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Axoclamp-2A Microelectrode Clamp Theory and Operation

1. all right while other ostensibly identical holders are not Therefore holders should be tested and selected The variability of the tip potentials may in some way be related to pressure developed when the microelectrode is pressed into the holder A narrow hole drilled into the side of the holder to relieve pressure might help 7 Q Using filling solutions with low pH or adding small concentrations of polyvalent cations like 4 may reduce the size of the tip potential Purves 1981 and therefore the magnitude of any changes i gt NE Interchangeability Any unity gain headstage in the HS 2 series can be used for MEI or 2 The equipment will not be damaged if headstages are exchanged while the AXOCLAMP 2A is switched on s Cleaning To clean salt spills from the input connectors wipe with a damp cloth Avoid spilling liquids on the headstage Input Leakage Current And How To Trim It To Zero All DC connected systems suffer from the problem of drift With changes in temperature and the passage of time the DC transfer functions of all semiconductor devices can drift by many millivolts away from their initial values major worry a microelectrode system is that the cumulative effects of drift in various parts of the circuit may lead to the development of a DC offset across the resistor Ro used to set the H Asaresult an undesirable DC leakage current is injected into the microelectrode Careful consider
2. 2 55 lt 55 5 5 26 4662 36 Tip Potentials Prevention nene Enn enne auf Interchangeability MX 37 1 tote eara best aai dad 37 Input Leakage Current And How To Trim To 7 37 38 DC Removal Tet 38 Inp t R sistances cT 39 HOLDERS wena trusted aE 39 Features 39 amp dates tdi us abu rs L E Unete E E E TEE 40 lop E sites 40 m 42 X n LINK UP XN POUR 42 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page MICROELECTRODES FOR FAST 5 43 Microelectrode Capacitance ssseeeeneeeeee ee 43 Microelectrode 5 44 Filling Solutions E dis 44 Recommended Reading 44 MODEL CELLS S eese sitet o E 45 The CLAMP 1 Model PER 46
3. Page 84 SPECIFICATIONS Outputs 10 V5 and Im are membrane voltage gain 10 and current recorded by microelectrode 1 and I are the continuous microelectrode 1 voltage and current V2 and 12 are microelectrode 2 voltage and current MONITOR is the output of the anti alias filter equals the input of the sampling device Gain 10 Baseline correction circuit automatically references Monitor trace to zero volts Gain of current outputs is 10 mV nA Maximum output level is 13V Current outputs indicate the true electrode current Output Lowpass Filter Cutoff 0 1 0 3 1 3 10 30 kHz Operates on Vm and Im Single pole filter Output impedances are 500 0 10 VOLTAGE CLAMP 10 90 Rise Time The following values were measured using 10 and 1 nF in parallel to model the cell 10 MQ resistors to model the microelectrodes and a 10 mV step command Rise Time in dSEVC mode 100 ps Rise Time in TEVC mode 30 ps Gain Maximum in dSEVC mode is 100 x H nA mV Maximum in cSEVC mode is 1000 x H nA mV Maximum in TEVC mode is 10 000 mV mV Range is 300 1 logarithmic scale Output compliance 25V Phase Shift Time Constant ms 0 02 0 2 2 20 200 Lead range ms 0 0004 00 4 0 4 0 40 0 400 Lag range ms 0 00 02 002 0 2 0 20 0 200 Anti Alias Filter Time constant range 0 2 100 ys RMP Balance Indicators Equal brightness indicates voltage clamping will be at resting membrane potential
4. 40 nA Set ME2 DC Current Command 5 nA Step Command or Ext ME2 Command 45 nA Use a headstage with the appropriate H xl is generally useful LINK UP When the AXOCLAMP 2A is used in dSEVC and DCC modes the voltage across the microelectrode rapidly switches up and down To an extent which depends on proximity a second microelectrode used in the same preparation will pick up some switching noise If the second electrode is used in a continuous mode the picked i noise can usually be removed by a lowpass filter If the second electrode is also used in a discontinuous mode e g when two interconnected cells in the same preparation are placed under dSEVC the pick up from one to the other can become a problem The two switching signals mix and a beat frequency signal appears at the difference frequency When both electrodes are switched at similar frequencies the beat frequency signal appears at a low frequency which cannot be filtered out Worse in an effort to clamp out the beat signal the clamping circuit passes beat frequency currents into the cell There are two ways to avoid this problem 1 Place an extensive grounded shield between the two electrodes This method has disadvantages The shield may be physically difficult to arrange and it may introduce sufficient capacitance at the headstage inputs to worsen the electrode performances 2 Use the Clock Link Up facility provided with each AXOCLAMP 2A to synchroniz
5. 17 Suggested Use n 217 Intracellular Balancing 0 0000000 18 BUZZ c 20 Remote BUZZ ena Se e EP E eodeavasuaunteas eeu ur 20 CALIBRATION SIGNAL quse IN be wa aD 21 CAPACITANCE NEUTRALIZATION AND INPUT CAPACITANCE 21 Primary ERR TERN TION NRI 21 SECOND AY esti seas pee Pee idi VAR equ 21 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC vi Page CREAR qu 22 COMMAND GENERATORS Leid m ter 22 Step Command Generator oe saws ee bube ne Sein Ve kN UN Fu E Qu YEN SR VN Rn XE 22 DE Command Generators 50e deo eire ed acts ba s 23 External Command Inputs 114 cole oeste eise ra ra eae eo ta ano o ne aUa a Sa E Fe dE 23 lanae ron E 23 CURRENT MEASUREMENT iet eene ei yste ce EUER NNI RATS 25 ip ee MODE RC Boxe bd MN 25 GROUNDING AND HUM eene 31 HEADSTAGES c URP MR DELETE OT 32 Meaning Of END XOU UN 32 Which Headstage To Use MMC ER 32 Capacitance Neutralization 34 Headstage Connectors NERIS UR 2 34 Tip Potentials 2 5 5
6. FIGURE 1 TWO PHASE MICROELECTRODE DECAY AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 16 DETAILED GUIDE TO OPERATIONS BATH PROBE Bath Potential Measurement In certain experimental circumstances it is desirable to make all voltage measurements relative to a reference point in the bathing solution rather than relative to ground These conditions may include precision measurements during changes of temperature or ion content of the saline or cases of restricted access from the extracellular space to the grounding point All measurements are normally made relative to the system ground However if an HS 2 headstage is plugged into the rear panel Bath Probe connector measurements by both MEI and ME2 are automatically made relative to the potential recorded by this headstage For optimum voltage clamp performance the bandwidth of the bath potential is limited to 300 Hz before it is subtracted from the potentials recorded by MEI and ME2 see Finkel amp Gage 1985 The bath microelectrode cannot be used for current passing The full bandwidth voltage recorded by the bath microelectrode is available at the VgATH OUT connector If there is no HS 2 headstage plugged into the Bath Probe connector a reference potential from an external amplifier can be subtracted by connecting a reference source to the IN connector Grounding It is quite uncommon to measure the bath potential Irrespec
7. However from a noise point of view the capacitance remains and causes an increase in high frequency electrode noise To optimize the response speed of low and medium impedance electrodes up to approx 300 0 when a driven shield is used the shield of headstages with H x0 1 and larger is driven from the capacitance neutralization circuit To optimize the headstage input resistance when a driven shield is used the shield of headstages with H x0 01 and smaller is driven from the output of the unity gain buffer inside the headstage If a shielded cable is being used and unusual electrode responses are observed try disconnecting the shield No shield drive is provided on the HS 2 x1MG x10MG and the HS 4 x1MG these headstages the case is grounded This is because they are primarily used for current passing in a two electrode voltage clamp TEVC In TEVC it is essential to minimize the amount of coupling capacitance between the voltage recording electrode and the current passing electrode This coupling can be minimized most conveniently if the case of the current passing headstage is grounded 3 Ground Output Connector The yellow ground socket of the MEI headstage is used for grounds the preparation Using this connection as the preparation ground minimizes hum Tip Potentials Detection During the passage of current the tip potentials of many electrodes change Changes in tip potential are indistinguishable from the mem
8. Input Leakage Current Adjustable to zero Input Leakage Current vs Temperature 10 fA C x0 0001 100 fA C x0 01 x0 1 1 H xl 10 pA 10 Offset Neutralization Range 0 500 mV Ten turn potentiometers Capacitance Neutralization Range HS 2L 1 to 7 pF HS 2M 2 to 20 pF HS 2MG 4 to 18 pF These values apply when headstage is used with microelectrode 1 amplifier With microelectrode 2 amplifier the maximum values are doubled T Buzz 2 Instantly increases capacitance neutralization to cause oscillation Operated by spring loaded pushbutton switch footswitch or by Remote Buzz Duration control latter allows the Buzz duration to be set in the range 1 50 ms Buzz Duration 1 50 ms when activated by the remote buzz control Clear Forces I max through the microelectrode Spring loaded toggle switch Bridge Balance Range 10 H MQ turn in Bridge mode 1 in cSEVC mode Ten turn potentiometers Digital Voltmeters Voltage Displays 1999mV Separate meters for Vi and V2 Current Displays 19 99 pA x 0 0001 1 999 nA 01 19 99 nA x 0 1 199 9 nA x1 1 999 mA x 10 Scaling i is set by miniature panel switches Display selections are Ih and Ivin Currents exceeding the digital display range can be measured on the BNC outputs AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC
9. MONITOR EEIE 48 NOISE IN DCC AND dSEVC 49 OFFSET CONTROLS Lii onc chi Fest leute vsu ds 50 OUTPUT BIETER iore eui tg tp d Maas ese ten ia THER 50 High Order Lowpass Filters For Low Noise Recordings 51 Rise Time Of High Order Filters 51 Note On Ultimate Rise OUTPUT IMPEDANCE AND PROTECTION 51 PANEL METERS 422000 8 51 Maa GR oerte p dr a a let ines 51 mV eesseeseesostocasosseceoosseecesoseteeesosssssosssessesoccssecessecsesosceeeseccosceseooooeee 52 dc HDD 52 PHASE s as eas UE ated ERE xu AR 52 POWER SUPPLY GLITCHES 53 POWER SUPPLY VOLTAGE SELECTION amp FUSE CHANGING 54 NOVERIT CR 54 Changing FUS coari saadanne RAEE eva aep kara ee Ee eae a Den basia 54 REMOTE teen darted 55 RMP BALANCE 5 57 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Vil viii Page SERIES RESISTANCE Ye
10. input socket to directly accept standard microelectrode holders 2 mm plugs are supplied with the headstages to connect wire leads if used In any mode level and step commands can be generated internally Level Commands one for voltage clamp and one for each microelectrode for a total of 3 are set on precision ten turn potentiometers The Step Command is set on a 3 4 digit thumbwheel switch and can be directed to either one of the microelectrodes or to the voltage clamp An indicator light for each microelectrode illuminates during current commands External command sources can be used simultaneously with the internal command sources Two dedicated Digital Voltmeters continuously display the microelectrode voltages while a third displays the current in the selected microelectrode or in a virtual ground circuit if used Front panel controls for each microelectrode and the virtual ground set the scaling of the current meter to suit the gain of your headstage A Digital Counter lets you know precisely what sampling rate you are using during single electrode voltage clamp or discontinuous current clamp Offset Controls are provided for each microelectrode and a variable Lowpass Filter is provided for the microelectrode used in single electrode voltage clamping As well an internally generated Calibration Signal can be superimposed onto each of the outputs Hence the output signals in many cases can be wholly conditioned within the A
11. rate at which the membrane capacitance can be charged Solutions There no perfect solutions for these two problems As always the best solution is to take steps to minimize R in the first place These include 1 In cSEVC arrange for the electrode resistance Re to be extremely small since Re Q In dSEVC eliminate R altogether by watching the Monitor output to make sure the transient decays completely before the next sample is taken 3 In TEVC keep the resistance of the grounding path low This includes the solution resistance the grounding electrode and a virtual ground if used Usually R is only a problem in TEVC if very large currents are passed AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 58 DETAILED GUIDE TO OPERATIONS Secondary solutions are the following 1 In cSEVC electronically subtract from the command voltage a voltage equal to the product of the membrane current and the presumed series resistance see cSEVC section This technique can begin to cope with both the error and the limited charging rate Unfortunately the compensation can rarely exceed 7096 before introducing instabilities Q The high frequency current noise is proportional to the gain but the clamp speed is limited by Since the membrane potential step response time is slow anyway it turns out that using some phase lag can significantly lessen the current noise without w
12. this extra compensation causes the headstage to go into high frequency oscillation If this is done while the tip of the microelectrode is pressing against the cell membrane the oscillation will often help the microelectrode impale the cell The exact mechanism is unknown but it may involve attraction between the charge at the tip of the microelectrode and bound charges on the inside of the membrane To use the FS 3 footswitches plug them into the 4 mm jacks on the back panel The red jack labelled 5 V is shared by the two footswitches There is one violet jack for each of the two footswitches Precise control of the duration of Buzz can be achieved by connecting a pulse generator to pin 15 of the Remote connector see Remote Section For some small cells a long duration Buzz can be deadly this case it may be helpful to use an external pulse generator connected to pin 15 of the Remote connector to gate the Buzz oscillation so that it is on for just a few milliseconds The hand held Remote Buzz generator see next page is designed to allow you to conveniently generate Buzz durations between 1 and 50 ms It is difficult to interpret the operation of Buzz by watching the 10 Vm trace This is because the x10 gain and lowpass filter on the 10 Vm output strongly affect the amount of headstage oscillation seen As a quick guide if the 10 Vm trace is unaffected then Buzz did not succeed so increase the Capacitance Neutralization setting If the
13. 10 V4 trace jumps then Buzz was successful The Buzz oscillation can be clearly observed on the V Cont output If a grounded shield adds a lot of capacitance to ME2 the Capacitance Neutralization range may be insufficient when an HS 2L headstage is used In this case it will be necessary to use an HS 2M headstage see Headstage Section Remote Buzz Installation Plug the Buzz control into the rear panel remot connector of the Axoclamp If you want to use some of the pins on the rear panel remote connector to remotely select the operating mode or activate the Clear currents you will have to remove the cover from the plug on the Remote Buzz unit and solder your ipis to the appropriate spare pins on this plug Use Set the desired Buzz duration on the Duration control of the Remote Buzz unit Press the button corresponding to the electrode you want to buzz Note that the Duration control is shared by the two electrodes For Buzz durations greater than 50 ms use the buttons on the front panel of the Axoclamp Neither the buttons on the front panel of the Axoclamp nor the footswitches use the duration set on the Remote Buzz unit AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS 21 CALIBRATION SIGNAL A calibration signal can be simultaneou on all of the voltage and current except for the duration of a HIGH signal on the Cal Ac
14. 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 71 Fig 13 An example of an incorrectly set dSEVC i e a false clamp a cell model Rm Cm Gain H sampling rate recording bandwidth and A A Filter were the same as in Fig 12 Phase Shift was at maximum lag Time Constant was 2 ms Capacitance Neutralization was under utilized A Top trace Membrane current Cal 1nA 4ms Note that this membrane current is much smaller and slower than the one in Fig 12 Middle trace Sampled membrane potential available at the 10 V output 10 4 ms Lower trace True membrane potential recorded by an independent electrode 10 mV 4 ms Note that the two voltage records are not the same The sampled membrane potential includes a large error due to the voltage across the microelectrode at the sampling time see B below B Multiple sweeps of the Monitor waveform This photo was taken with the cell held at 50 mV from rest This was done because when the cell was held at rest with the considerable amount of phase lag used the noise current pulses were too small to allow the adequacy of the decay to be seen The voltage transients did not decay to a horizontal baseline at the times the samples were taken arrow therefore the samples included some of the IR voltage drop across the microelectrode AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT
15. Blank Stops clamp from responding to new inputs for the duration of a HIGH control signal on the BLANK ACTIVATE input Used to reject stimulus artifacts Series Resistance Compensation Operates in cSEVC mode Value set on Bridge potentiometer External input at 100 mV V can be used in TEVC mode AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC SP CIFICATIONS Page 85 SAMPLING CIRCUIT Rate 500 Hz to 50 kHz Operates in DCC and dSEVC modes only Counter 3 digit display to 99 9 kHz max Blanked in continuous modes Sample Clock Logic level trigger output at the sampling rate Sample Acquisition Time lps 10 V step to 0 1 INTERNAL COMMANDS Note Commands from all sources sum linearly Voltage Clamp Step Command 1199 9 mV Set on thumbwheel switch Activated by a HIGH control signal on the STEP ACTIVATE input or by a front panel Switch Voltage Clamp Holding Position Range 200 mV transmembrane potential Ten turn potentiometer Current Clamp Step Command 199 9x H nA Set on thumbwheel as above DC Current Command d 100 x H nA Ten turn potentiometers EXTERNAL COMMANDS Sensitivities Ext VC command 20 mV V Series resistance compensation 100 mV V Ext ME 1 microelectrode 1 command 10 x H nA V Ext ME 2 microelectrode 2 command 10 x H nA V Input Impedance 22 kQ Max Input Voltages 30 V for voltage clamp commands 60 V for current clam
16. FEBRUARY 1990 AXON INSTRUMENTS INC Page 72 DETAILED GUIDE TO OPERATIONS 1 nA 10 mV 20 mV 4 ms 40 ps FIGURE 13 INCORRECTLY SET UP dSEVC i e FALSE CLAMP IN A CELL MODEL AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 73 Fig 14 Current and potential during dSEVC using the same suction electrode model used in Fig 10 Differences were Gain was 0 7 nA mV Phase Shift and A A Filter were both off Sampling rate was Upper trace Lower trace Noise 50 kHz a Membrane current Sampled membrane potential recorded from the 10 V4 output The current noise in the 3 kHz bandwidth was 80 pA peak to peak nA 10 mV ms FIGURE 14 CURRENT AND POTENTIAL RECORDING DURING dSEVC USING A SUCTION ELECTRODE MODEL AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 74 DETAILED GUIDE TO OPERATIONS Minimum Sampling Rate and Maximum Gain If the sampling rate is too slow the dSEVC will become unstable This is because the long current passing period allows the the membrane potential to charge right through and past the desired potential before the clamp has an opportunity to take a new sample of potential and adjust the current accordingly The larger the cell membrane capacitance Cm the slower the sampling rate fs which can be used for a given average gain The stability criteri
17. FEBRUARY 1990 AXON INSTRUMENTS INC HS gt MEL 145 gt MEL Sturt Zad Ma de segel 0 Ym c B 7 SLY Uy mede gt Sam ple elk Tg S4 Rech Yo 520 KH S poed See ee povus Please fold out so that you may refer to this page while reading the manual Medel CUI m DEC f Bridas ad Aa P9 p a Sha Av 23 REAR PANEL DR HARVEY J KARTEN MOM UNIVERSITY OF CALIFORNIA SAN OR 0409 ba JOELA 22093 0608
18. OPERATIONS 2 Shield Drive Connector The Shield drive is connected to the gold plated qian socket and to the case of the HS 2 x1L x0 1L x0 01M and x0 0001M headstages This drive is protected against continuous short circuits however for best frequency response the case must not be grounded In general this necessitates using an insulated mounting for the headstage such as the rod provided The shield connection is provided primarily for driving the shield of microelectrodes prepared for deep immersion see notes in Microelectrodes for Fast Settling section It may also be used for driving metal objects near the input or even the hutch in which the preparation is housed It can be used for driving the shield of a coaxial cable used to connect the microelectrode to the input although it is not recommended that the microelectrode be connected in this way If not used the shield socket is simply left unconnected There are two reasons why we do not recommend using shielded cable to connect the microelectrode to the headstage 1 The leakage resistance of shielded cable can degrade the input resistance when used with ion sensitive and other high impedance electrodes If shielded cable is used it should have teflon as the insulating material separating the shield and the inner conductor 2 Shielded cables add significant input capacitance The shield drive circuit mostly removes the effect of this capacitance on electrode response speed
19. a millivolt or less of Vm This requires a period of up to 9 electrodes time constants Te At the end of the voltage recording period a new sample of Vm is taken and new cycle begins The actual voltage used for recording purposes is the sampled voltage As illustrated in the bottom timing waveform the sampled value of Vm moves in small increments about the average value The difference between Vm ave and is the steady state error of the clamp which arises because the gain Gr of the CCS is finite The error becomes progressively smaller as Gr is increased The duty cycle used in dSEVC is current passing for 30 of each cycle and voltage recording for 70 of each cycle The cycling rate sample rate must be chosen so that there are ten or more cycles per membrane time constant This enables the membrane capacitance to smooth the membrane voltage response to the current pulses When optimally adjusted the circuit enables the first steady state measurement of voltage to be taken 1 to 2 cycle periods after the onset of a membrane conductance change or a change in the command voltage AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page65 Two controls not shown in the Figure are the Anti Alias Filter and the Phase control The Anti Alias Filter is a single pole filter between the output of the unity gain headstage A1 and SH1 see Fig 7 It can be used
20. and DCC modes use the largest feasible H value This is because the current passing response is best with low values of Ro A limitation on using large values is that as Ro becomes smaller the input leakage current of the headstage becomes more prone to increase with time and temperature see Input Leakage Current discussion later in this section A further limitation on using large values is that if see Table 1 is dese than the microelectrode resistance Re the high frequency noise is worse The H sets the current passing sensitivity in Bridge and DCC modes and the Gain in SEVC modes Hence it should be chosen for sensitivities suitable for your cell These sensitivities are listed in Table 1 above If Re gt gt Ri a Smaller value should be favored AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 34 DETAILED GUIDE TO OPERATIONS Capacitance Neutralization Range HS 2 Series headstages are available with L or M suffixes representing low and medium ranges respectively of Capacitance Neutralization see Table 3 The increased Capacitance Neutralization range is a trade off against microelectrode noise The HS 2L has the lowest noise close to the theoretically predicted thermal noise of the electrode The HS 2M has about 25 extra noise TABLE 3 HS 2L HS 2M Cap Neut Range in MEI Slot 1 to 4 pF 2 to 12 pF in ME2 Slot to 11 pF 2 to 35 pF Headstage Connectors Ther
21. current pulse either because of redistribution of ions in the tip or because of the distributed nature of the capacitance through the wall of the microelectrode see Fig 1 The final stages of the decay may often be so slow that additional delay introduced by a filter used to prevent aliasing an Anti Alias Filter causes insignificant worsening of the dynamic response The Anti Alias Filter can be used by the experimenter to trade off the noise recorded in DCC and dSEVC modes against the dynamic response That is increasing the Anti Alias Filter setting decreases the noise but can lead to instability in dSEVC and can make it more difficult in DCC to balance the response to a current step The Anti Alias Filter also has an effect in the continuous modes It acts as a lowpass filter on the voltage recorded by ME1 Thus the effects during TEVC and cSEVC the same as those due to a slow voltage recording microelectrode Using the Anti Alias Filter in these modes is not recommended Rotating the Anti Alias Filter control clockwise logarithmically increases the amount of filtering In the fully counterclockwise position the filter time constant is 0 2 us and the discontinuous clamp responses unaffected the fully clockwise position the filter time constant is 100 ys There is a maximal reduction in noise but the maximum sampling rate which can be achieved is severely limited to about 1 kHz or less Fast phase J Slow phase
22. is the small horizontal board in the left side of the instrument 4 For 115 V operation slide S2 to the left towards the label 115 For 230 V operation slide S2 to the right towards the label 230 5 Replace the top cover 6 Re connect the power cord 7 Mark the new operating voltage on the identification plate on the rear of the instrument Changing The Fuse The AXOCLAMP 2A uses a 0 5 A 250 V slow acting 5 x 20 mm fuse on both voltage ranges Before changing the fuse investigate the reason for its failure To change the fuse 1 Disconnect the power cord 2 Use a screwdriver or something similar to lever out the fuse holder 3 Discard the fuse from the active slot i e the slot which places the fuse closest to the inside of the instrument 4 Shift the spare fuse from the spare slot i e the slot which places the fuse towards the outside of the instrument to the active slot 5 Re connect the power cord AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 55 REMOTE Some of the front panel functions can be activated via the Remote connector at the rear of the Clamp These are Mode selection Buzz and Clear Possible uses of this facility include using a computer to select the Modes and using hand operated or foot operated switches for Buzz and Clear so that these functions can be used by the experimenter without moving from the m
23. period then scales the samples to yield the average current for the whole period Im output is smoothed by the output filter see the Output Filter section The current in ME2 is labelled L The gain of the current measurement circuits depends on the headstage current gain H It is 10 H mV nA The whole current into the bath can be separately measured using a virtual ground headstage See the Virtual Ground section DCC MODE Description In Discontinuous Current Clamp DCC mode MEI is cyclically used to pass current The voltage recorded at the tip of ME1 is memorized by a sample and hold circuit inbetween each current passing period after all transient voltages due to current passing have decayed Thus the membrane potential can be recorded independently of IR voltage drops across the electrode The advantage of DCC mode compared with Bridge mode is that it is tolerant of small changes in microelectrode resistance The disadvantage is that DCC mode is noisier than Bridge mode During DCC mode ME2 can be used for continuous current passing _ The principles of operation are outlined in the block diagram and timing diagram of Fig 4 and in the following discussion The voltage recorded by the microelectrode V1 is buffered by a unity gain head stage A1 begin the discussion assume that V is exactly equal to the instantaneous membrane potential Vm Switch S2 briefly closes thereby enabling the voltage on the h
24. set In response to a positive current pulse the membrane potential began to charge up Before the membrane potential reached its final value the current pulse was terminated and the membrane potential exponentially decayed to its final value The traces in B were recorded at a sweep speed which was fast compared with the membrane time constant hence the membrane responses look like straight lines top trace shows the voltage recorded when no Bridge Balance was used The response was dominated by the IR voltage drop across the electrode In the middle trace the Bridge Balance was optimum and in the bottom trace it was slightly overused When the Bridge is correctly balanced the resistance of the microelectrode can be read directly from the dial The sensitivity is 10 H MQ per turn AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 18 DETAILED GUIDE TO OPERATIONS The Bridge Balance controls operate on the 10 V output and on the output On the 10 V4 output the Bridge Balance control saturates when the IR voltage drop exceeds 600 mV referred to the input Intracellular Balancing The traces in Fig 2 were all recorded with the electrode inside the cell Since the electrode response and the oscilloscope sweep speed were fast compared with the membrane time constant as in Fig 2B the correct Bridge Balance setting was easy to see even through the electrode was inside the cell It is s
25. step command Monitor 10 V4 and Im For maximum stability switch the Phase Time Constant to 20 or 200 ms Increase the Gain for the best response on both Vm and Im Sometimes lower current noise can be achieved for the same step response with the Phase Time Constant on 0 2 or 2 ms Before switching to these values reduce the Gain since the stability margin is lower for smaller values of the Time Constant Advance the Bridge potentiometer to speed up the current and voltage settling times An example of a cSEVC set up in a cell model is shown in Fig 10 The cell model was 300 MQ 33 pF and the electrode was modeled by a 3 MQ resistor Because of Re there was limit to how fast the membrane capacitance could be charged This can be seen from the duration of the capacitance transient in the upper trace The clamping electrode 1 records the true membrane potential as well as the IR drop across itself thus the step response of the recorded voltage middle trace is faster than the true membrane potential lower trace recorded by an independent electrode As discussed in the Series Resistance section the time course of the true membrane potential corresponds to the time course of the membrane current AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 61 FIGURE 9 SIMPLIFIED SCHEMATIC OF cSEVC AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS
26. these two methods will often succeed in lowering the resistance of blocked microelectrode tips When used while the tip of the microelectrode is pressing against the membrane Buzz and Clear may also cause the microelectrode to penetrate the cell AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC FEATURES HEADSTAGES COMMAND GENERATORS OUTPUTS Page 5 Unity voltage gain HS 2 headstages are available in several current gains These cover the range of cell input impedances from less than 1 Mf to greater than 1 GQ Ultrahigh input impedance versions are also available for ion sensitive electrodes High speed and low noise are achieved by using bootstrapped power supplies for the input circuit of each headstage These bootstrapped power supplies are derived from special high voltage circuits so that the headstages will not be saturated by the large voltages that may occur during the passage of current through high resistance microelectrodes Capacitance Neutralization is also derived from high voltage circuits so that fast responses are not degraded during large input signals Current in each microelectrode is continuously measured during both voltage clamp and current clamp This measurement does not include currents from sources other than the microelectrode e g hum ionophoresis the other microelectrode and indicates zero if the microelectrode blocks Headstages have a gold plated 2 mm 0 08
27. to reduce noise at a given sampling frequency The output of the Anti Alias Filter can be observed on the Monitor output In practice it is this voltage not V1 which has to decay to Vm before a sample is taken The Phase control alters the frequency response of the differential amplifier A2 It can be used to compensate for the complicated frequency characteristics of a real cell The Gain control alters Gr Refer to the Specifications for its operating range While MEI is occupied by the dSEVC it is still possible to independently use ME2 For example ME2 could be used for recording from and stimulating other cells which make connections to the cell being voltage clamped E MEE AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 66 DETAILED GUIDE TO OPERATIONS vollage recording 0 volts current passing ME1 current clomp 63 current passing 5 voltage recording sample sample 4 M M M Vin ove FIGURE 11 SEVC BLOCK DIAGRAM AND TIMING WAVEFORMS AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 67 Suggested Use Use two oscilloscopes To the main one connect the 10 V and Im outputs Trigger this oscilloscope from the source
28. used as a dual channel microelectrode probe or as a microelectrode voltage clamp Voltage cla ping is a powerful technique for the control of membrane potential and for the investigation of processes a fecting membrane conductance Voltage clamping has traditionally been performed using two intracellular microelectrodes and the AXOCLAMP 2A can be used for this purpose The AXOCLAMP 2A can also be used for discontinuous single electrode voltage clamping dSEVC and for continuous single electrode voltage clamping CSEVC single electrode voltage clamp SEVC is more convenient to use than a two electrode voltage clamp TEVC in very small cells and cells which cannot be visualized A particular advantage of a dSEVC is that the voltage drop due to current flow through the series component of cell membrane resistance R is not clamped In addition for both types of SEVC instabilities due to coupling capacitance and coupling resistance between two microelectrodes do not arise The disadvantages of a dSEVC compared with a TEVC are that the response speed is slower the maximum achievable gain is lower and the noise in the current and voltage records is greater The design of the AXOCLAMP 2A reduces these disadvantages towards their theoretical minimums thereby allowing single electrode voltage clamping to be performed in the many situations where conventional voles clamping is not suitable A cSEVC is as low in noise as a TEVC but has a sev
29. will momentarily fall to zero because the PAR Ing contacts will pass through an open circuit state DC Command Generators Separate DC command generators are provided for VC 1 and ME2 The DC command for VC is called Holding Position It allows the membrane potential holding position during voltage clamp to be shifted to a value in the range 3 200 mV It is always operative during voltage clamp Before the voltage clamp mode is selected the Holding Position potentiometer is used to RMP Balance see the RMP Balance section The Holding Position potentiometer is deliberately not calibrated because the exact setting depends on the adequacy of the clamp gain Instead the holding position should be read directly from the digital voltmeter displaying Vm ten turn locking dial is used so that once set the Holding Position potentiometer can be locked to prevent accidental changes The MEI and ME2 DC commands are called DC Current Command Each is controlled by a precision ten turn dial and can be switched by a toggle switch from depolarizing to hyperpolarizing or off OFF An LED illuminates whenever the toggle switch is in the or position It also illuminates if the Destination switch is turned to the microelectrode in question and the Step Command generator is activated either by the Ext Cont switch or by a logic HIGH level on the Step Activate input The current is scaled by the If the Step Command and the DC
30. 28 DETAILED GUIDE TO OPERATIONS During the current injection period a square pulse of current proportional to V is injected into the electrode Because of this current V rises The rate of rise of Vi is limited by the parasitic effects of capacitance through the wall of the glass microelectrode to the solution and capacitance at the input of the buffer amplifier The final value of V reached consists mostly of the IR voltage drop across the micro electrode resistance Only a tiny fraction of V consists of the membrane potential recorded at the tip After 30 of one cycle has elapsed the voltage recording period begins when S1 changes over to the 0 volts position Passive decay occurs because the input of the CCS is 0 volts and thus its output current is zero Sufficient time must be allowed during the voltage recording period for V to decay to within a millivolt or less of Vm At the end of the passive decay period S2 is again briefly closed and a new sample of Vm is taken to begin a new cycle The actual voltage used for recording purposes is the sampled voltage The sampled membrane potential is connected to the 10 V output The V Cont output is the instantaneous electrode voltage The instantaneous current into the microelectrode is monitored by a differential amplifier A3 output of A3 is taken to an averager not shown which samples smooths and scales the current pulses and connects the average value to the Im output Du
31. Current Command are used simultaneously the total command is their sum External Command Inputs Three external command inputs are provided These are for setting the voltage clamp command Ext VC Command the current clamp command in ME1 Ext MEI Command and the current clamp command in ME2 Ext ME2 Command These inputs are active simultaneously with the internal command generators and do not depend on the position of the Destination switch The sensitivity of Ext VC Command is 20 mV V The sensitivity of the Ext MEI ME2 Command is 10 x H nA V The external command inputs are DC connected Therefore when using the Ext ME1 and ME2 Command inputs any deviation from zero volts of the external signal source while it is in its off state will cause a DC current to flow in the electrode This can be avoided by using 1 A very high quality external source which puts out a true zero voltage level in its off state or which can be trimmed to do so 2 isolated external source Mixing Commands Complex command waveforms can be generated by appropriately mixing the Step Command the DC Command and the Ext Command For example the command waveform in Fig 3 can be used to establish the current injected into 1 by setting the Destination switch to the MEI position and using the ME DC Command and the Ext Command input AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 24 7 D
32. DR HARVEY J KARTEN MD UNIVERSITY OF CALIFORNIA SAN DIEGO DEPARTMENT OF NEUROSCIENCES 0608 9500 GILMAN DRIVE LA JOLLA 92093 0608 February 1990 AXOCLAMP 2A MICROELECTRODE CLAMP THEORY AND OPERATION Check ma No Lnd Sale ws Written for Instruments Inc by Alan Finkel Ph D Copyright 1988 1990 Axon Instruments Inc No part of this manual may be reproduced stored in a retrieval system or transmitted in form or by any means electronic mechanical photocopying microfilming recording or otherwise without written permission from Axon Instruments Inc QUESTIONS Call 415 571 9400 Part Number 2500 000 REV B Printed in U S A Spes Dane T Itm Doreen cl ap amp TL Yo clar Lit ev P in ep quus MET bonne sd DA ey fy SEVE E yt V Comman le A p 13 ae H QUT T Timer u Step Aliut Use A empl hade p h wll hayer Y frre Telegv nte 2552 Te lt pp Vor iii COPYRIGHT THE CIRCUITS AND INFORMATION IN THIS MANUAL ARE COPYRIGHTED AND MUST NOT BE REPRODUCED IN ANY FORM WHATSOEVER WITHOUT WRITTEN PERMISSION FROM AXON INSTRUMENTS INC VERIFICATION THIS INSTRUMENT IS EXTENSIVELY TESTED AND THOROUGHLY CALIBRATED BEFORE LEAVING THE FACTORY NEVERTHELESS RESEARCHERS SHOULD INDEPENDENTLY VERIFY THE BASIC ACCURACY OF THE CONTROLS USING RESISTOR CAPACITOR MODELS OF THEIR ELECTROD
