane resistance of the cell model to 100 MQ see chapter 6 Set the holding current to 0 5 nA using the HOLD potentiometer 34 setting 50 reading 0 50 nA and the HOLD current polarity switch 34 to Make sure that the ELECTRODE RESISTANCE test Ret 12 is not active The POTENTIAL display 10 should read 50 mV according to Ohm s law The voltage at POTENTIAL OUTPUT BNC 43 should be 500 mV UU Uo Oe Remember The voltage at POTENTIAL OUTPUT is the membrane potential multiplied by 10 I Apply a test pulse of 0 5 nA to the cell model by giving a voltage step of 0 5 V to CURRENT STIMULUS INPUT 1 nA V 33 The length of the test pulse should be at least 30 ms d You should see a potential step of 500 mV amplitude at POTENTIAL OUTPUT BNC 43 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 1 e O mV remember that the display is r
47. c Circuits of Cortical Layer IV Spiny Neurons J Neurosci 23 2961 2970 I 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 Cerebr Cortex 16 223 236 I 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 Ld Weiss T Veh R W amp Heinemann U 2003 Dopamine depresses cholinergic oscillatory network activity in rat hippocampus Eur J Neurosci 18 2573 2580 Tracer injection and intracellular recording d Poulet J F amp Hedwig B 2006 The cellular basis of a corollary discharge Science 311 518 522 I 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 I Dodt H U and Zieglg nsberger W 1994 Infrared videomicroscopy a new look at neuronal structure and function Trends Neurosci 19 11 453 458 I 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 USA 101 16333
48. ckel 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 J Neurophysiol 91 1596 1607 Ld Wolfram V amp Juusola M 2004 The Impact of Rearing Conditions and Short Term Light Exposure on Signaling Performance in Drosophila Photoreceptors J Neurophysiol 92 1918 1927 version 2 0 page 69 SEC 05X User Manual 13 2 Books I Boulton A A Baker G B and Vanderwolf C H eds 1990 Neurophysiological techniques Basic methods and concepts Humana Press Clifton New Jersey I Cole K S 1968 Membranes ions and impulses University of California Press Berkely CA I Ferreira H G and Marshall M W 1985 The biophysical basis of excitability Cambridge University Press Cambridge I Frohr F 1985 Electronic control engineering made easy An introduction for beginners Siemens AG Berlin and Munich I Horowitz P and Hill W 1989 The art of electronics Cambridge University Press NY J Jack J J B Noble D and Tsien R W 1975 Electric current flow in excitable cells Claredon Press Oxford I Kettenmann H and Grantyn R eds 1992 Practical electrophysiological methods Wiley Liss New York I Neher E 1974 Elektrische Me technik in der Physiologie Springer Verlag Berlin I Numberger M and Draguhn A eds 1996 Patch Clamp Technik Spektrum Akad
49. col is used to rapidly compensate the microelectrode Figure 13 shows the compensation scheme of a sharp microelectrode immersed 3 mm into the cerebrospinal fluid Here the increase in speed can be seen clearly 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 What 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 2 0 page 32 SEC 05X User Manual fe gt BIA tg gt 2fs ie gt 2ff Sia fe upper cutoff frequency of the microelectrode fsw switching frequency of the SEVC 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 dASEVC 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 Similar settings are currently used for recordings in many labs The principle of operation in switch
50. ctivate INPUT 31 31 33 BNC connectors for an external CURRENT STIMULUS INPUT in CC mode Sensitivity 0 1 nA V 31 or 1 nA V 33 32 Toggle switch to activate INPUT 33 34 HOLDING CURRENT nA potentiometer and polarity switch 10 turn digital control that presets a continuous command signal HOLDING CURRENT X XX nA maximum 10 nA for CC Polarity is set by switch to the left of the control 0 is off position version 2 0 page 17 SEC 05X User Manual 35 CURRENT OUTPUT connector BNC connector providing the CURRENT OUTPUT signal after passing the CURRENT FILTER see 13 and the CURRENT OUTPUT SENSITIVITY switch see 16 BNC output connector monitoring the setting of CURRENT OUTPUT SENSITIVITY V uA switch 16 Resolution 1 V STEP i e 3V indicate a GAIN of 0 5 BNC output connector monitoring the setting of CURRENT FILTER Hz switch 13 Resolution 1 V STEP e 5 V indicate a filter frequency of 10 kHz Switch 39 to set the amplifier into the LINEAR mode The LINEAR mode is indicated by the LINEAR MODE LED 38 above green x1 red x10 Note When measuring in LINEAR x10 mode several changes to the scaling of displays inputs and outputs apply Please see chapter 8 2 for detailed information 40 SWITCHING FREQUENCY kHz display LED Display for the SWITCHING FREQUENCY in kHz in discontinous VC or CC mode 41 MODE SELECT TTL DHC TTL connector see 8 42 FREQ MON
51. d in the diagram in the middle it is slightly overcompensated The lower diagram shows a well balanced bridge compensated version 2 0 page 30 SEC 05X User Manual undercompensated potential mV 6 8 50 100 150 200 250 300 350 400 time ms overcom pensated potential mV 6 time ms compensated potential mV 3 fe 2 at 1 Ceremonies et ym ER ea PRN AP LIER OA EEL DEE 1 Bee 3 T T T T T T T O 50 100 150 200 250 300 350 400 time ms potential Figure 12 Tuning of the BRIDGE BALANCE using 100 MQ resistor version 2 0 page 31 SEC 05X User Manual 7 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 14 Microelectrode selection As depicted in chapter 7 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 electrod
52. dache Wellmann C Gr tsch S 2014 Mechanisms of coordination in distributed neural circuits encoding coordinating information J Neurosci 34 5627 39 I Smarandache Wellmann C Weller C Mulloney B 2014 Mechanisms of coordination in distributed neural circuits decoding and integration of coordinating information J Neurosci 34 793 803 version 2 0 page 65 SEC 05X User Manual I Stein W Eberle C C amp Hedrich U B S 2005 Motor pattern selection by nitric oxide in the stomatogastric nervous system of the crab Eur J Neurosci 21 2767 2781 _I Zhang C Guy RD Mulloney B Zhang Q Lewis TJ 2014 Neural mechanism of optimal limb coordination in crustacean swimming Proc Natl Acad Sci U S A 111 13840 13845 Recordings from plant cells I Raschke K 2003 Alternation of the slow with the quick anion conductance in whole guard cells effected by external malate Planta 217 651 657 I 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 I 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 I Beckers U Egelhaaf M
