Something is wrong with configuration with sensitivity setting (either input or output sensitivity).

You are seeing 2.5 nA of currents, that is why too much for a cell. 100-200 pA of current is enough to depolarize a cell.

Also axopatch 200 is not very good in current clamp mode.

Dear Elina,

Looks like the settings in your configurations are not correct. As Oliver mentioned above the readings on the current and voltage channels are way above normal levels. For example the voltage change is around 1000 mV, that is just not possible. I think what you should do is just current clamp your cell at resting membrane potential (rMP) and read the rMP on your amplifier. If rMP is far from action potential firing at rest -60 or -65 mV, then just inject small constant depolarizing currents till you bring the rMP to around firing thereshold, then you shoudl see action potentials of around 70-90 mV amplitude. See my publications below for examples.

I think you do need to go through your configuration list in the program  and re-configure everything from start. You may need someone with experience to that as the list is quite a long one.

best wishes, Refik

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Dear Elina,

First news are good - you use the model cell in an appropriate mode - whole cell, because the resistance is 500 MOhm, which is common for model cell :)

1.) 1st weird thing to me - huge command currents (lower graphs). Check your protocols (scripts) for stimulation, make sure that correct values/units are specified there. As I remember there is no stim. scailing option in Axopatch 200B, but check it either way.

You didn't see AP from living cell cause it was already dead because of such a huge currents.

2.) Certainly, you cannot see AP from model cell - it's just the simple box with 3 circuits to check the correctness of your set up settings, test protocols, etc. Usually, it has different resistances and capacitances to model the behavior of the cell at rest.

3.)There is no holding current (command current) according to your photos, that is not good. You must set it before starting you stim. protocol. In the living cell after membrane rapture you must hold the cell all the time (set holding voltage in VC mode and holding current in CC mode).

4.) As I see, there is no capacitance compensation (neither fast nor slow), that is not good as well.

5.)I would suggest you to follow advices from Refik and Olivier.

Here is a rough plan. Attach your model cell to the headstage, compensate the electrodes voltage offset using the test pulse in bath mode. Switch to the gigaseal mode (on cell ), set the holding voltage (command voltage) to -60-70mV, using higher gain (10-20mv/pA) compensate for fast capacitance (get rid off those fast spikes). Swith to whole cell mode, using 100pF range compensate for slow capacitance (make the line staight). Memorise the holding current (on the Axopatch digital display). Switch off the holding voltage (cause after you switch to current clamp it will become the holding current and usually lethal) quickly switch the mode to current clump and set the holding current as you have memorised it before. Now you are in the current clamp mode. The tricky point is not to kill your cell with currents. For the first time you can forget about another tricky thing - series resistance compensation. For a small cells with low initial Rs (5-25 MOhm) and short protocols you don't need it, especially if you have no need to analyse the AP shape (kynetics).

Now you can start your protocol (script).

Good luck!

Dear all! Thank you very much for your answers and advises!

Yesterday I spent all day communicating with my amplifier and finally I suppose I got something that it should be. Please find attached my new results from model cell in voltage and current clamp.  The main problem was of course with scaling. The automatic scaling assistant worked incorrect and it took some time to adjust values.

Special thanks to Tigran. Your protocol was very helpful and I realized that completely forgot about capacitance compensation (may be since I used to work with this magic buttons "C-fast" and "C-Slow" ))

Also I would like to ask you should I hold any holding current while recording action potentials, I mean before it? Am I right understood that after switching to current clamp I should holding membrane potential at -70 mV but already with help of injected current (in case of model cell it was -130 pA)? Should I also do it during action potentials recording before applying a testing current steps? That was my question. :)

P.S. Тигран, говорите ли Вы по-русски? И могу ли я в дальнейшем задавать Вам вопросы, если появятся? Было бы здорово!

You are welcome.

Short answer to all of your questions: Yes.

Now the longer one.

In whole cell mode one should clamp the cell at resting potential all the time  right after your pipette get inside. And here is why. After you break through the membrane pipette solution diffuse into the cell and, since its chemical composition cannot 100% match cytoplasm, after several seconds real resting potential changes accordingly. It happens because patchclamp pipettes have a "huge" tip (recall conventional 2.5-5 MOhm Rpip for whole cell). Pipettes for classic intracellular recordings have tips way more thinner (a few dozens of MOhms) and in that case such changes are neglectable in a short time interval.

To compensate this distortion you must hold (clamp) the cell at normal resting potential all the time after making whole cell (whether it idle or during the protocol). Also in CC mode: inject the holding current and as much as it was needed in VC mode to hold the cell at resting potential. Eventually, in CC mode the output baseline must be around -70mV as it is in physiological conditions, while in VC output baseline is the holding current (e.g., -130pA).

P.S. I thnk it's better to use English on ResearchGate question service, but feel free to ask me questions in Russian using private messages.

To be sure that your "current steps from -100 pA to 200 pA" is correct do the following. 

1) Connect model cell to the head stage in the "Bath" configuration. 

2) Create a new protocol in iClamp mode where you current goes from 0 pA to -100 pA for approximately 10 seconds.  

3) Switch major from display in your Axon 200 amplifier to measure the current.

4) Run you protocol and watch the current in the Axon amplifier display (not at the Clamplex on your computer). If the displays shows shift of the current from zero to -100 pA, then your setting is correct. Otherwise, you need to fix your settings in Clampex -> Configure -> Lab bench.

5) You may do the same as in (4) for +200 pA current step, just to be sure that all your currents are at appropriate levels. 

Good luck!