33. ENTS INC Page 2 INTRODUCTION This page is intentionally left blank AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC FEATURES 3 FEATURES AXOCLAMP 2A is a complete microelectrode current and voltage clamp for intracellular investigations It combines state of the art single electrode voltage clamping two electrode voltage clamping and two complete bridge amplifiers into one instrument Precision command voltages meters filters offsets and many other features are built in to give you unprecedented flexibility discontinuous single electrode voltage clamping bath potential measurement and compensation continuous single electrode voltage clamping internally generated precision command voltages two electrode voltages clamping automatic clamping at resting membrane potential discontinuous current clamping offset compensation two complete bridge amplifiers rejection of stimulus artifacts high speed headstages output bandwidth selection low noise low hum operation calbration signal on outputs push button selection of operating mode electrode buzz computer selection of operating mode electrode clear two digital meters for voltage display hands free operation of buzz and clear digital counter for display of sample rate anti alias filter 3 input digital meter for current display phase control separate current measuremen
34. ES AND CELL MEMBRANES DISCLAIMER THIS EQUIPMENT IS NOT INTENDED TO BE USED AND SHOULD NOT BE USED IN HUMAN EXPERIMENTATION OR APPLIED TO HUMANS IN ANY WAY AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC iv Illustrations of the rear panel view of the AXOCLAMP 2A are shown on the fold out page at the rear of the manual AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DR HARVEY p KARTEN UNIVERSITY OF CA UNIVERS LIFORNIA SAN Di DEPARTMENT OF NEUROSCIENCES von 500 GILMAN DRIVE LA JOLLA 22093 0608 TABLE OF CONTENTS Page INTRODUCTION CREATE DUE Iva eaten 1 FEATURES PI 3 PRES CERERI 3 GLOSSARY oponen sec nec tut M 9 QUICK GUIDE TO OPERATIONS 1 DETAILED GUIDE TO OPERATIONS et deba ccesincsas 15 ANTI ALIAS FILTER divas po TR OE 15 BATH PROBE Ra obo v elio ET 16 Bath Potential 16 Grounding MURDER m Mutin 16 HERO 16 BRIDGE MODE ea US ERR anerian uude eue 17 Description A A A A A 425AA AA A AA A e
35. ETAILED GUIDE TO OPERATIONS EXT COMMAND external sine wave in this example STEP COMMAND set on thumbwheel DC CURRENT COMMAND set on pot FIGURE 3 SUMMATION OF COMMANDS This figure shows the command potential that would result if all command sources were switched on one at a time and left on AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 25 CURRENT MEASUREMENT The current injected down each microelectrode is independently measured The measurement is true Thus if the electrode blocks the measured current falls to zero even though a current command may exist Two current outputs apply to ME1 Im is the membrane current while Ij Cont is the instantaneous current in MEl In continuous modes Bridge cSEVC and even TEVC Im and I Cont are identical However in discontinuous modes i e DCC and dSEVC Im and 1 Cont are different I Cont switches from zero to some finite value at the sample rate This is because for 30 of each period MEI is used for passing current while for the remaining 7096 of each period no current is passed and the IR drop due to the previous current is allowed to passively decay see DCC and cSEVC sections On the other hand Im is the true membrane current It is recovered from the instantaneous electrode current by a circuit which samples the current pulses retains the samples during the passive decay
36. INC Page 62 DETAILED GUIDE TO OPERATIONS cSEVC Compared With Whole Cell Patch Clamp The simplified schematic in Fig 9 shows that cSEVC is similar to whole cell clamping using the patch clamp technique However the implementation is very different In the patch clamp technique the voltage clamp is established by a specialized headstage containing a virtual ground circuit In the cSEVC technique the headstage is a general purpose unity gain buffer and the voltage clamp circuit is located in the main unit This difference is significant In the dedicated virtual ground headstage much less circuitry is involved and thus nonidealities of the electronics have much less effect Thus for fast events the patch clamp technique is considerably better than cSEVC the other hand for slow and moderate events the techniques become comparable Fig 10 Current and potential recorded during cSEVC Cell model was 300 0 33 pf Electrode was 3 MQ Bandwidth was 3 kHz for all traces H of HS 2L headstage was x0 1 Clamp gain was 3 3 nA mV Voltage command was 10 mV step Phase Time Constant was 0 2 ms Phase Shift was full lag A A Filter was off Capacitance Neutralization was minimum Upper trace Membrane current Charging time was limited by Re Middle trace Potential recorded by clamping electrode ME1 and available at the 10 Vm output Includes IR drop across MEI Lower trace True membrane potential recorded by an _ elec
37. Im which flows through it This voltage drop is equal to the product of Im and the resistance Re1 of MEI To keep the error due to small it is necessary that Rei be much smaller than the membrane resistance Rm Thus a 3 MQ microelectrode would be appropriate for use with a 300 MQ cell The voltage across MEI can be partially compensated by using the Bridge potentiometer Note that the range of the Bridge potentiometer is ten times less in cSEVC mode than in Bridge mode The reduced range is indicated by a small LED It is not normally possible to compensate more than about 70 of the electrode resistance without introducing oscillations During cSEVC Re has the nature of a series resistance R R is discussed in the Series Resistance Section j Important Note Anti Alias Filter The Anti Alias Filter is not recommended for use in cSEVC mode The reasons why are the same as those given in the TEVC Section Suggested Use In Bridge mode set the Capacitance Neutralization control for the best step response Set the Gain and Anti Alias Filter to minimum values Switch the Phase control off Switch off all current commands j Use the Holding Position control to yield equal brightness in each of the two RMP Balance lights At this setting the command potential during voltage clamp will be equal to the resting membrane potential RMP Lock the Holding Position control if desired Switch into cSEVC mode Set up a repetitive
38. J Neurobiol 6 411 422 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC lj A 2 REFERENCES This page is intentionally left blank AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC WARRANTY 1 WARRANTY We warrant every AXOCLAMP and every headstage to be free from defects in material and workmanship under normal use and service For 12 months from the date of receipt we will repair or replace without cost to the customer any of these products that are defective and which are returned to our factory properly packaged with transportation charges prepaid We will pay for the return shipping of the product to the customer If the shipment is to a location outside the United States the customer will be responsible for paying all duties taxes and freight clearance charges if applicable Before returning products to our factory the customer must contact us to obtain a Return Merchandise Authorization number RMA and shipping instructions Failure to do so will cause long delays and additional expense to customer Complete a copy of the RMA form on the next page and return it with the product This warranty shall not apply to damage resulting from improper use improper care improper modification connection to incompatible equipment or to products which have been modified or integrated with other equipment in such a way as to increase the time or di
39. Note Coupling 76 Saturation During The Capacitance 79 Choosing the Microelectrode Resistances 79 TRIGGERED CLAMPING E 79 TROUBLE SHOOTING coru do er e a ge ET 80 UNITY GAIN RECORDING THIRD POINT eee 80 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC VIRTUAL GROUND CURRENT MEASUREMENT 10 AND T OUTPUTS oe beat SPECIFICATIONS D itu td REFERENCES es RMA FORM isnt POLICY STATEMENT E SERVICE Bassa eda kde COMMENT FRONT AND REAR PANEL Fr pa l2 nel Mire T On TTE Canin ne see aks e TL 1 lue fee t e AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC This page is intentionally left blank AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC INTRODUCITON Page 1 INTRODUCTION The AXOCLAMP 2A Microelectrode Clamp can be
40. O ring by the sharp back end of the electrode you can smooth the electrode edges by rotating the back end of the electrode in a bunsen burner flame Cleaning For lowest noise keep the holder clean Frequently rinse the holder with distilled water If heavier cleaning is required briefly wash in ethanol or mild soapy water Never use methanol or strong solvents AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 41 Filling electrodes Only the taper and a few millimeters of the shaft of the electrode should be filled with solution The chlorided tip of the wire should be inserted into this solution Avoid wetting the holder since this will increase the noise Silver Chloriding The HL 2 17 holders are supplied with a Ag AgCl pellet that should give you many months of DC stable recordings The silver wire is surrounded by a Sylgard sealed teflon tube This ensures that the electrode solution only contacts the Ag AgCl pellet SYLGARD TEFLON TUBING Ag AgCl PELLET WP Ag WIRE En It is not practical to make a pellet small enough to fit inside the shaft of the narrow glass electrodes used in the and HL 2 12 holders therefore these holders are supplied with a piece of 0 25 mm silver wire It is up to you to chloride the end of this wire as required Chloriding procedures are contained in many electrophysiology texts e g Purves 1981 Typic
41. O OPERATIONS 10 foot 3m cable ET Remote 15 pin connector EXTERNAL SWITCH WIRING DIAGRAM AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 57 RMP BALANCE The two indicator lights for monitoring resting membrane potential RMP are used in two ways Before switching into a voltage clamp mode the Holding Position potentiometer is adjusted until the two lamps are equally dim nulled This ensures that when a voltage clamp mode is selected the membrane potential will be held within a few millivolts of RMP When adjusting the Holding Position control before voltage clamping the sensitivity of the null point is affected by the Gain During voltage clamp the RMP Balance lights provide a quick indication of when the cell is being held at its resting level That is the RMP Balance lights are nulled at this point SERIES RESISTANCE Origin A resistance in series with the membrane can arise a number of different ways In cSEVC R would mainly be due to the resistance of the suction electrode In dSEVC R would be due to a slow microelectrode response In TEVC R would be due to the tissue the bathing solution and the grounding electrode Problem The voltage recording microelectrode ME records the voltage across and Rm thus the recorded membrane potential is in error due to the IR voltage drop across R In addition R limits the maximum
42. Phase shift Modifies frequency response of voltage clamp amplifier Compensates for nonideal phase shifts of membrane Potentiometer adds phase advance lead or phase delay lag Switch selects range Anti Alias Filter Used in DCC or dSEVC modes to reduce noise of electrodes that have fast and slow settling characteristics D6 STEP COMMAND GROUP Uses D A converter to generate precision command voltage Destination Switch Selects voltage clamp or either microelectrode as target for command Commands are mV or nA respectively Thumbwheel Switch Sets magnitude with 0 0596 resolution Ext Cont Off Switch Cont position activates step command Ext position thumbwheel switch is off unless logic level HIGH applied to rear panel Step Activate input Off position overrides logic input Indication When destination is a microelectrode and step command is activated lamp in microelectrode DC Current Command Section illuminates D7 RATE GROUP Counter indicates sampling rate cycling rate in DCC and dSEVC modes Potentiometer adjusts rate from 500 Hz to 50 kHz lt AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC N 5 UA us x Page 14 QUICK GUIDE TO OPERATIONS D8 INPUTS AND OUTPUTS Vm Im Output Bandwidth switch s lects 3 dB frequency of single pole lowpass on Im and 10 V outputs Current 1 voltmeter displays DC current from either microel
43. THIRD POINT In normal operation both ME1 and ME2 can be used for unity gain recording and current passing A third point in the preparation can be recorded from if virtual ground current measurement is not being used To do so a unity gain headstage HS 2 is plugged into the Virtual Ground connector on the rear panel The voltage recorded appears on the IVIRT output No current can be passed via the HS 2 headstage used in the Virtual Ground connector When plugged into the Virtual Ground connector the input capacitance of the unity gain headstage is 4 pF VIRTUAL GROUND CURRENT MEASUREMENT A Virtual Ground headstage can be used to ground the preparation bath All of the current flowing into the Virtual Ground input is measured and a voltage proportional to the current is provided at the IVIRT output The output gain is 10 mV nA when the virtual ground output attenuation VG is x1 1 mV nA when VG is x10 and 100 mV nA when VG is 0 1 A Virtual Ground headstage is not required for normal use of the AXOCLAMP 2A because built in current measurement circuits are provided for each microelectrode However in TEVC mode the current output of the Virtual Ground headstage has slightly less high frequency noise than the output of the built in current measurement circuit The Virtual Ground circuit measures all currents into the preparation bath hence special care must be taken to ensure that conducting connections to the preparation bath do not act as
44. XOCLAMP 2A to suit your recording apparatus B AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 6 REMOTE CONTROL MODEL CELL GENERAL FEATURES Six outputs are conveniently located at the front panel for connecting to your oscilloscope These outputs are repeated at the rear panel where the other outputs the inputs and the headstage connectors are also located Hands free operation of Buzz is possible using the footswitches supplied with every AXOCLAMP 2A Selection of the operating mode can be made remotely for computer sequencing of experiments All AXOCLAMP 2As have a Buzz oscillator to assist in cell penetration The d ration of the Buzz oscillation is normally equal to the time that the front panel switch is pressed Practically the shortest duration that this switch can be pressed is about 100 ms For small cells 100 ms Buzz oscillation sometimes damages the cells immediately after penetration The Remote Buzz Duration Control supplied with the AXOCLAMP 2A is a hand held control that contains a trigger switch to buzz either electrode and a duration control for setting the Buzz duration in the range 1 50 ms An appropriate duration can be found for most cells that is sufficiently long to allow penetration of the membrane but short enough that the cell is not damaged after penetration Every AXOCLAMP 2A is supplied with a CLAMP 1 model cell This model cell plugs directly into
45. Y 1990 AXON INSTRUMENTS INC Page 70 DETAILED GUIDE TO OPERATIONS Important Note If the Phase controls are used it is possible to find false settings of Capacitance Neutralization or the Anti Alias Filter and Phase which together give a seemingly fast step response to Vm whereas in fact the step response in the cell is much slower This situation arises by underutilizing the Capacitance Neutralization or overutilizing the Anti Alias Filter so that the Monitor waveform fails to decay adequately when the voltage sample is taken The electrode voltage sampled has the nature of an IR drop across a series resistance Rs see Series Resistance Section Normally this would make the clamp unstable but by introducing phase lag stability can be re imparted although without any reduction of the voltage error This false condition only arises if the Capacitance Neutralization setting is altered after the Phase control has been switched in There are two ways to guarantee that this false condition will not occur 1 Don t use the Phase controls 2 If the Phase controls are used be sure to conscientiously observe the Monitor waveform to make sure that the decay to a horizontal baseline is complete at the end of each cycle An example of a false clamp is shown in Fig 13 The recorded value of Im is always a true measure of the membrane current even during this false setting Only the Vm record is erroneous The danger of this false condition i
46. ally the chlorided wire will need to be replaced every few weeks Heat smoothing the back end of the electrode extends the life of the chloride coating by minimizing the amount of scratch damage Another way to protect the AgCI coating is to slip a perforated teflon tube over the chlorided region The chlorided region should be ne enough so that the electrode solution does not come in contact with the bare silver wire Glass Dimensions Use the HL 2 12 holders for glass from 1 0 to 1 2 mm outside diameter OD The optimal dimensions are 1 15 mm OD and 0 5 mm ID Use the HL 2 17 holders for glass from 1 5 to 1 7 mm outside diameter OD The optimal dimensions 1 65 mm OD and 1 1 mm ID For other glass dimensions you can drill out the bore of the HL 2 12 holder AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 42 2 DETAILED GUIDE TO OPERATIONS IONOPHORESIS A When 2 is not used for intracellular penetrations it can be used for ionophoresis set the retaining and pulse currents 1 Set the desired retaining current on the ME2 DC Current Command control 2 Switch the Destination switch to ME2 Set the Step Command equal to the desired pulse current minus the retaining current Connect a pulse generator to the Ext ME2 Command input to set the desired pulse current minus the retaining current eg retaining current 5 nA ejection current
47. ample and hold circuit SH1 samples Vm and holds it for the rest of the cycle The sampled membrane potential is compared with a command voltage Ve in a differential amplifier A2 The output of this amplifier becomes the input of a controlled current source CCS if the switch S1 is in the current passing position The gain of the CCS is Gr The CCS injects a current into the microelectrode which is directly proportional to the voltage at the input of the CCS irrespective of the resistance of the microelectrode The period of current injection is illustrated at the start of the timing waveform 651 is shown in the current passing position during which a square pulse of current is injected into the microelectrode Because of this current V rises The rate of rise is limited by the parasitic effects of the capacitance through the wall of the glass microelectrode to the solution and the capacitance at the input of the buffer amplifier The final value of V mostly consists of the IR voltage drop across the microelectrode due to the passage of current I through the microelectrode resistance Re tiny fraction of V consists of the membrane potential recorded at the tip S1 then switches to the voltage recording position When the input of the CCS is 0 volts its output current is zero and V passively decays During the voltage recording period V decays asymptotically towards Vm Sufficient time must be allowed for V to reach within
