We are making whole-cell patch clamp recordings from mouse (and human) fast-spiking interneurons using Axopatch 200B amplifiers. We see a sharp overshoot after action potentials (see red trace in the image) which we assume is an artefact caused by pipette capacitance correction? Could anyone confirm this? What is best practice when making current clamp recordings using a Axopatch 200B? Should one use both pipette capacitance correction and 100% series resistance correction? We are trying to characterise the intrinsic properties of the neurons but it seems like pipette capacitance correction is making a huge difference. Any help would be much appreciated.
Currently we do the following before begining our recordings:
At the moment we do this:
1) in cell attached mode we use pipette capacitance correction to remove capacitve transients
2) we break through into whole cell mode
3) we run a short current step and find the correct series resistance of the cell with 100% correction
We then perform our recordings (such as current steps)
Hi Joseph,
Which current clamp mode of Axopatch 200B amplifier do you use for your recordings?
I suggest to use I-Current Fast mode. The large afterhyperpolarization-like artifact usually can be seen at I-Current Normal mode in events with high kinetics as AP of InN, especially if recordings are done at temperatures higher than RT (i.e. ~30 C).
Also you can try to re-adjust Cp in CC mode. It is still active and can be adjusted.
I can see from your pic on time point 0.10 that it is either not fully compensated or overcompensated.
Best regards,
Mir
Thanks Mir, this is super useful! Yes reading the manual our next idea was to try use I-Clamp Fast.
Regarding what you see at time point 0.10, does the bridge balance correction not cause this artefact (it's a bit independent of the pipette capacitance correction) - see attached pics?
Following your and other's advice we might also readjust (or reduce) Cp in CC mode.
Thanks again for your help though!
Joe
Hi Joseph
You are doing the right things and also getting good advise as above (Miroslav N Nenov). But... I would suggest that if you have a bridge amplifier in the lab (eg. Axoclamp 2B) that you use that instead of the 200B. Patch clamp amplifiers have the potential to introduce some distortions to fast waveforms, so if you have a bridge amplifier you will probably be better off. see this paper:
Article Action potentials recorded with patch-clamp amplifiers: Are ...
And I think that with Multiclamp this is not a problem anymore.
All the best
This brief article is nominally about correcting for pipette capacitance with a MultiClamp amplifier, but might be of interest to you, because the article also uses the Axopatch 200B as a comparison, and therefore explains a bit about the 200B circuitry.
http://mdc.custhelp.com/app/answers/detail/a_id/18716/
If you have not already done so, perhaps you can find helpful information by searching the Molecular Devices help database for Axopatch 200B articles, for example:
http://mdc.custhelp.com/app/answers/detail/a_id/16552
http://mdc.custhelp.com/app/answers/detail/a_id/16603
http://mdc.custhelp.com/app/answers/detail/a_id/16592
Hello
Kindly look at links here could help you
Article Errors in the measurement of voltage-activated ion channels ...
Article Expanding Neurochemical Investigations with Multi-Modal Reco...
Article Direct Measurement of Specific Membrane Capacitance in Neurons
Good Luck
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs