What are the steps to use the Axoclamp-2B for whole-cell patch clamp in continuous single-electrode voltage clamp (csevc) mode?

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1 What are the steps to use the Axoclamp-2B for whole-cell patch clamp in continuous single-electrode voltage clamp (csevc) mode? The Axoclamp-2B only performs csevc with ME1. The best overall headstage for this purpose is the HS-2A x0.1lu. For more information regarding the choice of headstages, see the Axoclamp-2B manual Reference section. Initial Amplifier Settings All controls refer to the ME1 controls, or to the controls in the VOLTAGE CLAMP section on the front panel of the AxoClamp. To begin, put the Axoclamp in BRIDGE mode. Turn the following settings on the amplifier to minimum (fully counter-clockwise): GAIN, PHASE LAG, PHASE LAG MULTIPLIER, CAPACITANCE NEUTRALIZATION, ANTI-ALIAS FILTER, and BRIDGE BALANCE. STEP COMMAND: +10.0; DESTINATION: ME1; Switch: EXT. Ensure that the H1 switch beneath the I(nA) meter is correctly positioned depending on the gain of the headstage. For an HS-2A x0.1 LU, the switch should be in the x0.1 position. Select Im on the I DISPLAY SELECT switch. Immediately to the left of the BRIDGE button is a switch for CONT. SEVC and DISCONT. SEVC mode. Put this switch to CONT. SEVC. The DC CURRENT COMMAND +/OFF/- switch should be OFF. Set the amplifier OUTPUT BANDWIDTH to 10 khz (also called the FILTER in the following sections). BNC Connections to the amplifier (assuming a Digidata digitizer) Amplifier Outputs: o Im to Analog In 0 o 10 Vm to Analog In 1 Amplifier Inputs: o EXT VC COMMAND (rear panel) to Analog Out 0 o STEP ACTIVATE (rear panel) to Digital Out 0

2 Model Cell Although the Axoclamp-2B was originally supplied with a choice of model cells, the PATCH-1U model cell is used for the following tests. This is the most appropriate model for whole-cell patch-clamp experiments. PATCH-1U models are available for purchase, and a description can be found in KB article #868. Other model cells may be used for this tutorial, but the details of the response may vary depending on the model cell parameters. Whole-cell voltage clamp procedure 1. Zero the Pipette Offset. Connect the PATCH-1U model cell BATH input to the headstage. Ground the model cell to the yellow socket on the headstage. Turn the INPUT OFFSET potentiometer on the front panel of the AxoClamp until the Vm meter on the amplifier reads 0 mv. 2. Measure the Pipette Resistance. When a TTL (5 V) pulse is applied to the STEP ACTIVATE input, a step pulse is delivered to the electrode as determined by the settings of the STEP COMMAND. Initially the amplifier is in BRIDGE mode, so that with the above settings, 1 na is applied to the pipette on ME1 for the duration of the TTL pulse. Therefore, use Clampex, a similar program, or a step-function generator to apply a short TTL pulse to STEP ACTIVATE. The resulting waveform should be a step in both current and voltage:

3 From this recording the pipette resistance can be calculated. Here, the model cell in the BATH position has a resistance of 10 mv / 1000 pa = 10 MΩ. 2. Set the Holding Position to Zero mv. Turn the HOLDING POSITION until the two RMP BALANCE lights are equally dim. The value on the HOLDING POSITION dial should be close to 5. This will set the holding command of the amplifier to 0 mv when the amplifier is switched to csevc mode. 3. Switch to csevc Mode. Press SEVC to put the amplifier into csevc mode. Turn the DESTINATION switch to VC(mV). Set the TTL pulse to STEP ACTIVATE to run repetitively, for example, at 10 Hz. Turn the pulses back on. The resulting output now resembles the pulses below.

4 (Sampling rate in Clampex = 20 khz, FILTER = 10 khz) Note that even though the STEP COMMAND is set to 10 mv, the resulting step is only ~7 mv. This is because the voltage clamp GAIN setting is set to its lowest value. The voltage clamp is unable to change the pipette potential to the desired command potential because the feedback loop gain is too low. 4. Neutralize the Pipette Capacitance. Increase the CAPACITANCE NEUTRALIZATION until the small overshoot on the current trace is removed, leaving a slightly rounded corner at the top of the current step. 5. Increase the Feedback GAIN. While repeatedly applying the test pulse, first increase the MULTIPLIER switch to 10. Now increase the GAIN slowly. The voltage step will increase, approaching 10 mv, while the current response also grows. Eventually the current becomes noisy, and then oscillates out of control. In order to achieve the optimal setting of GAIN, adjust the PHASE LAG and CAPACITANCE NEUTRALIZATION in turn, while increasing the GAIN and observing the trace. In general, as the trace becomes noisy and begins to show oscillation,

5 increase the PHASE LAG slowly until the trace begins to overshoot; then increase CAPACITANCE NEUTRALIZATION to reduce the overshoot. After adjustment of GAIN, PHASE LAG, and CAPACITANCE NEUTRALIZATION: (GAIN = 3, PHASE LAG = 0.3 x 10, CAP. NEUTRALIZATION adjusted; FILTER = 10 khz, Sampling = 20 khz) GAIN must be set high enough so that the amplifier can maintain the command potential. Therefore, the optimal settings of GAIN, PHASE LAG, and CAPACITANCE NEUTRALIZATION for a specific experiment must be chosen based upon the parameters of the pipette. Larger currents (lower pipette resistance) will require higher GAIN. 6. Form a Seal. In a real experiment, at this point the pipette would be advanced on the cell, a holding potential applied to the pipette, and a GΩ seal formed. As the seal forms, a test pulse is delivered to measure the seal resistance; with Clampex, this is easily done in the Membrane Test (or Seal Test in pclamp 8 and 9). The increasing resistance during seal formation can destabilize the voltage clamp. To avoid this problem, reduce the GAIN to 0.7 before beginning seal formation, and set MULTIPLIER to OFF (thereby turning off PHASE LAG). The voltage clamp may temporarily fail at this low GAIN, but as the seal forms, the current decreases and the clamp will return to the command potential. For example, the PATCH setting on the PATCH-1U model cell models a 10GΩ seal. The figure below shows its response to a 10 mv test pulse.

6 (GAIN = 0.7; MULTIPLIER = OFF; CAPACITANCE NEUTRALIZATION adjusted; FILTER = 10 khz; Sampling = 20 khz; HOLDING POSITION = -50 mv) The seal resistance is too high to accurately measure because of the noise in the current trace. 7. Break into the Cell. At this point the portion of cell membrane sealed to the pipette is ruptured, typically by further application of suction. This establishes the whole-cell voltage clamp. Before breaking into the cell, make sure that the GAIN setting is 0.7 or lower, to avoid oscillations which could damage the cell. For the purposes of this tutorial, switch the PATCH-1U model cell to CELL position. With the test pulse off, turn HOLDING POSITION until Vm is 50 mv. Alternatively, Clampex may be used to set the holding potential. The response to a 10 mv step is shown below.

7 (GAIN = 0.3; MULTIPLIER = OFF; HOLDING POSITION = -50 mv; FILTER = 10 khz, Sampling rate = 20 khz) Once again, since the GAIN is low, the amplifier is unable to keep the holding potential at 50mV, or apply a square voltage command. Set the MULTIPLIER to 10. Now increase the GAIN slowly, while observing the step response. As you see overshoots in the voltage response, increase the PHASE LAG. It may also be necessary to adjust the CAPACITANCE NEUTRALIZATION to achieve the best response, similar to that shown below.

8 (GAIN = 50; PHASE LAG = 0.95x10; CAP. NEUT. Adjusted; FILTER = 10 khz; Sampling = 20 khz) The appropriate final GAIN setting depends on the currents to be measured. Larger currents require higher GAIN so that the membrane potential will remain clamped. For this reason, it is recommended that Vm be recorded together with Im, in order to verify the validity of the voltage clamp. 8. Apply Series Resistance Correction. The current response is somewhat slow because the series resistance of the pipette is not corrected. In csevc mode, the BRIDGE BALANCE control acts to correct series resistance. While applying a repetitive test pulse, increase the BRIDGE BALANCE until the current response becomes sharper and the voltage step shows a slight overshoot. Increasing the series resistance correction too far, however, will result in severe oscillations that can damage a cell. Only increase the series resistance correction (BRIDGE BALANCE) until ringing is observed following the step on the voltage trace; then, reduce series resistance slightly to give a margin of safety.

9 With the model cell, the following traces show the response before (black) and after (red) series resistance correction was applied. (Filter = 10 khz, Sampling = 20 khz) In this case, BRIDGE BALANCE was advanced to 0.7. In csevc mode, with H = x0.1, this indicates that 0.7/0.1 = 7 MΩ of series resistance is corrected. Looking back at the measurement of the pipette in the bath, 7 MΩ / 10 MΩ = 70% of the series resistance was corrected in this case. This is the typical maximum series resistance compensation possible with the Axoclamp-2B. With a real cell, determining the percentage compensation is more complex, because the series resistance is not identical to the pipette resistance measured in the bath. Instead, the true series resistance must be measured in BRIDGE mode after breaking into the cell, and then compared to the corrected series resistance value. For details, see KB article # Choose a FILTER Setting. At this point, the cell is voltage clamped. Depending on the experiment, the FILTER can be set to a lower corner frequency in order to remove some

10 high frequency noise. The appropriate FILTER setting depends on the type of current being measured.