48. and 2 capacitively isolating the transformer primaries and secondaries i Nevertheless some power supply glitches do get through These can cause transients to appear on the voltage and current outputs which may corrupt Tp for example during fluctuation analysis The completely effective way to gain immunity from mains glitches is to eliminate them at the source Most glitches are due to the switching on and off of other equipment and lights on the same power supply circuit Precautions to be taken include Avoid switching equipment and lights on or off while recordings are being made Water baths heaters coolers etc should operate om zero crossing relays 3 RFI filters should be installed in vitich oroducing equipment In most circumstances occasional transients on the a are inconsequential and therefore no precautions have to be taken AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 54 DETAILED GUIDE TO OPERATIONS POWER SUPPLY VOLTAGE SELECTION amp FUSE CHANGING Supply Voltage The AXOCLAMP 2A can work from all international supply voltages The two input ranges are 1 115 For 100 Vas to 125 Va operation 0 230 V For 200 Vac to 250 Vac operation To change the supply voltage setting 1 Disconnect the power cord Q Remove the top cover 3 Locate the slide switch labeled S2 at the back of the power supply board The power supply board
49. antennae which pick up hum Saline filled tubes act as excellent antennae To prevent them carrying hum long saline filled tubes should have the saline pathway broken by an air filled drip near the preparation More complete explanations and instructions are provided with the VG series of headstages AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 81 10 Vm AND Im OUTPUTS The 10 V4 output is proportional to ten times the membrane potential Vm It is derived from the potential V1 recorded by ME1 Initially V is amplified then depending on the operating mode one of two techniques is used to derive the 10 V signal from the amplified V signal In Bridge mode the Bridge Balance technique is used to counter the effect of voltage drop IR voltage drop across ME1 during current passing so that only the membrane potential measured at the tip is passed to the 10 V output In DCC dSEVC mode samples of the amplified V signal are taken after the IR voltage drop across ME1 due to the previous current pulse has completely decayed Only the sampled values are passed to the 10 Vq output The maximum recording range of the 10 V4 output is 600 mV referred to the input This range is centered on the zero value set by use of the Input Offset control In Bridge mode this range includes the IR drop even though the IR drop may not be seen because the Bridge Balance is c
50. ass current without large changes in resistance Microelectrode Capacitance get fast settling it is essential to minimize the transmural capacitance CU from the inside of the microelectrode to the external solution is usually 1 2 pF per mm of immersion Two applications requiring different approaches are discussed here Target Cell Near Surface Of Solution In an isolated preparation C can be reduced by lowering the surface of the solution as faras possible see note below Precautions must be taken to prevent surface tension effects from drawing a thin layer of solution up the outer wall of the microelectrode If this film of saline is allowed to develop will be much worse that otherwise Because the film of saline has axial resistance the contribution to C will be very nonlinear and the voltage decay after a current pulse will either be biphasic as in Fig 1 or if it is monophasic it will not be very fast even when capacitance neutralization is used To prevent the saline film from developing the electrode should be coated with a hydrophobic material This can be done just before use by dipping the filled microelectrode into a fluid such silicone oi or mineral oil Another Sylgard or Q dope airplane glue can also be used to build up the wall thickness of the electrode thereby reducing The selected material should be painted onto the electrode to within 100 ym of the tip Note For a long slend
51. ation to this problem has been applied throughout the design of the AXOCLAMP 2A and the overall DC offset has been made as insensitive as possible to the drift in the integrated circuits As well special low drift integrated circuits have been used in all critical positions The magnitude of the DC leakage current increases with increases in H This normally introduces no greater error in the DC offset voltage developed across the microelectrode or the cell membrane nuns larger Hs are usually used with lower resistance cells and microelectrodes AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 38 DETAILED GUIDE TO OPERATIONS Before leaving the factory the DC offset voltage of each HS 2 headstage is trimmed so that the input leakage current is no more than 100 pA for 10 10 pA for H xl 1 for 01 1 pA for H x0 01 10 fA for H x0 0001 These input current levels are very low and cause negligible shifts in the cell membrane potential when the headstages are used with the recommended ranges of cell input resistances see Table 2 The shift in is calculated from input current x Rin If you ever suspect that the input current has grown to a level where Vm is significantly affected it can be re adjusted by the following procedure 1 Switch off all current commands and disconnect any external current commands 2 Remove the plastic cap from the access hole in
52. aturation is that not all of yf the 30 V appears across the microelectrode Some of it appears across the current sensing resistor Ro see Fig 4 in the headstage If Re Ry then 15 V appears across the microelectrode but if Re lt Ro the voltage across the microelectrode is even less To overcome this a headstage designated HS 4 is available which has a relay inside it to automatically link out whenever TEVC mode is selected There are two advantages to using the HS 4 headstage The first is that even in the linear operating region the time to establish a step voltage change is quicker and the second is that larger step changes can be established without entering the nonlinear i e saturating region The disadvantage is that the HS 4 headstage must be used in conjunction with a virtual ground current measurement headstage This is because the normal built in current monitors need in order to operate Because it requires a virtual ground headstage as well we do not normally recommend the HS 4 headstage unless the experimental circumstances demand it Contact Axon Instruments for more details Choosing the Microelectrode Resistances If large currents must be passed such as may occur daring large depolarizations of excitable cells then the resistance of ME2 should be as low as possible If low noise recordings are required which would be necessary for resolving small transmitter activated currents from the background noise then
53. brane potential and can therefore represent a serious source of error To prevent this error the following checks should be made 1 While the microelectrode is outside the cell set the offset to zero In bridge or DCC mode pass a constant current into the bath for about 10 seconds The current magnitude should be the same as the maximum sustained current likely to be passed during the experiment When the current is switched off the recorded potential should return to zero within a few milliseconds at most Some electrodes either return very slowly to zero potential or not at all These electrodes should be discarded AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 37 2 Once the experiment is in progress occasionally check the resistance of the microelectrode Changes in tip potential are usually accompanied by changes in electrode resistance Note that the tip potential changes described in this section are happening with a slower time course than the ones described in the Anti Aliasing section The causes of these slow changes in tip potential are unknown Potentials Prevention Not much can be done to prevent tip potentials from changing but the following may be helpful 1 Sometimes the slow changes in tip potentials are worse when standard microelectrode holders with an embedded AgCl pellet used instead of an Ag AgCl wire Some holders
54. chievable The Capacitance Neutralization setting of ME1 should not be altered during voltage clamp unless there is reason to believe the resistance of MEI has altered Extremely Important Note Coupling Capacitance The most significant factor in achieving a good two electrode voltage clamp is adequate prevention of interactions between the two electrodes Coupling capacitance as low as 0 01 pF can destablize the response at high gain settings a To minimize the coupling capacitance it is essential that a grounded shield be placed between the two microelectrodes and their headstages to prevent signals in ME2 being picked up by ME1 should extend between the two electrodes to within a millimeter of the surface of the solution It is possible to coat ME2 with a conductive paint which is then grounded This procedure works well but has a minor disadvantage in that it vastly increases the capacitance at the input of the ME2 headstage which may affect the high frequency measurement of Iz unless the capacitance neutralization of ME2 is properly set AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 77 Fig 15 shows the detrimental effects of only a small amount of coupling capacitance The traces in Fig 15A show the membrane current and voltage responses in a cell model when an extensive grounded shield was placed between the two electrodes In Fig 15B a 2 3 mm wide gap in
55. cle used in the AXOCLAMP 2A has been chosen to give a good compromise between aliased noise and dynamic performance Finkel amp Redman 1984b With this duty cycle the increase in noise due to aliasing is a factor of about two The two contributions to noise discussed above lead to a factor of four or more deterioration in noise To keep the deterioration as small as this the experimenter should aim to do the following 1 Keep the real value of Cin as small as possible so that only minimal capacitance neutralization must be used Avoid using coaxial cable to connect the microelectrode to the headstage Q Either increase the Anti Alias Filter setting at a given cycle rate or increase the cycle rate at a given setting of the Anti Alias Filter so that the amount of aliased noise is minimized Finally the amount of noise recorded can be reduced to some extent by using as much output filtering as possible However the output filtering should never be increased to the extent that dynamic information e g rise time is lost Usually output filtering at f 10 is a good compromise The best way of reducing noise in the records is by averaging repetitive signals This well known procedure reduces the noise by the square root of the number of averages without affecting the time course of the signal Notwithstanding the comparatively poor noise performance of discontinuous single electrode voltage clamps compared with two electrode voltage clamps t
56. de potential might be 200 mV though the experimenter does not see this potential because of the offset compensation To compensate several pF of input capacitance the gain of the capacitance neutralization circuit might be 2 Thus 400 mV would be fed back to C resulting in 200 mV across it If the dielectric resistance of Ca were 10 Q the guaranteed minimum of high quality capacitors there would be 2 pA ii through the capacitor AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 39 To eliminate this source of leakage current a DC removal circuit removes the DC voltage from across Cp The DC removal circuit operates with a 1 s or 10 s time constant There may be a transient shift in the electrode voltage while the Capacitance Neutralization control is being adjusted The DC voltage is also removed from the shield drive Input Resistance The input resistance of the headstages is predomh ntly related to Ro circuit inside the AXOCLAMP called a constant current source CCS controls the voltage across Ro Ideally the voltage across Re is independent of the electrode voltage The accuracy of the CCS in controlling the voltage across Ro is preset at the factory Extremely stable components are used in the CCS so that the accuracy will not fes with time In general the CCS is effective to one part in 10 so that the input resistance is 104R Other possible factors
57. dman S J 1985 Optimal voltage clamping with a single microelectrode In Voltage Clamping with Microelectrodes ed Smith et al Williams amp Wilkins Baltimore Finkel A S amp Redman S J 1983 A shielded microelectrode suitable for single electrode voltage clamping of neurons in the CNS J Neurosci Meths 9 23 29 Hamill O P Marty A Sakmann B amp Sigworth F J 1981 Improved patch clamp techniques for high resolution current recording from cells and cell free membranes patches Pfl gers Arch 391 85 100 Johnston D amp Brown 1983 Interpretation of voltage clamp measurements in hippocampal neurons J Neurophysiol 50 464 486 e 1981 Microelectrode Methods for Intracellular Recording and Ionophoresis London Academic Press Sachs F amp McGarrigle 1980 An almost completely shielded microelectrode J Neurosci Meths 3 151 157 Schwartz T I amp House Randall C 1970 small tipped microelectrode designed to minimize capacitive artifacts during the passage of current through the bath Rev Sci Inst 41 515 517 Suzuki K Rohligek V amp Fr mter E 1978 quasi totally shielded low capacitance glass microelectrode with suitable amplifiers for high frequency intracellular potential and impedance measurements Pfliigers Arch 378 141 148 Wilson W A amp Goldner M M 1975 Voltage clamping with a single microelectrode
58. e are three teflon insulated 2 mm 0 08 inch sockets in the headstage see diagram These are standard diameter sockets 1 Microelectrode Input Connector The red socket is the microelectrode input The connection between the microelectrode and this socket should be kept as short as possible Some excellent methods are i Solder a silver silver chloride wire directly to one of the 2 mm plugs supplied Use the wire to connect to the microelectrode which supported on a separate mounting LA ETT ii For greater mechanical stability use an HL 2 series microelectrode holder from Axon Instruments iii Plug a standard microelectrode holder 2 mm plug directly into the input socket The teflon input socket should allow enough clearance for most standard holders iv Use a BNC type microelectrode holder This requires HLB 2 adaptor from Axon Instruments AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Leakage current trim access AXON INSTRUMENTS white MODEL O Mounting rod GAIN SHIELD SERIAL Shielded cable gold P GROUND Connected to case yellow Notes Model may be HS 2L HS 2M or HS 4M Gain refers to headstage current gain H HS 2 and HS 4 HEADSTAGE CONNECTION DIAGRAM AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 35 Page 36 2i DETAILED GUIDE TO
59. e can be continuously altered from a low value of 500 Hz to a high of 50 kHz This enables you to take advantage of the decrease in noise and response times occurring when faster sampling rates are used AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 4 CURRENT CLAMPING FEATURES The sample clocks of two AXOCLAMP 2A s can be synchronized in a Master Slave configuration This is useful in experiments in which two cells in the same preparations are independently voltage clamped using dSEVC Linking the two clocks prevents the generation of spurious signals which would otherwise appear at harmonics of the difference in the two clocks frequencies Output compliance in TEVC mode is 30 V This reduces the chance of saturation while the membrane capacitance is charging after a step change in voltage To further minimize the chance of saturation during TEVC a relay switched headstage HS 4 is available to automatically bypass the current sensing resistor inside the headstage The HS 4 headstage must therefore be used in conjunction with a virtual ground current monitor VG 2 The HS 4 headstage is recommended only when large ultra fast voltage steps in big cells must be established Another unique control is a Resting Membrane Potential RMP Balance Indicator which enables you to preset the clamp offset so that when you switch into voltage clamp mode the cell membrane will automatically be clam
60. e potential recorded by a third independent electrode Middle trace is membrane potential recorded by MEI and clamped by the voltage clamp circuit Bottom trace is the membrane current A added phase shift B Phase control set to Center Frequency time constant of 0 2 ms Phase shift on 4 The true membrane potential response is unaffected but the membrane current noise is greatly reduced to a level consistent with the slow membrane potential response AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 59 FIGURE 8 HOW SERIES RESISTANCE R AFFECTS VOLTAGE CLAMP PERFORMANCE AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 60 DETAILED GUIDE TO OPERATIONS SEVC MODE CONTINUOUS Continuous single electrode voltage clamping 5 is one of two single electrode voltage clamp modes In cSEVC current passing and voltage recording are performed simultaneously as shown in the block diagram of Fig 9 voltage recorded by the microelectrode buffer A1 is compared in a high gain differential amplifier A2 to a command potential V The output of A2 acts to keep the difference at its input ej very small Hence Vj is clamped equal to Vc The circuit clamps the voltage across the microelectrode 1 as well as the membrane potential V m The voltage across MEI is non zero because of the current
61. e potentiometer At the center position of the potentiometer the net change to the frequency characteristics of the voltage clamp circuit is nil The Time Constant switch changes the maximum lag and lead values as listed in the Specifications In some preparations no phase lag or lead is required If this is so the Time Constant switch should be switched to the Off position i With an RC cell model the best voltage clamp will be achieved when no Phase shift is used AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS 53 Use The Phase controls can be used during voltage clamp to compensate for the frequency characteristics of membranes which are not well modeled by parallel resistance and capacitance Both the membrane voltage and current step responses should be improved by using the Phase controls If only the membrane voltage step response is improved it is likely that there is a resistance R in series with the membrane See the Series Resistance Section for a discussion of this problem In some cases using some phase lag will reduce the current noise during voltage clamp See the Sections on each type of vole clamp for more details POWER SUPPLY GLITCHES The AXOCLAMP 2A has been designed to minimize the effects of power si pply transients glitches This is achieved by 1 taking the incoming power a radio frequency interference
62. e rules above is the best start The final hum level can often be kept t less than 100 pV peak to peak referred to Vm One technique that should not be used to reduce the hum is the delicate placement of cables so that a number of competing hum sources cancel out Such a procedure is too prone to accidental alteration DR HARVEY J KARTEN M D UNIVERSITY OF CALIFORNIA SAN DIEGO DEPARTMENT OF NEUROSCIENCES 0608 9500 GILMAN DRIVE LA JOLLA 92093 0608 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 32 DETAILED GUIDE TO OPERATIONS HEADSTAGES The HS 2 unity gain headstage buffers the high impedance of the microelectrode making the potential recorded by the microelectrode available to the rest of the circuitry It also provides the means for injecting current into the microelectrode and for neutralizing the input capacitance The Meaning Of H A precision resistor Ro in Fig 4 inside the headstage sets the headstage current gain H Choosing the H depends on the cell to be clamped see below The particular value of H used affects the Bridge Balance range the sensitivity to current commands the sensitivity of the current monitors and the gain in SEVC mode The effects are clearly marked on the front and rear panels and since they always appear in multiples of 10 they are easy to calculate For your convenience Table 1 summarizes these effects Note that voltage commands durin
63. e their sampling clocks 15 pin connector on the rear panel enables the sampling clock circuits of two AXOCLAMP 2As to be linked by cable One AXOCLAMP 2A becomes the Master and the other the Slave which is which is determined by the orientation of the cable After Link up whenever both AXOCLAMP 2As are in DCC or dSEVC modes the Slave s sampling clock is overridden by the Master s In all other combinations of operating modes the two AXOCLAMP 2As remain fully independent For example if the Slave is in DCC or dSEVC modes but the Master is in neither the Slave s sampling clock is re enabled AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Fel Page 43 By forcing both AXOCLAMP 2As to sample synchronously the beat frequency problem is eliminated At the instant that both AXOCLAMP 2As sample their electrode voltages there will be no pick up from one electrode to the other because the voltages across both electrodes must have decayed to near zero in order for the clamps to operate Clock Link Up only affects the sampling clocks All other functions of the two AXOCLAMP 2As remain fully independent MICROELECTRODES FOR FAST SETTLING The key to discontinuous voltage and current clamping with a single microelectrode is the character of the microelectrode itself microelectrode voltage must settle rapidly after a current pulse and the microelectrode must be able to p
64. e used interchangeably I h Im Lag Lead MEI ME2 Re Rm Rin SEVC TEVC Vi V2 VG Vin Vion Total input capacitance of the headstage due mainly to the microelectrode and any connecting cable Input capacitance of cell Continuous single electrode voltage clamp Discontinuous current clamp Discontinuous single electrode voltage clamp Sampling rate rate for switching from current passing to voltage recording in DCC and dSEVC modes The average gain during dSEVC The instantaneous gain of the controlled current source during dSEVC Headstage current gain Continuous current flow in microelectrode 1 Current flow in microelectrode 2 Membrane current flow High frequency cut High frequency boost Microelectrode 1 Microelectrode 2 Electrode resistance Resistance in series with membrane Resting membrane potential Input resistance of cell membrane Single electrode voltage clamp Two electrode voltage clamp Continuous voltage recorded by microelectrode 1 Voltage recorded by microelectrode 2 Voltage Clamp Virtual ground output attenuation Membrane potential recorded by microelectrode 1 Voltage at the input of the sample and hold amplifier SH1 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 10 GLOSSARY This page is intentionally left blank AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC QUICK GUIDE TO OPERATIONS z
65. e which is driven to the electrode potential When the switch is in the BATH position both electrode resistors are connected to ground This is a convenient position for practicing bridge balancing techniques and offset adjustment 5 When the switch is in the CELL position both electrode resistors are effectively intracellular In Bridge or DCC mode you should see exponential voltage responses to steps of current In dSEVC mode you should be able to clamp the cell at gains of up to 0 8 nA mV using an HS 2 x0 1 headstage at sampling rates up to 8 kHz In TEVC mode use one of the following electrode combinations 1 two x0 1 headstages two x1 headstages or a x1 headstage for ME2 and a x0 1 headstage for ME1 The electrode resistances in this model cell are too large for you to practice cSEVC AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 47 pe CONNECT MEl CONNECT 1 HEADSTAGE GRDUND FIGURE 6B CLAMP 1 MODEL CELL AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 48 DETAILED GUIDE TO OPERATIONS MONITOR The Monitor output is used to check the settling characteristics of the voltage at the input to the sample and hold device This is advisable during DCC and dSEVC and the notes on these two modes should be consulted for details The Monitor signal i
66. echnique known as bootstrapping fixes the voltage drop across Cini to a constant value thereby preventing current flow neuer Cin The effective value of Cis is thus reduced to well below its real value i Secondary A commonly used technique known as capacitance neutralization is used to negate and the effective remnant of capacitance neutralization circuit attempts to inject into the headstage input a current which it anticipates will be required to charge and discharge Cin during signal changes To use the capacitance neutralization circuit the voltage response to a current step should be observed on an oscilloscope Advance the capacitance neutralization control as far as is possible without introducing overshoot in the step response This setting is optimal for current passing and is also optimal for recording potentials at the tip of the microelectrode It is important to recognize that the capacitance neutralization circuit is not more than 90 effective even for ideal microelectrodes This is because of the finite frequency responses of the headstage amplifiers AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 22 DETAILED GUIDE TO OPERATIONS and the capacitance neutralization circuit and also because Cin does not behave ideally as a linear lumped capacitor Consequently the amount of Ci that the circuit must neutralize should be kept as small as possible To this end avoid
67. ectrode or virtual ground if used Switch used to select meter input Decimal point set on Hj H2 or VG switches All BNC inputs and outputs located on rear panel Frequently used outputs repeated on front panel Im output I Cont Output Ip output output 10 Vm output V Cont output Monitor output V2 output Sample Clock output Output Cal Activat input Step Activate input Blank Activate input Ext VC Command input Ext ME1 Command input Ext ME2 Command input R Comp input VBaarH IN input D9 REMOTE Clear D10 CLOCK LINK UP Membrane current recorded by MEI ME current equals Im in Bridge cSEVC and TEVC modes current Virtual ground current Membrane potential recorded by ME1 gain of 10 Instantaneous ME1 potential No Bridge Balance Input of sample and hold amplifier Should be observed on second oscilloscope during DCC and dSEVC modes ME2 potential Includes Bridge Balance Logic level pulses at the sample rate used to trigger monitor oscilloscope Potential recorded by bath electrode Logic HIGH on this input puts calibration voltage proportional to thumbwheel setting onto voltage and current outputs Logic HIGH activates Step Command Logic HIGH activates Blank During Blank Vm prevented from updating Thus stimulus artifacts are rejected Voltage on this input converted into voltage clamp command Voltage on this input converted
68. er microelectrode we regard 200 ym or less as a low solution level 500 um is tolerable 1 mm or more is regarded as deep For a microelectrode which tapers steeply i e a stubby microelectrode deeper solutions can be used with less loss of performance When working with very low solution levels there risk of evaporation exposing the cells to the air unless a continuous flow of solution is provided across or through the preparation If evaporation is a problem one way to overcome it is to float a layer of mineral oil on the surface of the solution If used this layer of oil will have the additional advantage of automatically coating the electrode as it is lowered into the solution AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Re Page 44 DETAILED GUIDE TO OPERATIONS Target Cell Deep In Solution In some preparations e g in vivo CNS the target cell is several millimeters below the surface of the solution In this case the more difficult procedure of guarding the electrodes may have to be used This involves coating the outside of the microelectrode with a metal layer and connecting this layer to the case socket of the unity gain headstage Depending upon the case socket is either connected to the capacitance neutralization circuit or to the unity gain output guarding procedure does not reduce C Instead it reduces the effect of C by controlling the voltage across it The metal guard
69. ere disadvantage in that the voltage drop across the microelectrode is clamped unless is made Since the required compensation is never perfect the cSEVC can only be used when the electrode resistance is very small compared with the cell input resistance These favorable ns can often be achieved by the whole cell patch technique Because of the AXOCLAMP 2A s advanced design it itself does not limit the achievable performance Instead the dominant factor affecting SEVC performance is the microelectrode Users of the AXOCLAMP 2A in either of the SEVC modes should be quick to question then adjust the microelectrode and its placement AXOCLAMP 2A is a sophisticated instrument Even experienced researchers are advised to read this manual thoroughly and to familiarize themselves with the instrument using model electrodes i e resistors and cells e g parallel RC before attempting experiments with real microelectrodes and cells We will be pleased to answer any questions regarding the theory and use of the AXOCLAMP 2A Any comments and suggestions on the use and design of the AXOCLAMP 2A will be much appreciated We would be most grateful for reprints of papers describing work performed with the AXOCLAMP 2A Keeping abreast of research performed helps us to design our instruments to be of maximum usefulness to you who use them Axon Instruments Inc AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUM
70. etting The dial markings not meaningful Calibrated dials are used for these controls because they have brakes to prevent accidental movement The normal procedure for using the Offset controls is to zero the voltmeter readings when the microelectrode is outside the cell All subsequent readings are then with respect to the potential of the extracellular solution OUTPUT FILTER Built in filters are provided to smooth the 10 V and Im outputs These are single pole lowpass filters Six 3 dB frequencies fi can be selected As well as reducing the noise a filter also slows the rise time of the filtered signal A single pole filter converts a step into an exponential There is no overshoot The time constant of the exponential is Tfi The 10 90 rise time of the exponential is te 2 27 The six available f s and the corresponding 7 s and t s are given in Table 3 TABLE 3 fi kHz 0 1 0 3 E 3 10 30 ps 1600 530 160 53 16 5 3 te us 3500 1200 350 120 35 12 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 51 High Order Lowpass Filters For Low Noise Recordings The order of a filter refers to the number of poles RC sections For example a third order filter has three poles Each pole attenuates the high frequency noise at 20 db decade During TEVC the current noise increases at 20 db decade above a frequency determined by the me
71. f most microelectrodes changes with time and with current passing R is affected not only by the magnitude of the current but also by its polarity In general microelectrodes of lower resistance are more stable during current passing than microelectrodes of higher resistance Filling Solutions The best filling solution to use depends on the preparation under investigation and the experience of the investigator Although KCI gives one of the lowest tip resistances for a given tip diameter it is not necessarily the fastest to settle after a current pulse K citrate is sometimes faster It is important to be aware that during current passing large amounts of ions from inside the microelectrode can be ionophoresed into the cell For example if current is passed by the flow of ion species A from the microelectrode into the cell then after 50 seconds of current at 1 nA or 1 second of current at 50 nA the change in concentration of A inside a cell 100 um in diameter is 1 mM If A is an impermeant ion the cell may swell due to the inflow of water to balance the osmotic pressure Recommended Reading A small book by Purves 1981 serves as an excellent general reference for microelectrode techniques AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS 45 MODEL CELLS We recommend that you practice using the AXOCLAMP 2A on an RC cell model The resistor provided wit
72. fficulty of servicing the product This warranty is in lieu of all other warranties expressed or implied Axon Instruments Inc AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC This page is intentionally left blank AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC WARRANTY RETURN MERCHANDISE AUTHORIZATION C 1 RETURN MERCHANDISE AUTHORIZATION RMA No Date of RMA Shipping check list 1 Package instrument with at least 3 inches of packing material all around 2 Enclose a completed copy of this form 3 Write RMA number on outside of package 4 Pre pay freight for door to door delivery Modei Serial No In warranty Outof warranty Customer s purchase order No not required for warranty repair DESCRIPTION OF PROBLEM Customer s Shipping Address Customers Billing Address Name Name Phone Phone Send completed form with merchandise to Axon Instruments Inc 1101 Chess Drive Foster City CA 94404 U S A Write RMA number on outside of package AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC RETURN MERCHANDISE AUTHORIZATION m HARVEY J KARTEN MD UNIVERSITY OF CALIFORNIA SAN DIEGO DEPARTMENT OF NEUROSCIENCES 9608 500 GILMAN DRIVE LA JOLLA 92093 0608 This page is intentionally left blank AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT
73. g microelectrode and the input noise of the buffer amplifier The excess noise becomes progressively larger as the microelectrode time constant is reduced In discontinuous systems the microelectrode time constant must be reduced more than in continuous systems so that after a current pulse the microelectrode voltage will decay to Vm within the time allotted for passive recording The excess noise due to optimizing the capacitance neutralization can vary from a factor of about two in a system where primary efforts have been taken to keep the input capacitance low to much larger factors in systems where large amounts of capacitance to earth and capacitance to shield are tolerated The second major reason for the deterioration in noise performance of discontinuous microelectrode clamps has to do with the sampling process As discussed in the section on the Anti Alias Filter sampling processes alias the noise in the input signal spectrum into a larger magnitude spectrum confined to a bandwidth equal to half of the sampling rate f The normal procedure used in digitizing systems to avoid aliasing is to reduce the bandwidth of the input signal to f 2 or below This is not possible in discontinuous microelectrode clamping because reducing the bandwidth of the microelectrode increases the time constant and therefore prevents adequate settling The amount of aliased noise depends in part on the current duty cycle used in the discontinuous clamp The 30 duty cy
74. g voltage clamp are not affected Which Headstage To Use The value required depends on the typical input resistances Rin of your cells recommended values are in Table 2 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 33 TABLE 1 How H affects control and measurement ranges x10 1 0 1 HO Ro 1 10 MQ 100 MQ Max Bridge Balance 10 MQ 100 MQ 1000 MQ Max Step Command 1999 nA 199 9 nA 19 99 nA Max DC Current Command 1000 nA 100 nA 10 nA Ext Command 100 nA 10 nA V 1 nA V Max Total Current 6000 nA 600 nA 60 nA I Output 1 mV nA 10 mV nA 100 mV nA Max Gain in dSEVC 1000 nA mV 100 nA mV 10 nA mV Max Gain in cSEVC 10000 nA mV 1000 nA mV 100 nA mV Max Gain in TEVC 10000 10000 10000 1 For x0 01 replace by GQ nA by pA in x10 column For x0 0001 replace by GQ nA by pA in x0 1 column For x100 replace MQ by nA by in x0 1 column 2 Measured with electrode resistance Re Ro TABLE 2 Recommended H values for various cell input resistances x10 for 300kQ0 H 3 MQ H 1 for 3MQ lt 30 MQ H x0 1 for 30MQ2 lt Ra lt 300 MQ H x0 01 for Rin o 300 MQ H x0 0001 for ion sensitive electrodes Some overlap in these recommendations is allowable The guiding principles are these 1 2 3 4 G For maximum sampling rates in dSEVC
75. h Page 11 QUICK GUIDE TO OPERATIONS The controls and operation of the AXOCLAMP 2A are very briefly described in this section Detailed explanations are given in the alphabetically organized Section E of this manual D1 HEADSTAGES 1 HS 2 Series HS2 series headstages are standard Two supplied with AXOCLAMP 2A All HS 2 headstages record voltage at unity gain 1 6 B DLC TEA zt Available in several headstage current gains H Front panel controls read directly in indicated units when xl values are powers of 10 Small values used with high resistance cells and electrodes Large H values used to pass large currents x10 0 1 0 01 for recording and clamping 0 0001 for ion sensitive electrodes Headstages normally supplied in L version low noise low capacitance neutralization range M version can be supplied to compensate large capacitance of grounded shield Red connector Microelectrode input Gold Connector Driven shield case Yellow connector Ground output 2 HS 4 Series Optional for current passing electrode ME2 in two electrode voltage clamp Requires VG 2 for current measurement Bypasses internal current setting resistor during two electrode voltage clamp so output voltage applied directly to electrode Supplied in L or M versions only When AXOCLAMP 2A is not in two electrode voltage clamp mode HS 4 operates same as HS 2 3 VG 2 Series Optional
76. h each headstage can be conveniently used to simulate the microelectrode and the RC cell model can be soldered directly to the free end see Fig 6 If two electrode voltage clamping is being practiced it is important to place a grounded shield between the model electrodes and between the headstages grounded shield 55 ME1 headstage z Notes 1 and Ro are resistors to simulate the microelectrodes 2 Rm and Cm are a resistor and capacitor to simulate the cell FIGURE 6A SUGGESTED CELL MODEL AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 46 DETAILED GUIDE TO OPERATIONS The CLAMP 1 Model Cell If you do not need to model your cell exactly the CLAMP 1 Model Cell shipped with your AXOCLAMP 2A is a convenient model to work with The cell and electrode components simulate a small to medium sized cell having an input resistance of 50 MQ a membrane time constant of 25 ms and electrode resistances of 50 0 See Figure 6B The case of the model cell is connected to ground Shielding between the two electrode resistors is effected by the body of the switch Install the model cell by plugging it into one or both of your headstages Connect the gold plated ground jack to the yellow jack on the back of the MEI headstage using the cable provided Do not make connection to the gold plated jack on the front of the HS 2 headstage this is connected to the headstage cas
77. he single electrode technique is extremely rewarding because it allows voltage clamps to be performed in preparations where two electrode voltage clamping is just not feasible As well the signal to noise ratio in many preparations during discontinuous single electrode voltage clamp is despite the above considerations adequate for data to be analyzed without averaging AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 50 DETAILED GUIDE TO OP RATIONS OFFSET CONTROLS The Offset controls compensate for the junction potentials in the experimental setup The offset compensation for the V2 output works by adding a DC voltage to the output Therefore it is called the Output Offset control i The offset compensation for the 10 Vm and V outputs is performed in the first stage of the recording circuit This is necessary so that after amplification of the input signal the full range of the sample and hold circuitry can be utilized The ME1 offset compensation should not be altered during voltage clamp because the voltage clamp circuitry will interpret the change i in the offset setting as a change in Vm To remind you of this important Characteristic the control is called the Input Offset For both controls the compensation range i is 500 mV The no compensation point is in the middle of the range of the multi turn dials Each turn of the dials is approximately 100 mV The dials can be locked after s
78. icroscope The selected functions are activated by HIGH logic levels applied to the appropriate pin New Modes are selected and kept after a HIGH level of 1 us or more in duration Buzz and Clear are activated for the duration of the HIGH level Using the Remote facility does not disable the front panel switches The pin connections for the Remote connector are as follows DIGITAL Ground 5 V output BRIDGE mode DCC mode SEVC mode TEVC mode CLEAR MEI CLEAR MEI Not used Not used Not used CLEAR ME2 CLEAR ME2 BUZZ ME2 BUZZ MEI fem eo ot mh pmo rtr NO ON DAL To use the Remote controls the external control signals can be wired to a 15 pin D type connector which can then be plugged into the Remote connector on the rear panel 5 V is provided for wiring up any remote switches you may use Do not short circuit this supply The Mode Select inputs pins 3 6 have 50 kf input resistances the other inputs pins 7 8 12 15 have input resistances The FS 3 footswitches provided with the AXOCLAMP 2A consist of a pair of normally open switches for activating Buzz of each electrode If footswitches are not convenient you can easily connect your preferred switches by following the wiring diagram below For remote operation of microelectrode 1 Buzz and microelectrode 2 Buzz AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 56 DETAILED GUIDE T
79. into ME1 current command Voltage on this input converted into ME2 current command Used to compensate voltage drop across membrane R during TEVC Not normally required See service manual for suggested circuit Bath potential recorded by other equipment subtracted from and if connected to this input Allows certain functions to be remotely activated by computer or switches These are Mode Buzz and Allows sampling clocks from two AXOCLAMP 2As to be synchronized This eliminates electrode cross talk when two AXOCLAMP 2As in dSEVC mode used to clamp two cells in same preparation Requires LU 1 link up cable AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 15 DETAILED GUIDE TO OPERATIONS ANTI ALIAS FILTER A property of all digital sampling systems is that noise in the input signal at frequencies greater than 0 5 of the sample rate fs is folded down to appear as extra noise the bandwidth from zero to 0 5 of f see section on noise This phenomenon is known as aliasing Aliasing be overcome by filtering the input signal before sampling thereby reducing the high frequency noise content However this filtering procedure degrades the dynamic response of the input signal and when used with an ideal microelectrode worsens the clamp performance The voltage across a real microelectrode often has a two phase decay after the end of a
80. lamp MEI is the voltage recording electrode and ME2 is the current passing electrode The output of the clamp is a voltage source in contrast to SEVC modes in which the clamp output is a current source which is connected to ME2 The voltage clamp gain control is marked in units of V V This refers to how many volts the output will change by for each volt of difference between Vin the membrane potential and the command potential For example when the gain is at its maximum value of 10 000 V V a 100 V difference between Vm and would cause the output to shift by 1 V If the resistance of ME2 was 10 0 there would be a current of 100 nA The best settings of the voltage clamp parameters are found by setting up the best possible response to a step change in V Usually the ability of the voltage clamp to follow a step change in command is identical to the ability of the voltage clamp to follow a step change in membrane conductance Finkel amp Gage 1985 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 76 DETAILED GUIDE TO OPERATIONS Factors affecting the voltage clamp response are The Gain control determines the steady state accuracy and the response speed The Phase control introduces a combination of phase lag and phase lead a zero in the voltage clamp amplifier The Holding Position control shifts the clamped membrane potential The Capacitance Neutralization se
81. layer must be insulated from the preparation solution For different approaches to this method see Schwartz amp House 1970 Suzuki Rohligek amp Fr mter 1978 Sachs amp McGarrigle 1980 and Finkel amp Redman 1983 Shielding the electrode introduces high frequency noise therefore it should only be done when absolutely necessary The amount of added noise is proportional to the amount of shield capacitance so only the minimum necessary length of microelectrode should be shielded Because of the distributed nature of the axial resistance of the microelectrode of the axial resistance of the metal layer and of Ci the shielding technique is not perfect In practice the effect of these nonidealities is to cause the step response of the microelectrode to overshoot even when the Capacitance Neutralization gain is unity For this reason the Capacitance Neutralization circuit has a minimum less than unity Microelectrode Resistance Another important aspect of the microelectrode is the tip resistance Re This Should be as low as possible consistent with good impalements of the cell There are two advantages associated with low values of Re Settling Time The decay time constant for the microelectrode voltage after a current pulse depends strongly on Re Hence lower Re values produce faster settling times As well high Re values are sometimes associated with a slow final decay even after C has been eliminated Stability Re o
82. ll only be seen when the Blanking period ends Usually this deviation is preferable to the situation that can occur if Blanking is not used If Blanking is not used the artifact picked up by MEI is treated by the voltage clamp circuit as an attempt by the cell to change its potential Therefore the voltage clamp circuit causes a current to be passed into the cell to clamp this presumed membrane potential change If the stimulus artifact is large the consequent current artifact can be large enough to damage the cell AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 17 The width of the Blanking period should be no longer than the minimum width required to cover the period of the stimulus artifact It is important not to Blank for longer than necessary since during Blanking no updating of Vm is allowed Even when Blanking is used attempts should still be made to minimize the artifact at the source BRIDGE MODE Description In Bridge mode the microelectrode voltages are monitored continuously and continuous currents can be injected down ME1 or ME2 Associated with the current flow I in a microelectrode is a voltage drop across the microelectrode which depends on the product of the current and the microelectrode resistance Re This unwanted IR voltage drop adds to the recorded potential The Bridge Balance control can be used to balance out this voltage drop so that only me
83. lly bypasses the current measuring resistor during two electrode voltage clamp mode In all other modes HS 4 headstage behaves like an HS 2MG headstage with xl Must be used in conjunction with a VG 2 virtual ground headstage VG 2 virtual ground headstage Measures total bath current The virtual ground output attenuation VG is available in three values specify with order x 0 1 1 standard and x10 The output ivin is 10 VG mV nA ORDERING INFORMATION When ordering please specify 1 Current gain H of two headstages provided 2 Gain and type of any extra headstages 3 Diameter D of headstage mounting rods Unless you specify otherwise the AXOCLAMP 2A will be supplied with one HS2L x1 and one HS 2L 0 1 headstage each with D 5 16 7 9 mm AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 88 SPECIFICATIONS This page is intentionally Icft blank AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC REFERENCES A 1 REFERENCES Brennecke R amp Lindemann B 1974 Theory of a membrane voltage clamp with discontinuous feedback through a pulsed current clamp Rev Sci Instrum 45 184 188 Finkel A S amp Gage P W 1985 Conventional voltage clamping with two intracellular microelectrodes In Voltage Clamping with Microelectrodes ed T G Smith et al Williams amp Wilkins Baltimore Finkel S amp Re
84. mbrane potential is recorded The term Bridge refers to the original Wheatstone Bridge circuit used to balance the IR voltage drop and is retained by convention even though the circuitry has been replaced by operational amplifier techniques The particular setting required to balance the Bridge is a measure o icroelectrode resistance In cSEVC mode the Bridge potentiometer compensates electrode IR voltage drop at one tenth sensitivity Suggested Use Set the Destination switch to 1 2 and externally trigger the Step Command generator so that pulses of current are repetitively injected into ME1 2 Alternatively derive the command for injecting current pulses by connecting a signal source to the Ext ME1 2 Command input Start with the Bridge Balance control set to zero Advance the dial until the fast voltage steps seen at the start and finish of the current step are just eliminated The Bridge is correctly balanced The residual transient at the start and finish of the current step is due to the finite response speed of the microelectrode No attempt is made to balance this transient since it covers a very brief period only and it is a useful indication of the frequency response of the microelectrode The transient can be minimized by correctly setting the Capacitance Neutralization The Bridge balancing procedure is illustrated in Fig 2 The trace in A was recorded in model cell when the Bridge Balance control was correctly
85. mbrane time constant Finkel amp Gage 1984 To adequately limit this noise the filter used for data display and storage should be at least 2 order and preferably 3 4 order Rise Time Of High Order Filters As a rule of thumb it can be noted that for lowpass multiple pole filters having less than 10 overshoot the 10 90 is time is within a few percent of t in a single pole filter having the same 3 dB frequency However the frequency specified for many multiple pole lowpass filters is the 3 dB frequency of the component lower order filters instead of being the 3 dB frequency of the complete filter Before using these filters it is advisable to check the 10 90 rise time of a step signal applied to the input Note On Ultimate Rise Time When a signal with 10 90 rise time t is through a filter with 10 90 rise time tz the rise time of the output signal is au zd ti t2 OUTPUT IMPEDANCE AND PROTECTION All outputs are by 560 0 output resistors All outputs can withstand a continuous short circuit to ground or any voltage in the 15 V range However in keeping with normal such short circuits should be avoided PANEL METERS Three digital panel meters DPMs are provided to continuous display the DC level of some of the important outputs These displays are mV This DPM indicates the membrane potential in all modes It is derived from the 10 Vm output maximum displayed val
86. n Oscillation helps cell penetration Footswitches supplied as standard accessories Bridge Compensates electrode voltage drop during current passing Resistance scaled by H read on ten turn dial Range automatically reduced tenfold during cSEVC Input Offset Adds 500 mV DC to electrode voltage at early stage Use to zero electrode voltage while extracellular DC Current Command injection of constant current Magnitude set on ten turn dial Polarity set on switch LED indicates when current injection activated Clear Passes large hyperpolarizing and depolarizing current to clear blocked electrodes or help cell impalement Voltmeter Indicates membrane potential Vm in mV D4 MICROELECTRODE 2 ME2 GROUP An independent intracellular extracellular electrometer similar to MEI Differences are Potential is labelled V2 Output offset adds 500 mV to electrode voltage in output stage AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC QUICK GUIDE TO OPERATIONS Page 13 D5 VOLTAGE CLAMP GROUP Gain Sets open loop gain during voltage clamp In SEVC modes output is current source Therefore gain is nA mV In TEVC mode output is voltage source Therefore gain is V V Holding Position Sets holding potential during voltage clamp Range 200 mV RMP Balance Lamps Null during Bridge or DCC so that when activated voltage clamp will be at resting membrane potential
87. n and maximum current capacity Imax H x0 0001 for ion sensitive electrodes H x0 01 for Rin greater than about 300 MQ x0 1 for Ria about 30 300 MQ x 1 standard for Rin about 3 30 MQ x10 for Rin about 300 to 3 These ranges are suggested for optimum performance Some overlap is allowable Imax 1000 x H nA 5 rms measured with a 10 kHz single pole filter in the measurement circuit 51 47 rms measured with 10 100 MQ source resistance and capacitance neutralization adjusted for a 10 1 kHz bandwidth and with a 10 1 kHz single pole filter in the measurement circuit Values are for H x1 x0 1 HS 2L headstage 16 54 ps for a voltage step applied to the input via a 10 100 MQ low capacitance resistor and 16 60 us for a current step into the same resistor Capacitance neutralization adjusted for zero overshoot Values are for H x1 x0 1 COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC SPECIFICATIONS Page 83 Working Input Voltage Range 13 V for transients and steady state protected to 30 V Input Resistance 1014 1015 0 x 0 0001 see note 1030 x 01 1020 H x0 1 101 Q Hz x1 1090 x 10 Note For the x0 0001 headstage the input resistance of each headstage is measured individually The unique test results are supplied with each x0 0001 headstage Input Capacitance Not relevant See 1 settling time and noise specifications
88. of a correctly set up dSEVC is shown in Fig 12 AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 68 DETAILED GUIDE TO OPERATIONS Fig 12 An example of a correctly set up dSEVC in cell model Rm was 100 MQ Cm was 33 pF Rej was 100 MQ Gain was 1 nA mV HS 2L headstage x0 1 Sampling rate was 7 kHz Voltage command was a 10 mV step No Phase Shift or A A Filter Capacitance Neutralization was optimum Recording bandwidth in A was 1 kHz A Top trace Membrane current Cal 4nA 1 ms Middle trace Sampled membrane potential available at the 10 Vm output Cal 10 mV 1 ms Lowertrace True membrane potential recorded by an independent electrode Cal 10 mV 1 ms Note that the two voltage records are identical because the Capacitance Neutralization was correctly set B Multiple sweeps of the Monitor waveform This photo was taken with the cell held at rest The current pulses vary from sweep to sweep because of the sampled voltage noise The important feature is that the voltage transients decay completely by the time the samples are taken arrow even for the largest transients Cal 20 mV 40 ys AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS 69 4 10 mV 20 mV 1 ms 40 us FIGURE 12 CORRECTLY SET UP dSEVC IN A CELL MODEL AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUAR
89. of the electronics is the right hand side looking from the front thick sheet of steel placed between the AXOCLAMP 2A and the radiating equipment can effectively reduce the induced hum Initially make only one connection to the AXOCLAMP 2A This should be to the oscilloscope from the V 10 V outputs Ground the ME1 headstage input through a 1 MQ resistor to the yellow ground connector After verifying that the hum levels are low start increasing the complexity of the connections one lead at a time Leads should not be draped near transformers which are inside other equipment In desperate circumstances the continuity of the shield on an offending coaxial cable can be broken Try grounding auxiliary equipment from a ground distribution buss This buss should be connected to the AXOCLAMP 2A via the yellow 0 16 inch 4 mm socket on the rear panel This socket is connected to the AXOCLAMP 2A s Signal ground i e the outer conductors of all the BNC connectors signal ground in the AXOCLAMP 2A is isolated from the chassis and power ground l 5 2 772 If more than headstage is used all the headstage cables should run from the AXOCLAMP 2A to the preparation in a bundle The bundle can be formed either by at twisting the cables together or by loosely tying them together Experiment While hum can be explained i in n theory e g direct pickup earth loops in practice the ultimate theory is the end result Following th
90. olding capacitor to charge up to the value of Vm S2 opens again after the sample period and Vm is held by buffer amplifier A2 interfaces to the recording apparatus This switch capacitor and buffer amplifier arrangement constitute an analog memory known as a sample and hold amplifier AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 26 DETAILED GUIDE TO OPERATIONS Immediately after the sample period the current injection period begins when switch S1 changes over from the 0 volts position to the current command voltage position This connects to differential amplifier A4 arranged so that its output is Vi V The voltage appearing across Ro is exactly equal to thereby forcing the current L into the microelectrode to be equal to V Ro Amplifiers A4 and 1 and resistor R constitute a controlled current source CCS which injects a current into the microelectrode directly proportional to the voltage at the input of the CCS irrespective of the resistance of the microelectrode or the voltage at its tip AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 27 58 0 volts Inside V CRO 0 volts S1 1 7 sample sample FIGURE 4 DCC MODE BLOCK DIAGRAM AND TIMING DIAGRAM AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page
91. ometimes useful to inject a brief small current pulse at the start of each oscilloscope sweep in order to continually check the Bridge Balance setting during the course of an experiment Figure 2 Illustration of Bridge balancing technique All traces were recorded from the 10 V4 output The model cell was 10 0 1 nF was 10 MQ Recording bandwidth 30 kHz Vertical calibration 20 mV referred to Vm A Response to 5 nA 10 ms current pulse Bridge correctly balanced Trace is membrane response only Cal bar 20 ms B Response to 5 nA 1 ms pulse Cal bar 1 ms Top trace Bridge balance used Fast voltage steps at start and finish of the current pulse are the electrode IR voltage drop Middle trace Bridge correctly balanced Trace is membrane response only Transient electrode response remains Bottom trace Bridge balance overused Negative going step is introduced by the Bridge Balance circuit AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS 19 Lo T4 Cell FIGURE 2 BRIDGE BALANCING PROCEDURE AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 20 DETAILED GUIDE TO OPERATIONS BUZZ When the Buzz switch or the footswitch is depressed the amount of Capacitance Neutralization is increased If the Capacitance Neutralization control is within a few turns of optimum
92. on is see Brenneke amp Lindemann 1974 Finkel amp Redman 1984 0 lt lt 2 Cm f For critical damping we require G zm 1 Cm fs Thus for a given if Cm is small f must be large As an example if G 1 nA mV and Cm 100 pF then f must be 10 kHz for critical damping If f is less than 10 kHz in this example the step response will overshoot and at 5 kHz the clamp will oscillate destructively If the sampling rate in this cannot be as great as 10 kHz because the microelectrode diam is too slow then a lower value of G will have to be used to maintain stability Clamp Error With finite gains in the voltage clamp circuit Vin does not quite follow V The error is Ve Vm Similarly if V is constant and the cell membrane conductance changes there is an error in the measurement of the current underlying the conductance change This error is similar in percentage to the voltage error Usually the gain of the voltage clamp circuit be increased so that e is 10 or less The percentage error depends on the frequency of the command signal or of the conductance change It is smallest for slow signals and DC and largest for the fastest signals Thus very fast transients such as the rising phase of synaptic currents will be clamped less well than slower transients such as the decay phase of synaptic currents Gain The clamp gain during dSEVC mode is given in nA mV This refer
