How long can neocortical neurons be recorded intracellularly in general?
I am doing voltage clamp on neocortical neurons. Previously when I was doing currently clamp, I was able to record for up to 2 hours (average 1 hour). However, when I was doing this voltage clamp, the cell can only last about 20 minutes. After about 20 minutes, my holding current would suddenly jump from several pA to several nA.
Some differences between my voltage clamp and current clamp recordings:
1). Extracellular ACSF: in voltage clamp, I have used low concentration of Ca2+ (0.25mM) and high Mg2+ (8mM) in contrast to current clamp (2.5mM Ca2+ and 1.2mM Mg)
2). Intracellular solution: in voltage clamp, I have included Qx314 to block Na+ current.
3). I did very large 4 seconds steps to make the cell reach 0mV (holding at -80mV). I have also done steps to make the cell reach -120mV. So there is a huge swing of voltage. I was actually able to do these large steps repeatedly without problem within that 20 minutes for a handful of cells.
4). Size of glass pipette electrodes: current clamp, usually about 5~7 MOhm. Voltage clamp, about 2~4 MOhm. I wonder if the size of the electrode matters.
Can any of the differences listed above make the cell die early and suddenly? What else can contribute to this problem? I also would like to know how long can people usually record neocortical neurons intracellularly. If there are any tips you can give me to improve my recording quality and duration, that would be great.
Thank you.
Hi Edward, I've never done this recordings with the ACSF you mention for voltage clamp. However, I think I can rule out at least some of the possibilities you suggest:
-Did you check the differences in osmolarity between your ACSF and intracellular solution for voltage clamp?
-The size of the recording pipette shouldn't be a problem. I've done recordings in LV pyramidal cells in Cingulate/Prefrontal cortex for more than 1 hour with 2.5-4 MOhm pipettes.
-Working with CsCH3SO3 internal solution I did recordings at both, -70 and +10 mV in the same cell for several minutes each (2x [15min @ -70mV; 15min @ +10 mV]), without a problem. So I think this shouldn't be an issue either. Anyway, how long are the hyperpolarization (-120mV) steps? This could be more challenging for the cells than the depolarization.
-I used TTx to block (extracellularly) sodium channels, but I think that at the end of the day is the same than blocking them intracellularly. However, I'm not sure about this.
Sorry I cannot be more helpful, I'm a newbie to electrophysiology.
Hope this helps.
Thanks for the reply, J. It's good to rule out at least some of the possibilities. My hyperpolarizing step is 500ms, following immediately after the large depolarizing step.
Then I think the depolarization step (given the duration of the pulse) is not the problem either. Hope anyone else can help.
Good luck with this!
Do you absolutely have to use the very low Ca? Can you use some specific blockers instead? My empirical evidence suggests low Ca creates lots of problems (harder to form gigaseal to start with so I assume membrane fluidity may be affected, but this is just a hunch).
There is no difference between the current clamp and voltage clamp internal solution besides QX-314? A higher resistance electrode will make a smaller hole in the plasma membrane. This is likely the reason why I have been able to hold cells for hours using sharp electrodes (70-100 MOhms).
What's the concentration of QX314 you used? I had this problem before with 10 mM QX314. I finally reduced it to 2mM, it still effectively blocks AP, and the holding current was much better. Even though I don't know why.
I am using 5mM QX314. The holding current is around 50pA to -80mV. Is this too much holding current? Also, could TTX applied extracellularly be better than QX314 internal?
Sorry, Olivier, I just saw your comment. Yes. There is no difference in the internal except the QX314. The pH and osmolarity are properly balanced after mixing all the salts and drugs.
Indeed, I used electrodes with sharper tips in current clamp, and it does seem to make the cell last longer (I guess less damage to the cell?). I am not using sharp electrodes in the current experiments, as that would require a different internal, right? In addition, since I am doing voltage clamp, would it be better to have electrodes with larger tips so that I can have better access to the cell?
Sure sure, if you are doing voltage clamp, better to have lower access resistance to improve quality of the clamp.
Did you try lowering the QX314 concentration as others mentioned?
I haven't tried that. But I am preparing to switch to TTX applied extracellularly instead.
Hi all,
Regarding the concentration of QX314, is 1mM sufficient to block the AP?
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