53. dard headstage 10 kHz bandwidth Rise time 10 90 lt 100 us for 50 mV step applied to a cell model Ret 5 MQ Rm 500 MQ Cm 22 pF duty cycle 25 switching frequency 30 kHz standard headstage 10 kHz bandwidth POWER REQUIREMENTS 115 230 V AC 60 W 1 25 A 0 63 A fuse SLOW DIMENSIONS 19 rack mount cabinet 19 483 mm wide 14 355 mm deep 5 25 132 5 mm high weight approx 9 kg version 2 0 page 73 SEC 05X User Manual 15 Index abbreviations 4 Absolute value optimum 56 accessories 11 AUDIO potentiometer 19 AVO method 56 basic settings 24 bias current adjustment 28 bridge balance 30 31 47 BRIDGE BALANCE potentiometer 14 BRIDGE BALANCE potentiometer and toggle switch 17 capacity compensation 34 CAPACITY COMPENSATION 17 cell model 24 connections and operation 26 description 25 clamp performance 55 closed loop system 54 control theory 54 CUR SENS MON 1 V 4 7 V 18 CURRENT nA display 15 CURRENT CLAMP unit 17 CURRENT FILTER Hz switch 15 CURRENT OUTPUT connector 18 CURRENT OUTPUT SENSITIVITY V nA switch 16 CURRENT STIMULUS INPUT 17 DHC mode 14 DISABLED RESET switch 16 dSEVC capacity compensation 36 duty cycle 55 Operation 9 operation principle 54 parameter tuning 56 principle of operation 32 33 Speed of Response 55 switching frequency 32 DURATION potentiometer 17 DUTY CYLE switch 15 electrical connections 24 electrode 29 artifacts 48 capacity compensation 32
54. de switched mode Therefore this technique is called discontinuous SEVC dSEVC 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 2 2 The discontinuous current and voltage signals are then smoothed and read at the CURRENT OUTPUT and POTENTIAL OUTPUT connectors 2 2 Principle of Operation current output record s1 K Vem D potential output el Figure 3 Model circuit of SEC systems version 2 0 page 8 SEC 05X User Manual current inject current free sample timing Vm d 2 e COS cut 1 d kh r V AT _ Voal Vout o_o Figure 4 Principle of dSEVC operation Figure 3 and Figure 4 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 an xl operational amplifier A1 in Figure 3 At this point the potential V A1 in Figure 4 is the sum of the cell s membrane potential and the voltage gradient which develops when current is in
55. e ms shown holding current 1 nA switching frequency 2 kHz version 2 0 SEC 05X User Manual 200 capacity overcompensated 200 400 voltage voltage mV 600 800 1000 i capacity undercompensated 100 150 voltage voltage mV 200 250 4 300 I I I l capacity well compensated 100 150 200 voltage voltage mV 250 300 350 4 400 time ms Figure 16 Tuning of the coarse capacity compensation Time course of the signal at ELECTRODE POTENTIAL OUTPUT rear panel is shown holding current 1 nA switching frequency 2 kHz A cell model was connected electrode resistance 100 MQ version 2 0 page 37 SEC 05X User Manual 120 capacity overcompensated 1 00 EL nl eel a a Fel Eh en ell eel el ll BL g i 1 1 potential eis current 100 150 200 20 300 voltage mV TOM ee ee E EI time ms capacity undercompensated O potential parenn current Li JAAMA W A AA N me N E Te D l voltage mV 0 l T l 4 A 4 0 50 100 150 200 250 300 120 time ms capacity well compensated 100 ee ee Ba ee ee ee ee ee ee aaa 80 60 potential current 40 voltage mV 0 50 100 150 200 250 300 time ms Figure
56. e 59 SEC 05X User Manual Ld Wolfram V amp Juusola M 2004 The Impact of Rearing Conditions and Short Term Light Exposure on Signaling Performance in Drosophila Photoreceptors J Neurophysiol 92 1918 1927 Capacitive transients in VC recordings I Sutor B amp Hablitz J J 1989 Excitatory postsynaptic potentials in rat neocortical neurons in vitro I Electrophysiological evidence for two distinct EPSPs J Neurophysiol 61 607 620 Leak subtraction _I Sutor B amp Zieglgansberger W 1987 A low voltage activated transient calcium current is responsible for the time dependent depolarizing inward rectification of rat neocortical neurons in vitro Pfliigers Arch 410 102 111 Double cell voltage clamp method J 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 JI Dhein S Wenig S Grover R Tudyka T Gottwald M Schaefer T amp Polontchouk L 2002 Protein kinase Calpha mediates the effect of antiarrhythmic pept
57. e cell model SEC MODA Passive cell model SEC MOD see chapter 6 Low noise low bias current headstage SEC HSP with a reduced current range 10 headstage 1 e maximal current is 12 nA Headstage for extracellular measurements SEC EXT Mini headstage set SEC MINI SE Filter for the EPMS system Data acquisition module Stimulus isolator module Iontophoresis module Pressure ejection module CellWorks hard and software oe od ss tbe veh Sos eb one pe dh version 2 0 page 11 SEC 05X User Manual Description of the Front Panel 3 2 FOVLSOVSH ow BOINVTVE 39048 30v1S0v3H 1NaNI SOINWIHES INAANO FNAdlNO INIAXNO FNOW O3H4 AIWULO AIWUL NIQIOH w WU A ALIAILISNAS LNdino LNIJYYNO ZH X31714 LNIXYND yoz 9 os ZH AONSNOAYS ONIHOLIAS AONVLSISAY TWILNALOd yd 39 SA DOPOA TLL 99 99 IJ oHa DA ZH 43114 YILNI LOd AEL 00 AOL 4 AS gos xe Z TLL OHA NOW OSes VIIN id LOATASSGOW ALT AS Aug x dWV19D ADVIIOA NOILVdddO 40 JGOW su 1SNOO SL oz 0 og YOIVYOJLNI L 002 oD ana 205 LSNOS JNU vo IDIA 0 Figure 5 SEC 05X front panel view page 12 version 2 0 SEC 05X User Manual In the following description of the front panel elements each element has a number that is related to that in Figure 5 The number is followed by the name in uppercase letters
58. e 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 Principles of the dSEVC technique are described in chapter 2 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 2 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 provide 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 proto
59. e 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 HEADSTAGE BIAS CURRENT control 24 Figure 5 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 1s 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 2 0 page 28 SEC 05X User Manual I 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 zero Watch the upper dig
60. easing 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 For further procedures to improve electrode performance see e g Juusola et al 1997 7 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 carefully to zero with the OFFSET control 25 Figure 5 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 compensation should be reached when the OFFSET control reads a value around 5 otherwise it is likely that the headstage or the amplifier is damaged version 2 0 page 29 SEC 05X User Manual 7 4 Bridge Balance in BR mode If current is passed through an electrode the occurring voltage deflection potential drop at Ret affects the recording of membrane potential in BRIDGE mode Therefore this deflection must be compensated carefully by means of the BRIDGE BALANCE control 21 23 This control is calibrated in MQ Wi
61. ed mode is shown below U sampling SwF npi fast compensation 10 kH ae y peg EA es 0 18 V e standara mee a compensation aoe do 1 1c HY 0 18 V U g 40 kHz 2 5 u l us sampling Figure 13 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 2 0 page 33 SEC 05X User Manual Important Artifact free dSEVC is only possible when the switching frequency and the capacity compensation can be adjusted such that the electrode potential is in a steady state during the sampling intervals see Figure 13 Microelectrode artifact settling 20 mV step capacitance optimally compensated 10 mv potential output from SEC during voltage step 40 ms Y CC mode VC mode membrane potential recorded by a second electrode optimal compensation undercompensated 20 mW step c
62. esponses of the cell to the test pulses should reflect the cell membrane resistance and time constant I Start the experiment in BR mode or I 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 overcompensated compensated undercompensated 5 mv 20 ms ee ns ee m 1 n 20 ms Figure 21 Adjustment of the bridge balance after penetrating a cell version 2 0 page 47 SEC 05X User Manual A uncompensated potential mV current nA 100 5 r 3 0 80 C Tom 7 stray i 2 5 V 60 REL ae 20 40 5 T L Cm 1 5 20 Vv a REL 1 0 0 ne Cay 20 0 5 40 T T T D 0 0 0 20 40 60 80 100 120 140 160 B time ms Cstray Compensated potential mV current nA 80 5 7 3 0 70 4 20 60 5 50 2 0 40 1 5 30 20 5 1 0 10 05 0 10 0 0 T 0 20 40 60 80 100 120 140 160 time ms Cstray and Vre compensated potential mV current nA 60 5 7 3 0 we Tom 50 2 5 40 2 0 T 30 we cm t 1 5 20 5 1 0 10 0 5 0 0 0 0 20 40 60 80 100 120 140 160 time ms potential current Figure 22 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 c
63. etinal ganglion cells without enzymes J Neurosci Meth 137 25 35 I 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 J Neurophysiol 92 3134 3141 I 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 I 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 Recordings from Crustacea J DiCaprio R A 2003 Nonspiking and Spiking Proprioceptors in the Crab Nonlinear Analysis of Nonspiking TCMRO Afferents J Neurophysiol 89 1826 1836 I DiCaprio R A 2004 Information Transfer Rate of Nonspiking Afferent Neurons in the Crab J Neurophysiol 92 302 310 _I 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 J Mulloney B Smarandache Wellmann C Weller C Hall WM DiCaprio RA 2014 Proprioceptive feedback modulates coordinating information in a system of segmentally distributed microcircuits J Neurophysiol 112 2799 809 L Smaran
64. eved 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 2 0 page 54 SEC 05X 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 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 dUm dt max Umaxr Cm Rea 1 1V s 10 3 mV ps 1a 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 d
65. f 250 us 4 Set the amplifier in CC mode and select a low switching frequency 1 to 2 kHz 41 Apply positive or negative current to the electrode using the HOLDING CURRENT control potentiometer 34 at the front panel 44 You should see a signal at the oscilloscope similar to that in Figure 15 Turn the COARSE CAPACITY COMPENSATION carefully clockwise until the signal becomes as square as possible lower diagram in Figure 15 version 2 0 page 34 SEC 05X User Manual Important If you use a model cell e g to train yourself 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 16 lower panel 4 Increase the switching frequency to at least 25 kHz The amplitude and shape of the signal should not change considerably version 2 0 page 35 200 100 100 200 voltage mV 300 400 500 600 100 150 voltage mV 200 250 300 voltage mV N O O Figure 15 Tuning of the coarse capacity compensation with an electrode resistance 100 MQ in the bath Time course of the signal at ELECTRODE POTENTIAL OUTPUT rear panel is SEC 05X User Manual capacity overcompensated voltage time ms capacity undercompensated voltage time ms capacity well compensated voltage 35 4 45 5 55 6 65 tim
66. he oscilloscope which displays the switching pulses of the ELECTRODE POTENTIAL output 9 see chapter 7 6 version 2 0 page 20 SEC 05X User Manual Grounding SEC instruments have two ground systems 1 the internal ground called INTERNAL GROUND represents the zero level for the recording electronics and is connected to the recording chamber and the BNC input output sockets 2 mains ground PROTECTIVE EARTH is connected to the 19 cabinet and through the power cable to the protection contact of the power outlet GROUND outlets are located on both headstages and on the front panel For both grounds there is an outlet on the rear panel GROUND black socket internal system ground PROTECTIVE EARTH green yellow socket mains ground 19 cabinet All SEC systems have a high quality toroid transformer to minimize stray fields In spite of this noise problems could occur if other mains operated instruments are used in the same setup The internal system ground GROUND sockets should be connected to only one point on the measuring ground of the recording chamber and should originate from the headstage The enclosure of the headstage is grounded Multiple grounding should be avoided and all ground points should originate from a central point to avoid ground loops 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 This connection can be dis
67. ide on gap junction conductance Cell Adhes Commun 8 257 264 I 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 L M ller A Lauven M Berkels R Dhein S Polder H R amp Klaus W 1999 Switched single electrode amplifiers allow precise measurement of gap junction conductance Amer J Physiol Cell 276 4 C980 C988 L 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 I 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 I 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 version 2 0 page 60 SEC 05X User Manual Simultaneous recordings with two SEC amplifiers I Haag J amp Borst A 1996 Amplification of high frequency synaptic inputs by active dendritic membrane processes Nature 379 639 641
68. inear mode introduces a series resistance error that is dependent on the magnitude of series resistance and current that flows during measurement Important The LINEAR mode x1 or x10 must not be used if two SEC amplifiers work in synchronized Master Slave configuration Important BRIDGE balance and Capacity Compensation work in LINEAR mode and can be used to minimize artifacts during electroporation 8 3 VCcCC mode optional General Description The Voltage Clamp controlled Current Clamp VCcCC or slow voltage clamp SLOW VC mode is used for performing accurate current clamp recordings in the presence of membrane potential oscillations The npi single and two electrode current and voltage clamp amplifiers npi SEC 05X 10OLX npi TEC 05X series have been modified in a way that slow membrane potential oscillations are exactly controlled by the voltage clamp module without affecting faster responses e g postsynaptic potentials PSPs and action potentials APs The response speed of the voltage clamp feed back circuit has been decreased by incorporation of electronic circuits with large time constants 1 10000 s In addition fast current stimuli e g for conductance measurements can be applied through the current clamp input CURRENT STIMULUS BNC connector Operation version 2 0 page 44 SEC 05X User Manual The VCcCC mode is controlled through two front panel elements located in the VC part of the front panel
69. ing 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 7 1 Problem 3 The system oscillates see also voltage clamp in chapter 7 7 Possible reason 1 The capacitance of the electrode is overcompensated Solution 1 Turn the COARSE CAPACITY COMPENSATION at the headstage and CAPACITY COMPENSATION potentiometer 27 Figure 5 to the leftmost positions and compensate the input capacitance again see chapter 7 6 Problem 4 With the cell model connected the Ret display does not show the correct value within a tolerance 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 CAPACITY COMPENSATION potentiometer 27 Figure 5 to the most left positions and compensate the input capacitance again see chapter 7 6 2 Contact npi version 2 0 page 53 SEC 05X 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 set point input as fast and as accurately as possible in the presence of incoming disturbances
70. input without smoothing and apply adequate identical pulses to the cell e g small hyperpolarizing pulses I 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 percent will be present Tuning the integrator I Reconnect the integrator to form the complete PI controller by turning the INTEGR potentiometer 5 Figure 5 on _I Apply adequate test pulses without filtering L Adjust the integrator time constant 5 Figure 5 to achieve the 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 1s used an external command input filter can be used to smooth the command
71. ital display and set the POTENTIAL output to zero with the OFFSET control I 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 I The upper digital display and the POTENTIAL OUTPUT BNC connector x10mV now show a voltage deflection which is proportional to the flowing output current bias current _J This bias current can be tuned to zero with the BIAS control 24 The current is zero when the voltage deflection is zero i e the meter shows zero J As a rule the current output CURRENT OUTPUT BNC 35 and the CURRENT DISPLAY 9 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 7 2 Electrode Selection Electrodes must be tested before use This is done by applying positive and negative current pulses Electrodes which show significant differences in resistance for current flow of opposite polarity rectification cannot be used for intracellular recordings By incr
72. jected 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 Veen 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 3 samples Vee and holds the value for the remainder of the cycle VsHi in Figure 4 The differential amplifier A2 in Figure 3 compares the sampled potential with the command potential Vcom 1n Figure 3 The output of this amplifier becomes the input of a controlled current source CCS in Figure 3 if the switch S1 Figure 3 is in the current passing position The gain of this current source increases as much as 100 uA V due to a PI proportional integral controller and improved electrode capacity compensation In Figure 3 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 Veen Figure 4 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 3 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 IsH
73. l coarse control for input capacity compensation Electrode connectors two gold plated SUBCLIC SMB with driven shields Driven shield output 2 3 mm connector yellow range 15 V impedance 250 Q Ground 2 3 mm connector black or headstage enclosure Holding bar diameter 8 mm length 100 mm Size 100x40x25 mm Headstage enclosure 1s connected to ground version 2 0 page 71 SEC 05X User Manual ELECTRODE PARAMETER CONTROLS Offset ten turn control 400 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 gt 100 kHz Rise time 10 90 Ret 100 MQ lt 10 us Rise time 10 90 Ret 10 MQ lt 8 US Electrode artifact decay switched modes 10 nA signal lt 500 ns Ret 10 MQ lt 1 5 us Ret 100 MQ CAPACITY COMPENSATION tuned with no overshoot ELECTRODE RESISTANCE TEST obtained by application of square current pulses 1 nA 10 mV MQ at the POTENTIAL OUTPUT display XXX MQ SWITCHED MODE PARAMETERS Switching frequency linear control ca 10 70 kHz Duty cycle 1 8 1 4 1 2 12 5 25 50 current injection CURRENT RANGE in SWITCHED MODE with duty cycle of 1 2 1 4 1 8 Standard headstage 60 nA 30 nA 15 nA SEC HSP headstage 6 nA 3 nA 1 5 nA SWITCHED MODE OUTPUTS Electrode potential max 12 V output impeda