93. onse of real membranes The controls are in two parts a potentiometer to shift from lead to lag and a 4 position switch to set the time constant Phase lead boosts the high frequency gain of the voltage clamp circuit In some preparations this can be used to sharpen the step response and improve the voltage clamping of fast conductance changes On the debit side use of phase lead increases the noise and can also cause high frequency oscillations To reduce the risk of oscillations the phase control circuit is arranged to always introduce some phase lag with the phase lead The added phase lag restricts the maximum increase in the high frequency gain to a factor of 2 achieved when the potentiometer control is turned to the extreme lead position Phase lag cuts the high frequency gain of the voltage clamp circuit This can be used to reduce the noise but at the same time it slows the response and introduces ringing In the extreme lag position the phase control circuit introduces pure lag The action of the Phase Shift potentiometer can be summarized as follows In the extreme lead position a combination of phase lead and lag is introduced such that the high frequency gain is doubled The amount of phase lead is gradually reduced by counterclockwise rotation of the potentiometer and falls to zero at the extreme lag position At the same time the amount of phase lag cutting the high frequency gain is increased by counterclockwise rotation of th
94. orrectly set The full 600 mV input referred cage is available in DCC and dSEVC modes irrespective of the current The In output is proportional to the membrane current In Bridge cSEVC and TEVC modes it is the continuous electrode current In DCC and dSEVC modes Im is found by E the c current nt during the Suter DARING period and multiplying by the duty cycle AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC SPECIFICATIONS Page 82 SPECIFICATIONS MODES Five main operating modes selectable by color coded illuminated push buttons remotely These are 1 Bridge 2 DCC Discontinuous Current Clamp 3 dSEVC Discontinuous Single Electrode Voltage Clamp 4 cSEVC Continuous Single Electrode Voltage Clamp 5 TEVC Two Electrode Voltage Clamp MICROELECTRODE AMPLIFIERS Two Channels Unity Gain Headstages Hum line frequency pickup Headstage Current Gain H Maximum Current Noise with grounded input Noise with a source resistance 1 Settling Time AXOCLAMP 2A THEORY amp OPERATION Standard is the HS 2L type HS 2M types are the same except 1 the noise is greater by about 20 2 the capacitance neutralization range is extended HS 2MG types are similar to the HS 2M types mom that the case is grounded instead of driven Less than 10 peak to peak grounded input Available in 5 values specify two with order Select on basis of cell input resistance Ri
95. orsening the response speed This is illustrated in Fig 8 Note that even though the recorded potential is made faster by using the Phase controls the true membrane potential and current are not speeded up 3 In TEVC electronically subtract from the command voltage a voltage equal to the product of the membrane current and the presumed series resistance To do this you would need to use an external potentiometer to find a fraction of Ib and feed it into the rear panel R COMP input What is the True Membrane Potential Time Course For an isopotential cell the time course of the true membrane potential is the same as that of the recorded membrane current The recorded potential which includes the voltage drop across Re may be much faster See Fig 8 for an illustration of this effect In a non isopotential cell for example a neuron with an and dendrites the true membrane potential recorded at the tip of the voltage recording electrode will in fact settle faster in response to a step voltage command than will the membrane current In this situation the presence of a series resistance will exaggerate the difference in time courses Fig 8 Membrane potential and current during TEVC Cell model was Rm 10 0 Cm 1 nF 300 KQ Ra Re 10 MQ For all traces recording bandwidth was 30 kHz Gain was 700 V V Vertical calibration 10 mV div Horizontal calibration 1 ms div ub B Upper trace is true membran
96. p commands CALIBRATION SIGNAL A pulse equal in magnitude to the setting on the thumbwheel switch is superimposed on the voltage and current outputs for the duration of a HIGH control signal on the CAL ACTIVATE input AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 86 SPECIFICATIONS BATH POTENTIAL COMPENSATION Signal recorded by bath headstage or by an external amplifier is automatically subtracted from the intracellular measurements If bath potential is not measured the system automatically reverts to using as the reference potential Standard headstages HS 2 work as bath headstages when plugged into the bath headstage connector VIRTUAL GROUND CURRENT MEASUREMENT A VG 2 virtual ground headstage can be plugged into the connector provided The current measured is the sum of all currents into the preparation The correct operation of the AXOCLAMP is not affected by the use or nonuse of virtual ground current measurement REMOTE Logic HIGH control signals activate BUZZ and CLEAR of each microelectrode and select between BRIDGE DCC SEVC and TEVC modes 15 pin connector MODEL CELL A model cell is provided with the AXOCLAMP 2A Electrodes are 50 0 cell is 50 MQ 500 pF A switch grounds the electrodes directly BATH mode or through the cell CELL mode Special plugs connect directly to the GROUNDING Signal ground is isolated from the chassis and
97. ped at its resting value irrespective of the clamp gain A remarkable BLANK facility can be used to force the voltage clamp system to ignore stimulus artifacts that would otherwise be picked up by the voltage recording circuit and result in large current artifacts which could damage the cell under clamp A Monitor output enables the input to the sampling circuit to be observed It is essential to observe this signal during dSEVC to ensure that the microelectrode voltage due to current passing has time to adequately decay at the end of each cycle An oscilloscope trigger signal at the sample rate is provided for use with the Monitor signal The AXOCLAMP 2A allows very fast discontinuous single electrode voltage clamping a test cell see specifications the 10 to 90 rise time is only 100 us In a real setup the response speed is limited by the microelectrode characteristics but membrane potential rise times without overshoot of less than 1 ms have been regularly achieved in a variety of cell types Two electrode voltage clamping is much faster Two controls for each microelectrode are devoted to clearing blocked microelectrode tips and assisting cell penetration is a Clear switch which can be used to force large hyperpolarizing or depolarizing currents through the microelectrode The other is a BUZZ switch which causes the mocroelectrode voltage to oscillate Depending on the microelectrode and the preparation one of
98. power ground CONTROL INPUTS Above 3 V is accepted as logic HIGH Below 2 V is accepted as logic LOW Inputs are protected to 15 V v HEADSTAGE DIMENSIONS Case is 2 25 x 1 14 x 0 87 57 2 x 29 0 x 22 1 mm Mounting rod is 4 102 mm long Available mounting rod diameters are 1 4 5 16 or 3 8 6 3 7 0 or 9 5 mm Specify required mounting rod diameter with order Input sockets for the microelectrode shield and ground are 0 08 2 mm diameter Cable length is 10 feet 3 m AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC SPECIFICATIONS Page 87 CASE DIMENSIONS 7 177 mm high 19 483 mm wide 12 5 317 mm deep Mounts in standard 19 rack Handles are included Net weight 18 lbs 8 kgs SUPPLY REQUIREMENTS Line voltage 100 125 Vac or 200 250 Vac User selectable by an internal switch Line Frequency 50 60 Hz Power 20 W Fuse 0 5 A slow 5x20 mm Line Filter RFI filter is included Line Cord Shielded line cord is provided ACCESSORIES PROVIDED Operator s amp Service Manuals 2 mm plugs for use with headstages Low capacitance test resistor for each headstage Spare globes for Mode switches Spare fuse Footswitches to operate Buzz of both electrodes Clamp 1 Model Cell Remote Buzz Duration hand held control OPTIONAL ACCESSORIES not required for normal operation HS 4 Relay Switched Headstage Miniature relay inside headstage automatica
99. rial and error Like Buzz the mechanism for impalement is unknown COMMAND GENERATORS Command levels for voltage clamp or current clamp can be obtained from the internal step command generator from the internal DC command generators and from external sources Step Command Generator The Step Command generator can be used either as a current clamp or voltage clamp command j depending on the position of the Destination switch If the Destination switch is used to select VC then the magnitude on the thumbwheel switch represents voltage clamp potential in mV s irrespective of the headstage current gain H If the Destination switch is used to select ME1 or ME2 then the magnitude on the thumbwheel switch represents the number of nA of current to be injected down ME2 respectively The current range is scaled by the H The maximum magnitude on the thumbwheel switch is 199 9 corresponds to depolarizing voltage shifts and currents corresponds to hyperpolarizing voltage shifts and currents AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 23 The tion for which the Step Command is activated can be made continuous by switching the Ext Cont Off toggle to Cont or externally determined by a logic HIGH level on the rear panel Step Activate input When rotating the thumbwheel switch in continuous mode be decisive If the switch is rotated slowly the purs
100. ring DCC mode the input to the CCS and the output of the ME1 current monitor are automatically scaled so that they represent the true membrane current even though the instantaneous current flows for only 30 of the time The cycling sampling rate must be chosen so that there are ten or more cycles per membrane time constant This enables the membrane capacitance to smooth the membrane voltage response to the current pulses Suggested Use Turn the Anti Alias Filter to the minimum value and switch to DCC mode Set the Destination switch to MEI and set up a repetitive square current command Observe Im and 10 Vm on the main oscilloscope Observe the voltage at the Monitor output on a second oscilloscope which need not be a high quality type with the gain at 100 mV div 10 mV div input referred Trigger this oscilloscope from the Sample Clock output on the rear panel Proceed to adjust the Capacitance Neutralization in one of two 1 For acceptable but not optimum Capacitance Neutralization advance the Capacitance Neutralization control until the square step at the leading edge of the 10 V4 response is first eliminated B For optimum Capacitance Neutralization switch the Step Command generator to continuous Advance the Capacitance Neutralization control until the Monitor waveform decays most rapidly to a horizontal baseline but without any overshoot These techniques are illustrated in Fig 5 The traces in Fig SA
101. s derived from V see Fig 7 After amplification by 10 is filtered by the Anti Alias Filter The output of the Anti Alias Filter is the input of the sample and hold device and the signal provided to the Monitor output A baseline correction circuit compensates for shifts in V so that Vmon always decays to zero This prevents from moving off the oscilloscope screen when the holding potential is shifted during voltage clamp BASEL INE CORRECTION Monitor output MI To rest Anti of circuit alias filter FIGURE 7 ANTI ALIAS FILTER amp MONITOR CIRCUIT AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 49 NOISE IN DCC AND dSEVC MODES The noise inherent in discontinuous microelectrode clamps discontinuous current clamp or discontinuous single electrode voltage clamp is four or more times worse than the noise in continuous microelectrode clamps bridge current clamp or two electrode voltage clamp when the discontinuous microelectrode clamps are adjusted for the same dynamic response and accuracy as the continuous microelectrode clamps There are two major reasons for this inherent deterioration in noise performance The first is to do with capacitance neutralization A fundamental property of all capacitance neutralization circuits is that they introduce noise in excess of what is contributed by the thermal noise of the recordin
102. s that most of the presumed membrane potential is in fact voltage drop across the microelectrode Which SEVC to use with a Suction Electrode In the previous section we discussed how a continuous SEVC can be planed by taking advantage of the low resistance of a suction electrode The problem with the cSEVC technique is the error introduced by Re which can only be partially overcome by series resistance compensation This problem can be completely avoided by using the dSEVC mode It turns out that the conditions when a suction electrode is used are ideal for dSEVC That is because Re is very small the electrode time constant is fast In addition the magnitude of the voltage transient across the electrode for a given current is proportional to Re and therefore small when Re is small This double advantage of low values means that the dSEVC can be cycled very rapidly without sampling error Fig 14 shows the result of a dSEVC in exactly the same cell model that was used in the cSEVC shown in Fig 0 The most significant difference in the set up besides the clamping mode used was the fact that no phase shift was used in the dSEVC Since the IR drop across the electrode was not sampled the recorded potential during dSEVC had the same time course as the membrane current and the true membrane potential recorded by an independent electrode not shown The disadvantage of the dSEVC mode was the additional current noise AXOCLAMP
103. s to how many nanoamps the output current will change by for each millivolt of difference between Vm the membrane potential and the command potential The value indicated on the front panel is the average value The average value depends upon the instantaneous gain during the current passing period and upon the duty cycle AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 75 SPACE CLAMP When interpreting the current measured during voltage clamp due consideration should be given to the adequacy of the spatial exterit of the membrane voltage control In general measurements of currents generated more than 0 1 electrotonic lengths from the point of the voltage clamp electrode s will be subject to significant error This problem is discussed in detail by Johnston and Brown 1983 TEN TURN POTENTIOMETERS The ten turn potentiometers used in the AXOCLAMP 2A are high quality wirewound types An inherent problem of wirewound potentiometers is that the wire elements tend to oxidize This condition is curable If a potentiometer becomes noisy the potentiometer manufacturer recommends rapidly spinning the knob 20 30 times between full clockwise and full counterclockwise This clears the oxide off the element and restores noise free operation E TEVC MODE Description In TEVC Two Electrode Voltage Clamp mode the AXOCLAMP 2A acts as a conventional voltage c
104. sce VEO na CREER EUN en inen EON Use DR a RE Sa 57 cec vu ve Da eoe e ade tt ua a banks CURVE tats caa 57 Probl MC M nus 57 SOLUTIONS 99 57 What is the True Membrane Potential Time Course 58 SEVC MODE CONTINUOUS redet e ERRAT 60 Important Note Anti Alias 60 Suggested Use eere teet o eee eese eese die aves acoge vado Ee eae 60 cSEVC Compared With Whole Cell Patch 62 SEVC MODE 5 08 2 22 264 Description sees Ee iu 64 lt 2 NEC E REINES 67 Important Note ete qa sn veta bsec ead eo qud 70 Which SEVC to use with a Suction 1 70 Minimum Sampling Rate and Maximum Gain tex cr bere bx ds ess 14 Clamp Sese Pot eade 74 74 SPACE 75 TEN TURN POTENTIOMETERS UE da Egg 75 jy 83H op cM TE 75 Description E M 75 SUS POS bed MSIE T P 76 Extremely Important
105. show that poorly adjusted Capacitance Neutralization during DCC mode is similar to poorly adjusted Bridge Balance during Bridge mode If the square step cannot be eliminated without overshoot on the Monitor waveform decrease the sample rate fa Set the Output Bandwidth to 1 5 or less of fs Reduce the noise on the 10 V4 and Im traces either by advancing the Anti Alias Filter or by increasing fs adjusting the capacitance neutralization where necessary AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS mE Page 29 Figure 5 cell Illustration of Capacitance Neutralization adjustment during DCC All traces were recorded with a model 10 0 1 nF was 10 MQ Cycling rate was 25 kHz A 10 Vm re Response toa 10 nA 1 ms current pulse Vertical calibration 20 mV referred t to Vm Horizontal calibration 1 ms B Vmon Output during the 10 nA current pulse Vertical calibration 40 mV referred to Vm Horizontal calibration 10 ps There are three pairs of corresponding traces Traces 1 Traces 2 Traces 3 Capacitance neutralization underutilized There was a fast step in Vm at the start and finish of the current pulse because decayed too slowly to reach its final value Capacitance neutralization optimum Vin shows the membrane response only Von decay was fast with no overshoot and easily reached the final value Capacitance neutraliza
106. t circuits for sampling clock synchronization each microelectrode model cell virtual ground current measurement VOLTAGE CLAMPING Voltage clamp with one or two microelectrodes your choice is dictated by the needs of your investigation the AXOCLAMP 2A does both Discontinuous Single Electrode Voltage Clamping dSEVC is based on the technique of sampling the membrane potential while zero current flows and then retaining this sampled value while current is injected into the cell This procedure is rapidly repeated to produce a smooth response Continuous Single Electrode Voltage Clamping uses a low resistance electrode to continuously record membrane potential and inject current The error caused by voltage drop across the electrode resistance can be partially reduced by series resistance compensation With Two Electrode Voltage Clamping TEVC one microelectrode is to continuously ee record membrane potential while the other is used to inject current E Dc MCCC LC CMM ET oa Gain of the voltage clamp amplifier is quickly set on a smooth acting nonlinear control phase response of the amplifier is altered from lead to lag by a Phase Shift potentiometer with a Center Frequency Switch to select the range A unique variable Anti Alias Filter helps reduce noise towards the theoretical minimum during dSEVC by slowing the response of the sampling circuit to suit the sample rate and the microelectrode response The Sample Rat
107. the headstage cover 3 Ground the headstage input via a resistor equal to 10 where Re is given in Table 1 On an oscilloscope at 2 mV div observe the 10V output through the filter set to 100 Hz Use the Offset control to center the trace on the screen 4 Now ground the headstage input via a resistor equal to Ro in Table 1 Observe the shift of the oscilloscope trace 5 Repetitively swap from grounding via Ro 10 to grounding via Ro Adjust the trim pot inside the headstage until there is no shift Note 1 For values of 1 GQ or more it is important to clean the surface of the resistor thoroughly to remove leakage pathways Depending on the reason for a trim being necessary the trim procedure may have to be repeated if the headstage is changed Warning If an external source is connected to the Ext MEI and ME2 Command input any time the source is non zero a proportional current will flow in the microelectrode Many external sources do not put out a true zero voltage when in the off state thus there may be an unwanted electrode current due to the fact that an external source is connected To avoid this use an external source in which you can adjust the off state voltage or use an isolated external source DC Removal One potential source of a small but variable input leakage current is due to DC current flow through the dielectric of the capacitor used for capacitance neutralization For example the electro