74. l 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 accurate recordings even of fast signals 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 effects 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 SEC 05X System 3 1 SEC 05X Components The following items are shipped with the SEC 05X system Y SEC 05X amplifier Y Headstage Y GND and DRIVEN SHIELD 2 6 mm banana plug connectors Please open the box and inspect contents upon receipt If any components appear damaged or missing please contact npi electronic or your local distributor immediately support npielectronic com version 2 0 page 10 SEC 05X User Manual Optional accessories Electrode holder set with one holder for sharp microelectrodes without port one patch electrode holder with one port and an electrode holder adapter SEC EH SET gt Activ
75. ll model 1 3 connectors for the headstage 1 electrode resistance 100 MQ 3 electrode resistance 5 MQ 2 GND ground connector to be connected to GND jack of the headstage 4 CELL switch for cell membrane representing a membrane of either 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 the electrodes are connected to a 1 GQ resistor simulating the formation of a GIGASEAL with a patch electrode version 2 0 page 25 SEC 05X User Manual Whole cell Patch sharp Microelectrode Driven Shield BNC connector G c woo _ q oaow _ c SMB connector Re 5 MQ Re 100 MQ SMB BNC adapter R GROUND RSEAL 1 KQ 1GQ Figure 10 Schematic diagram of the passive cell model 6 2 Connections and Operation Connections Ld Turn POWER switch of the amplifier off a For simulation of an experiment using a patch electrode 1 Connect the BNC jack of the cell model to the BNC connector Perr of the headstage b For simulation of an experiment using a sharp electrode I 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 1 Connect GND of the cell model to GND of the headstage I Do not connect DRIVEN SHIELD version 2 0 page 26 SEC 05X User Manual Simulation of elect
76. ls and Book Chapters about npi Single electrode Clamp Amplifiers 58 DD BOOR erates races ere N A T nae 70 14 SEC 05X Specifications Techical Data asn E E 71 15 F a o Qe etertere tee eee re eer eet eee en ern ee eee aC Meee ere ere 74 version 2 0 page 3 SEC 05X User Manual About this Manual This manual should help the user to setup and use SEC systems correctly and to perform reliable experiments If you are not familiar with the use of instruments for intracellular recording of electrical signals please read the manual completely The experienced user should read at least chapters 1 3 7 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 Vv optional components with In some chapters the user is guided step by step through a certain procedure These steps are marked with WI 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 Rei electrode resistance SwF switching frequency Tcm time constant
77. lysis of neurons within deep layers of the brain J Neurosci Meth 67 121 131 Selection of switching frequency electrode time constant capacity compensation J Juusola M 1994 Measuring complex admittance and receptor current by single electrode voltage clamp J Neurosci Meth 53 1 6 _J 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 Ld Weckstr m M Kouvaleinen E amp 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 I Dietrich D Clusmann H amp T Kral 2002 Improved hybrid clamp resolution of tail currents following single action potentials J Neurosci Meth 116 55 63 Voltage clamp controlled current clamp I Peters F D Czesnik A Gennerich amp Schild D 2000 Low frequency voltage clamp recording of voltage transients at constant average command voltage J Neurosci Meth 99 129 135 I Pfeiffer K amp French A S 2009 GABAergic excitation of spider mechanoreceptors increases information capacity by increasing entropy rather than decreasing jitter J Neurosci 29 10989 10994 version 2 0 page 58 SEC 05X User Manual J Rien D Kern R amp Kurtz R 2011 Synaptic transmission of graded membrane potential ch
78. me 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 Finkel A S amp Redman S J 1985 Optimal voltage clamping with single microelectrode In Voltage and Patch Clamping with Microelectrodes eds Smith T et al Williams amp Wilkins Baltimore version 2 0 page 56 SEC 05X User Manual 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 procedure 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 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 I Use the command
79. membrane I Release pressure from the pipette Now forming of the seal is indicated by the voltage deflections getting much larger I If the seal does not form apply gentle suction to the pipette until a gigaseal is established b Figure 24 1 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 24 and you read the expected membrane potential I Read the membrane potential and if necessary readjust BRIDGE BALANCE and or CAPACITY COMPENSATION as shown in Figure 21 and Figure 22 using current stimuli that do not activate ion channels or transporters version 2 0 page 49 SEC 05X User Manual _I Start the experiment in BR mode or I 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 i J cell membrane b 10 mV gigaseal formed C whole cell configuration established 5 mV 25 ms Figure 24 Approaching the cell forming a gigaseal and establishing the whole cell configuration version 2 0 page 50 SEC 05X User Manual 10 Tuning VC Performance A notorious problem in
80. micro openings in polyimide films Med Biol Eng Comput 41 233 240 _I 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 I St ckl A Sinz F Benda J Grewe J 2014 Encoding of social signals in all three electrosensory pathways of Eigenmannia virescens J Neurophysiol 112 2076 91 I Torkkeli P H Sekizawa Ss amp French A S 2001 Inactivation of voltage activated Na currents contributes to different adaptation properties of paired mechanosensory neurons J Neurophysiol 85 1595 1602 I 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 J 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 d Walz H Grewe J Benda J Static frequency tuning accounts for changes in neural synchrony evoked by transient communication signals 2014 J Neurophysiol 112 752 65 I Wang J Ye
81. nce 250 Q Switching frequency TTL output impedance 250 Q CURRENT OUTPUT 0 1 nA V 10 nA V selectable by rotary switch output impedance 0 Q current display X XX nA POTENTIAL OUPUT Sensitivity x10 mV output impedance 0 Q potential display XXX mV CURRENT CLAMP Inputs 1 nA V 0 1 nA V input resistance gt 100 kQ HOLD X XX nA ten turn digital control with 0 switch max 10 nA BRIDGE balance ten turn digital control together with range toggle switch 10 MQ position XX X MQ 100 MQ position XXX MQ Noise BRIDGE MODE 400 UVpp 1 pApp with 100 MQ resistance at 10 kHz bandwidth VOLTAGE CLAMP Input 10 mV 40 mV input resistance gt 100 kQ HOLD XXX mV ten turn digital control with 0 switch max 1000 mV GAIN 100 nA V 10 WA V ten turn linear control Noise potential output lt 500 u Vpp current output lt 300 pApp version 2 0 page 72 RESPONSE SPEED SEC 05X User Manual measured at a cell model Ret 100 MQ Rm 100 MQ Cm 100 pF duty cycle 25 switching frequency 40 kHz standard headstage 10 kHz bandwidth potential output lt 500 uVpp current output lt 200 pApp measured at a cell model Ret 5 MQ Rm 500 MQ Cm 22 pF duty cycle 25 switching frequency 40 kHz standard headstage 10 kHz bandwidth Rise time 10 90 lt 350 us for 50 mV step applied to a cell model Ret 100 MQ Rm 100 MQ Cm 100 pF duty cycle 25 switching frequency 30 kHz stan
82. nly with an appropriate specified type STATIC ELECTRICITY Electronic equipment is sensitive to static discharges Some devices such as sensor inputs are equipped with very sensitive FET amplifiers which can be damaged by electrostatic charge and must therefore be handled with care Electrostatic discharge can be avoided by touching a grounded metal surface when changing or adjusting sensors Always turn power off when adding or removing modules connecting or disconnecting sensors headstages or other components from the instrument or 19 cabinet TEMPERATURE DRIFT WARM UP TIME All analog electronic systems are sensitive to temperature changes Therefore all electronic instruments containing analog circuits should be used only in a warmed up condition i e after internal temperature has reached steady state values In most cases a warm up period of 20 30 minutes is sufficient HANDLING Please protect the device from moisture heat radiation and corrosive chemicals version 2 0 page 5 SEC 05X User Manual 2 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 8 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 operational amplifiers and an innovative method of capacit
83. nough 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 I 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 I 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 I Muller A Kukley M Stausberg P Beck H Muller W amp Dietrich D 2005 Endogenous Ca2 Buffer Concentration and Ca2 Microdomains in Hippocampal Neurons J Neurosci 25 558 565 I 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 I 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 I Okabe
84. ode the mode of operation can be set by a TTL pulse applied to the MODE SELECT TTL BNC DHC Dynamic Hybrid Clamp discontinuous MODE selection rotary switch LED indicators remote selection by TTL 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 connector with driven shield Driven shield output 2 3 mm connector yellow range 15 V impedance 250 Q Ground 2 3 mm connector black 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 lt 10 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 red range 15 V impedance 250 Q Ground 1 mm connector black or headstage enclosure Size 71x38x20 mm Mounting plate 71x54 mm 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 contro
85. odel by giving a voltage step of 0 2 V to VC COMMAND INPUT 45 The length of the test pulse should be at least 30 ms 1 You should see a potential step of 200 mV amplitude at POTENTIAL OUTPUT 43 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 1 e 30 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 version 2 0 page 42 SEC 05X User Manual 8 Special Modes of Operation 8 1 Dynamic Hybrid Clamp DHC Mode optional General Description The Dynamic Hybrid Clamp DHC mode is used for investigations of ionic conductances in voltage clamp VC mode following action potentials in current clamp CC mode In CC mode an action potential is detected by a spike detector and triggers a timing unit This timing unit sets a delay for triggering the SEC being in CC mode The SEC switches from CC mode to VC mode with the actual membrane potential as holding potential Operation The DHC mode is set through the MODE OF OPERATION switch 8 at the front panel When the switch is set to DHC the amplifier is in CC mode and the membrane potential is fed into sample and hold electronics If a TTL pulse 5 V is applied to the MODE SELECT TTL BNC connector 41 the SEC is switched to VC mode The COMMAND INPUT for voltage cl
86. of the cell membrane VreL potential drop at Rex version 2 0 page 4 SEC 05X User Manual 1 Safety Regulations VERY IMPORTANT Instruments and components supplied by npi electronic are NOT intended for clinical use or medical purposes e g for diagnosis or treatment of humans or for any other life supporting system npi electronic disclaims any warranties for such purpose Equipment supplied by npi electronic must be operated only by selected trained and adequately instructed personnel For details please consult the GENERAL TERMS OF DELIVERY AND CONDITIONS OF BUSINESS of npi electronic D 71732 Tamm Germany 1 2 3 4 5 GENERAL This system is designed for use in scientific laboratories and must be operated only by trained staff General safety regulations for operating electrical devices should be followed AC MAINS 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 o
87. 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 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 so