108. the input sockets of the headstages A switch allows the CLAMP 1 model cell to be configured as a BATH mode two 50 0 electrodes to ground b CELL mode two electrodes connected to a 50 MQ 500 pF cell The CLAMP 1 model cell can be used to test and practice using bridge current clamp discontinuous current clamp single electrode voltage clamp and two electrode voltage clamp It is a useful tool to use while learning the operation of the AXOCLAMP 2A and subsequently to verify the correct operation of the AXOCLAMP 2A and the recording pathway A third HS 2 headstage can be used extracellularly to record bath potential bath potential is then subtracted from the potentials recorded by the two intracellular microelectrodes to compensate for shifts in bath potential due to changing of solutions or temperature VG 2 Virtual Ground headstage may be used to measure total bath current Generally the built in current monitors are more useful since they yield the microelectrode currents separately without any interfering currents e g from ionophoresis Since both microelectrode amplifiers are complete one microelectrode can be used for ionophoresis while the AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC FEATURES QUALITY FURTHER INFORMATION AND ORDERING Page 7 other is used intracellularly Internally generated hum due to the built in power supply has been prevented by
109. the resistance of MEI should be as low as possible If two headstages with different Hs are used the one with the larger and therefore greater current passing ability should be used with ME2 TRIGGERED ee In some experiments i it is desirable to switch into lads clamp only when a specific event threshold is reached For example it may be desirable to switch into voltage clamp when the unclamped action potential goes above a predetermined level To do this an external device must be used to detect the event and signal its occurrence by putting out a logic HIGH The logic HIGH is then applied to pin 5 or 6 of the Remote connector on the rear panel of the AXOCLAMP 2A The AXOCLAMP 2A will then remain in voltage clamp mode until the logic HIGH is removed from pin 5 or 6 and a separate logic HIGH applied to pin 3 or 4 of the Remote connector AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 80 DETAILED GUIDE TO OPERATIONS TROUBLE SHOOTING It has been our experience at Axon Instruments that the majority of troubles Feported to us have been caused by faulty equipment connected to our instruments If you have a problem please disconnect all instruments connected to the AXOCLAMP except for the oscilloscope and one headstage Ground the headstage through the original test resistor supplied by Axon Instruments If the problem persists please call us for assistance UNITY GAIN RECORDING
110. the shield caused high frequency oscillations and noise The back end of the electrodes were 40 50 mm apart When the gap size was increased further the clamp went unstable Figure 15 The destabilizing effects of coupling capacitance Traces were recorded in a cell model Rm 10 MQ Cm I nF Ret Ro 10 MQ Gain 1000 V V recording bandwidth 10 kHz Phase Shift and Anti Alias Filter both off 10 mV step command Upper traces are 12 the memorane current Lower traces are Vm Electrodes were 40 50 mm apart MIS E A Extensive grounded shielding between the two electrodes B 2 3 mm gap in the grounded shield caused high frequency oscillations AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 78 DETAILED GUIDE TO OPERATIONS 200 nA 10 mV 100 ps FIGURE 15 AN EXAMPLE OF THE DESTABLIZING EFFECTS OF COUPLING CAPACITANCE AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS 44 79 Saturation During Capacitance Transient The output voltage of the AXOCLAMP 2A main unit during TEVC is 30 V This is usually sufficient to drive the current through ME2 required to charge the membrane capacitance during a step voltage change However for large steps in some cells the saturate and the time required to establish the step change will be longer than necessary Part of the reason for the s
111. tion overutilized fast steps Vm reappeared this time because of overshoot and ringing in Vmon Note that unlike a Bridge circuit the effect of two much compensation can put either a positive or a negative step on Vm positive in this example depending on which cycle of the ringing in Vmon is sampled AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 30 DETAILED GUIDE TO OPERATIONS FIGURE 5 HOW TO SET THE CAPACITANCE NEUTRALIZATION DURING DCC MODE AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 31 GROUNDING AND HUM A perennial bane of electrophysiology i is line frequency pickup noise often referred to as hum Hum can occur not only at the mains frequency but also at multiples of it The AXOCLAMP 2A has inherently low hum levels less than 20 peak to peak take advantage of these low levels great care must be taken when integrating the AXOCLAMP 2A into a complete recording system The following procedures should be followed 1 Q 3 4 5 6 Only ground the preparation bath by directly connecting it to the yellow ground connector on the back of the ME1 headstage or to a vier ground headstage if used Place the AXOCLAMP 2A in a position in the rack where transformers in adjacent equipment are unlikely to radiate into its electronics The most sensitive part
112. tivate Activate input For voltage outputs the magnitude of the Cal signal is igal is direct equal to the setting of the Step Command thumbwheel switch For example 123 4 will put 123 4 mV on the voltage outputs For current outputs the magnitude of the Cal Signal is 10x the setting of the Step Command thumbwheel switch For example 019 6 will put 196 mV on the current outputs The equivalent current depends In this example the Cal signal of 196 mV would correspond to 19 6 nA for H x1 1 96 nA for H x0 1 etc Suggested Use At the start of a recording sequence briefly activate Cal After a short interval activate the Step Command The Cal signal will be a permanent record of the command voltage or current CAPACITANCE NEUTRALIZATION AND INPUT CAPACITANCE The Capacitance at the input of the headstage amplifier is due to the capacitance of the amplifier input itself Cini plus the capacitance to ground of the microelectrode and any connecting lead Cin combined with the microelectrode resistance Re acts as a lowpass filter for signals recorded at the tip of the microelectrode Two techniques may be used to increase the recording bandwidth Primary A special technique is used in the headstages to keep the contribution to Cin from the input amplifier as small as possible This consists of adding the input signal voltage to the power supply voltages used by the input stages This t
113. tive of whether or not the bath potential is measured the preparation bath should be grounded by directly connecting it to the yellow ground connector on the back of the MEI headstage or to a virtual ground headstage if used BLANKING A common problem when using stimulating electrodes is that some of the stimulus is directly coupled into the recording microelectrode This can saturate subsequent high gain amplifiers and the coupling capacitors of AC circuits The saturation effects may take tens or hundreds of milliseconds to subside The best way to minimize or even eliminate this artifact is at the source by using small stimuli isolated stimulators placing an grounded shield between the stimulating electrodes and the microelectrodes etc Often though it is not possible to reduce the artifact to manageable levels AXOCLAMP 2A can circumvent the effects of the stimulus artifact by Blanking At the moment the logic level of the Blank Activate input goes HIGH the value of Vm is sampled and saved For the duration of the HIGH signal this saved value is used instead of the actual potential In voltage clamp modes the voltage clamp current during the Blanking period will be held at the level which existed at the start of the period A small deviation from the command potential may develop during the Blanking period as a result of comparing the command to the sampled value of Vm instead of the instantaneous value of Vm This deviation wi
114. trode Time course is the same as that of the membrane current Noise The current noise the 3 kHz bandwidth was 55 pA peak to peak If the gain was reduced so that the capacitance transient took 1 5 ms to settle the current noise fell to 12 pA peak to peak noise looks worse in the photo due to blooming of the photographed oscilloscope trace AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC DETAILED GUIDE TO OPERATIONS Page 63 3 nA 10 mV 1 ms FIGURE 10 CURRENT AND POTENTIAL RECORDING DURING cSEVC CELL MODEL AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 64 DETAILED GUIDE TO OPERATIONS SEVC MODE DISCONTINUOUS Description In discontinuous single electrode voltage clamp dSEVC mode the tasks of voltage recording and current passing are allocated to the same electrode Time sharing techniques are used to prevent interactions between the two tasks The principles of operation have been published Brenneke amp Lindemannn 1974 Wilson amp Goldner 1975 Finkel amp Redman 1984 and are outlined in the block diagram and timing diagram of Fig 11 and in the following discussion A single microelectrode ME1 penetrates the cell and the voltage recorded V1 is buffered by a unity gain headstage 1 begin the discussion assume that at this moment V is exactly equal to the instantaneous membrane potential V A s
115. tting of MEI affects the voltage clamp response The Capacitance Neutralization setting of ME2 affects the current monitoring circuit at high frequencies and also has a small effect on the voltage clamp response ET The Anti Alais filter slows the microelectrode response and should not be used in TEVC mode Suggested Use In Bridge mode set the Capacitance Neutralization of each microelectrode for the best step responses This is important but not critical and in order to be tolerant of changes in the microelectrodes resistances which might occur during TEVC it is suggested that Capacitance Neutralization should be slightly underutilized Use second order or better lowpass filter to remove the high frequency noise from 1 see Output Filter section Set the Gain and Anti Alias Filter to minimum values Switch the Phase control off Switch off all current commands Use the Holding Position control to yield equal brightness in each of the two RMP Balance lamps At this setting the command potential during voltage clamp will be equal to the resting membrane potential RMP Lock the Holding Position control if desired Switch into TEVC mode Set up a repetitive step command Monitor both 10 V4 and 15 Increase the Gain as far as possible without causing overshoot in the step response In cells whose membranes do not cause the same phase shift 90 as a parallel RC cell model the Phase control can be used to increase the maximum gain a
116. ue is approximately 600 mV which is the value which will typically be s seen when the ME1 headstage input is open circuit AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 52 DETAILED GUIDE TO OPERATIONS V2 mV This DPM indicates V2 in all modes The maximum displayed value is 1999 mV signals are indicated by a partially blanked display and or to indicate polarity I nA This DPM can display one of the following currents ls D orlymr current to be displayed is chosen using the I Display Select switch Three small switches are used to change the decimal point location so that the display can be read directly in nA for the headstage being used The HCG switch is active when the I Display Select switch is in the Im position the switch is active when 13 is selected the VG switch is active when Ivirt is selected To use turn the switch to the gain of your headstage PHASE A voltage clamp is a negative feedback circuit and as such it requires a 90 phase shift within the circuit Ideally this phase shift is supplied by the capacitance of the membrane In practice membranes introduce significantly less than 90 phase shift see discussion by Finkel amp Gage 1984 The frequency response of the voltage clamp circuit can be modified by the Phase controls The voltage clamp circuit can thereby be adjusted to compensate for the nonideal phase resp
117. used to time the command signals To the second one which need not be a high quality type connect the Monitor output Set the gain to 100 mV div 10 mV div input referred Trigger this oscilloscope from the SAMPLE CLOCK output on the rear panel Set up a repetitive current pulse in Bridge Mode Balance the electrode voltage drop as shown in Fig 2 in the Bridge Section Set Gain and Anti Alias to minimum Switch Phase Shift off i e set Center Frequency to OFF Switch to DCC mode Proceed to optimally set Capacitance Neutralization as described in the DCC Section method B and illustrated in Fig 5 Set the Output Bandwidth to 1 5 or less of f Switch off the current pulse Use the Holding Position control to achieve equal brightness in each of the two RMP Balance lights Switch to SEVC Set up a repetitive 10 mV step command Increase the Gain as far as possible without causing overshoot or instability in the step response Reduce the Gain slightly below the maximum value to get a safety margin Introduce Phase lag or lead if by doing so the step response of both the current and the voltage can be improved Increase the Anti Alias Filter while checking t the settling characteristics on the monitor waveform The noise on Vm and Im may be reduced by this procedure Only use as much Anti Alias as is consistent with stability Set the Anti Alias Filter back to minimum before using a new electrode An example
118. using a specially constructed low radiation transformer by placing the supply well away from the rest of the circuitry and by using internal shielding The incoming power is filtered to remove radio frequency interference RFI The excellence of the components and construction will be obvious to you from the high quality of the cabinet and controls Precision ten turn potentiometers and reliable switches abound But the high quality is more than skin deep gold plated connectors ar used throughout ultralow drift operational amplifiers are used in all critical positions 1I C s are socketed for easy maintenance and the circuit designs and operation have been well tested in laboratories throughout the world this adds up to low noise low drift reliable and accurate operation And the excellence does not stop with the hardware We also provide a detailed operator s manual that serves as a handbook of procedures for microelectrode users A separate service manual is also supplied The AXOCLAMP specification sheet contains complete technical details and ordering information Please call the factory for answers to any questions you may have AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 8 FEATURES This page is intentionally left blank AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC GLOSSARY Page 9 GLOSSARY AXOCLAMP and AXOCLAMP 2A ar
119. using long lengths of shielded cable to connect the microelectrode to the input If possible plug the microelectrode holder directly into the input Use shallow bathing solutions Avoid having grounded objects near the electrode Do not ground the headstage case If metal objects such as the microscope must be very near the electrode they may be disconnected from ground and connected to the gold shield socket in the headstage This technique can improve the microelectrode response speed However it may be that in DCC and dSEVC modes there will be an increase in the amount of switching noise picked up by independent recording electrodes if used See also the section titled Microelectrodes for Fast Settling CLEAR There is one Clear switch for each microelectrode It is used to pass up to 600 x H nA down the microelectrode and correspond to depolarizing and hyperpolarizing currents respectively The Clear switch is used for two purposes 1 When the microelectrode tip resistance goes high this condition can often be cleared by rapidly toggling the Clear switch from to Because of the large current passed this should only be done extracellularly 2 Sometimes microelectrode tips press against the cell membrane but fail to penetrate A quick flick of the Clear switch will often force the microelectrode to penetrate Whether to use hyperpolarizing or depolarizing current depends on the preparation and must be determined by t
120. virtual ground headstage measures total bath current Not required for normal operation Required in two electrode voltage clamp if HS 4 headstage used Virtual Ground output attenuation VG specifies the sensitivity Smaller VG is more sensitive used for low currents AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC Page 12 QUICK GUIDE TO OPERATIONS D2 MODE GROUP Illuminated pushbuttons reconfigure AXOCLAMP 2A for different operating modes BRIDGE Two conventional microelectrode amplifiers DCC Discontinuous current clamp on microelectrode 1 SEVC Single electrode voltage clamp on microelectrode 1 Discontinuous SEVC dSEVC uses time sharing technique electrode switches repetitively from voltage recording to current passing Continuous cSEVC is analogous to whole cell patch clamp electrode simultaneously does voltage recording and current passing TEVC Two electrode voltage clamp Microelectrode 1 does voltage recording Microelectrode 2 does current passing ee re Se ee NS unc Cont Discont Switch and lamps operate only in SEVC mode D3 MICROELECTRODE 1 ME1 GROUP Complete intracellular extracellular electrometer Capacitance Neutralization Neutralizes electrode input capacitance Clockwise rotation reduces effective input capacitance and speeds response Overutilization oscillates headstage Buzz Deliberate overutilization of capacitance neutralizatio
121. which would decrease the input resistance are minimized For example the field effect transistor FET input of the headstage is referenced to the input voltage rather than to ground This technique is known as bootstrapping Thus the effective resistance of the input is much greater than the already high resistance of the FET Leakage current and resistive loading through the insulation of the input Socket are minimized by using Teflon insulation and by driving the case with the DC input voltage HOLDERS Features The HL 2 series holders have been designed for low noise mechanically stable microelectrode recordings with or without suction The body of the holders are made out of polycarbonate for lowest noise and easy cleaning Maintenance is simple because the holder can be fully disassembled for cleaning and parts replacement i Mechanical stability of the electrode is assured several ways For example as the electrode cap is closed the O ring is forced into a special recess and pulls the electrode firmly back into the holder so that its end presses tightly against the electrode seat The holder mates firmly with the special teflon connectors on the HS 2 HS 4 and VG 2 series 2 mm diameter pin is used for the electrical connection The holders are designed to emerge along the long axis of the headstage right angle dines can be purchased if it is necessary for the holder to emerge at 90 from the headstage A BNC to A
122. xon adaptor HLB 2 can be purchased if you wish to use third pary BNC style holders AXOCLAMP 2A THEORY amp OPERATION COPYRIGHT FEBRUARY 1990 AXON INSTRUMENTS INC f Page 40 DETAILED GUIDE TO OPERATIONS Parts The various parts of the holders are shown in the exploded view ELECTRODE PIN ELECTRODE CAP RECESS SEAT SEAT PIERCED SEAL PIN CAP Five spare O rings and one spare pierced seal are provided with each holder Additional O rings pierced seals pins and Ag AgCl pellet assemblies can be purchased from Axon Instruments HL 2 12 holders use a plain Ag wire and O rings with a 1 2 mm hole HL 2 17 holders use a Ag AgCl pellet assembly and O rings with a 1 7 mm hole To replace the silver wire insert the nonchlorided end through the hole of the pierced seal and bend the last 1 mm over to an angle of 90 Press the pierced seal and the wire into the pin seat Push the large end of the pin down onto the bent over wire and into the pin seat This assures good electrical contact Screw the pin cap down firmly but without excessive force Use Insertion of electrode Make sure the electrode cap is loosened so that pressure on the O ring is relieved but do not remove the electrode cap Push the back end of the electrode through the electrode cap and O ring until it presses against the electrode seat Gently tighten the electrode cap so that the electrode is gripped firmly To minimize cutting of the

Axoclamp-2A Microelectrode Clamp Theory and Operation

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