88. or is connected in the VCcCC mode in a way that fast current stimuli can be applied to the electrode The condition for such recordings is a ratio of gt 1 1000 between current pulse duration and VCcCC time constant Slow long lasting current signals or DC such as the HOLDING current will be removed by the action of the VCcCC system In the fast VC mode the current clamp input is disconnected automatically In this way using the VCcCC mode fast current stimuli can be used e g to monitor conductance changes version 2 0 page 45 SEC 05X User Manual 9 Sample Experiments In the following the basics of experiments are described either using a sharp or a patch electrode It is assumed that all connections are built as described in chapter 5 Before starting remove the cell model 9 1 Sample Experiment using a Sharp Microelectrode to imine elec C stray cell OK m ground Ro Ci R ground Figure 20 Model circuit for intracellular recording using a sharp electrode Cm membrane capacitance Cstray electrode stray capacitance Ra access resistance Rm membrane resistance 1 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 Ld Make the basic settings see chapter 5 Again It is of major importance that SEC O5X systems are used only in warmed up condition 1 e 20 to 30 minutes after turning power on I Adj
89. ponse 8 overshoot Nes 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 PI 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 PI Controller is used 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 Figure 25 Tuning VC according to 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 I 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 INTEGRATOR TIME CONST 5 Figure 5 to OFF I Switch to VC mode and apply uniform test pulses to the cell model Ld The controller is now in P mode proportional only Watch the potential output and increase the GAIN so that no overshoot appears Ld Turn the integrator on
90. rode in the bath _I Set switch 4 Figure 9 to the lower position I Set switch 5 Figure 9 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 _I Set switch 4 Figure 9 to the lower position I Set switch 5 Figure 9 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 9 for an experiment with sharp electrodes or the 5 MQ electrode connector 3 Figure 9 for simulating an experiment with patch electrodes I Switch the CELL membrane switch see 4 Figure 9 to the desired position CELL 1 or CELL 2 J Turn all controls at the amplifier to low values less than 1 and the OFFSET in the range of 5 zero position and the OSCILLATION SHUTOFF in the DISABLED position _J Turn POWER switch of the amplifier on Now you can adjust the amplifier see below and apply test pulses to the cell model 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 In the upper position the CELL membrane
91. round and is linked to the bath via an agar bridge or an Ag AgCl pellet The headstage is attached to the amplifier with the headstage cable see 1 version 2 0 page 21 SEC 05X User Manual Figure 7 and a 12 pole connector The headstage is mounted to a holding bar that fits to most micromanipulators Note The shield of the SMB 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 et Headstage ea hh pt a i Figure 7 Standard headstage electrode holder optional and electrode holder adapter optional of the SEC 05X The standard headstage consists of the following elements see Figure 7 1 Headstage cable to amplifier 2 Coarse capacity compensation potentiometer 3 Holding bar 4 GROUND Ground connector 5 ELECTRODE SMB connector for microelectrode 6 DRIVEN SHIELD connector version 2 0 page 22 SEC 05X User Manual 4 2 Low noise Headstage SEC HSP The low noise low bias headstage range 12 nA see also Optional accessories in chapter 3 1 has an external capacity compensation and a BNC electrode holder connector Electrode holder External capacity compensation Headstage with mounting plate Figure 8 Low noise headstage with electrode holder
92. rupted to avoid ground loops see Ogden 1994 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 4 Headstages 4 1 Standard Headstages The SEC 05X comes with the standard headstage range 120 nA for connecting glass electrodes with high resistances or patch 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 measurements is also available see chapter 4 2 The electrode filled with electrolyte is inserted into an electrode holder optional see Figure 7 which fits into the electrode holder adapter optional see also Optional accessories in chapter 3 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 O1 is available contact npi for details GROUND provides system g
93. s rete Ae wg T nee cee cece na ne haere ince cheat 43 6 25 dinear Mode optiona oi siostisotaancia eu eta iae a E 43 General DESC PU ON eyar a a S 43 OPEO a E onaneae eee 43 Sa NCCC C mode Opona eean E E E 44 Re Me aT Sei Oi na 44 Opera a rere eer ersten a a E a men Arr er reer ren ren tre 44 C rentekhmp Mputa a EEEE EEan 45 OD SOMIPle EXPE MOMS asisetisssseaceucceiieh assacetadesiasniaesiewuatenecececestetacesuise ie ueiitanatenetecnetsietuncetec 46 9 1 Sample Experiment using a Sharp Microelectrode ccccccccsssssseseeceeeeeeaeeeseeeeeees 46 9 2 Sample Experiment using a Patch Electrode ccecccsccccccceceeseseeecceeeeeeaaaeseeeeeees 49 10 Tonne VYE PEnOnna NCO na e E E E Ree ht or erent 51 Ge Oey eo OB 67 Tg 6 fo 21 8 0 a ne em re eer oe ri ree a ne er tee ee ne eee eng et 51 TUNS Procedir ig sat a at acetic ee Accu ea aise chal eee a beets er 32 1 Trouble S OO CA ea E A 53 12 VA 0 8c 1 6 1 EU ee my Te eR a et een CMI E E n nm ae Peseta rere 54 12h OLY Ol OD era MO Mictan snmica Yeieu aaa sasleate AA 54 version 2 0 page 2 SEC 05X User Manual 12 2 Speed of Response of SEC Single Electrode Clamps cccccccccceccceeeeeesesseeeeeeeeees 55 12 3 T ning Procedures fort VC Controllers sise ita tivesundiadta tie niswhanedttntd ER E 56 Pracuieal pC AON aa ven asda a a teens hes cue bohcee 56 13 Ve Ene nE o S tectacet E E E ent steer E aust ce reee eee setae ees cee erect eet 58 13 1 Papers in Journa
94. sistance and must not produce offsets Try to keep electrode resistances as low as possible Keep cables short Check regularly whether cables and or connections are broken Make sure that silver wires for the electrodes are properly chlorinated 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 97 2 the absolute 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 2 0 page 51 SEC 05X User Manual Response to a command variable Response to a disturbance variable step Linear optimum LO aperiodic response P Controller l N pire oO goe slow response no overshoot Piscine slow response large deviation Absolute value optimum AVO PI Controller i fastest response 4 overshoot E e slow response slight deviation Symmetrical optimum SO Unsmoothed com mand variable Pl Controller l fast response 43 overshoot Smoothed com mand variable slow res
95. smooth muscle hyperpolarization in inner ear artery J Physiol 564 475 487 J 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 I Juusola M and Hardie R C 2001 Light Adaptation in Drosophila Photoreceptors I Rising Temperature Increases the Bandwidth of Reliable Signaling J Gen Physiol 117 27 41 J 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 I Kohling R Koch U R Hamann M amp Richter A 2004 Increased excitability in cortico striatal synaptic pathway in a model of paroxysmal dystonia Neurobiol Dis 16 236 245 version 2 0 page 67 SEC 05X User Manual I Kurtz R Beckers U Hundsdorfer B Egelhaaf M 2009 Mechanisms of after hyperpolarization following activation of fly visual motion sensitive neurons Eur J Neurosci 30 567 577 I 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 J Neurophysiol 94 2772 2784 I Leger J F Stern E A Aertsen A amp Heck D 2005 Synaptic integration in rat frontal cortex shaped by network activity J Neurophysiol 93 281 293 I Marsicano G Goode
96. th the cell model connected or the electrode in the bath the BRIDGE BALANCE control is turned on clockwise until there is no artifact on the POTENTIAL OUTPUT see Figure 12 I Make the basic settings at the amplifier see chapter 5 1 Connect a cell model or immerse the electrode into the bath as deep as necessary during the experiment _I Apply current pulses to the electrode either using an external stimulator via the CURRENT STIMULUS INPUT connectors 31 33 Figure 5 L Watch the POTENTIAL OUTPUT at the oscilloscope and adjust the BRIDGE BALANCE as shown in Figure 12 using the BRIDGE BALANCE potentiometer 23 Figure 5 After adjustment you should see a straight voltage trace without artifacts caused by the potential drop at Ret Important BRIDGE BALANCE must be tuned several times during an experiment since most parameters change during a recording session see Figure 11 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 compensated undercompensated 5 my 20 ms 20 ms Figure 11 Adjustment of the bridge balance after cell penetration n BR mode Figure 12 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 an
97. topoulos 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 I DeBock F Kurz J Azad S C Parsons C G Hapfelmeier G Zieglgansberger W amp Rammes G 2003 a2 Adrenoreceptor activation inhibits LTP and LTD in the basolateral amygdala involvement of Gio protein mediated modulation of Ca t channels and inwardly rectifying K channels in LTD Eur J Neurosci 17 1411 1424 I Dodt H Eder M Frick A amp Zieglgansberger W 1999 Precisely localized LTD in the neocortex revealed by infrared guided laser stimulation Science 286 110 113 I 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 77558 7568 version 2 0 page 63 SEC 05X User Manual I Huang K P Huang F L Jager T Li J Reymann K G amp Balschun D 2004 Neurogranin RC3 enhances long term potentiation and learning by promoting calcium mediated signaling J Neurosci 24 10660 10669 J 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 Lut
98. ust HEADSTAGE BIAS CURRENT to zero if necessary see chapter 7 1 I Reconnect the CURRENT STIMULUS INPUT and or the VC COMMAND INPUT put an electrode into the electrode holder and attach it to the headstage 1 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 7 2 compensate the potential offset see chapter 7 3 compensate the input capacitance see chapter 7 6 and measure the electrode resistance using switch 12 Figure 5 L Apply current steps to the CURRENT STIMULUS INPUT and adjust the BRIDGE BALANCE to suppress all artifacts on the POTENTIAL OUTPUT see chapter 7 4 1 Now the system is preadjusted for measurements in BR mode Find a cell version 2 0 page 46 SEC 05X User Manual L 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 _I Apply a BUZZ to the electrode I If you are lucky the tip of the electrode is now inside the cell J If necessary readjust BRIDGE BALANCE and or CAPACITY COMPENSATION as shown in Figure 21 and Figure 22 using current stimuli that do not activate ion channels or transporters J You can read the membrane potential and apply current pulses to the cell After penetration the voltage r
99. uty cycle 1 8 1 4 or 1 2 The maximum current is 15 nA 30 nA or 60 nA With the maximum current determined electronically by the current source for Re lt 100 MQ the maximum speed of response can be calculated as dUn dt max Imax Cm 2 For a given command step Ucom the shortest time tr to reach this level can be calculated as tr Ucom dUn dt max 3a The maximum voltage change AUmax which can be achieved in a given period of time At 1s Examples Cm 300 pF Rm 50 100 MQ a Ra 5 MQ b Ra 100 MQ 0 05mV us duty cycle 1 8 Equation 2 dUm dt max 0 1 mV us duty cycle 1 4 0 2 mV us duty cycle 1 2 1 ms duty cycle 1 8 Equation 3a t 0 5 ms duty cycle 1 4 Ucom 50 mV 0 25 ms duty cycle 1 2 version 2 0 page 55 SEC 05X User Manual 12 3 Tuning Procedures for VC Controllers The initial settings using GAIN only guarantee a stable clamp that 1s not very accurate and not fast enough 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 Which method one should follow depends on the type of experiment 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
100. y 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 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 2 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 related 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 comm
101. z B 2002 The endogenous cannabinoid system controls extinction of aversive memories Nature 418 530 533 _I 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 I 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 I Rammes G Steckler T Kresse A Schutz G Zieglgansberger W amp 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 I 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 and hippocampal plasticity J Neurosci 24 10117 10127 Ld 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 J Neurophysiol 91 1596 1607 I Yeckel

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