I'm relatively new to patch clamp, but I've been working extensively on optimizing our recording parameters for studying sEPSCs in adolescent rat CA1 pyramidal cells. My biggest hurdle has been consistent cell health in my slices. Some days, cells go to gigaohm almost instantly upon releasing positive pressure and applying minimal negative pressure. Other days, I cannot get a seal above 250 MOhm. I am currently using Jonathan Ting's NMDG and HEPES based solutions since I have had better luck with this method than traditional cold sucrose slicing. Osmolarity and pH are always properly adjusted (300-310 mOsm; pH 7.3-7.4), and solutions are well saturated with carbogen prior to slicing and recording. Each animal undergoes transcardial perfusion before slicing. I have tried slicing in both frontal and horizontal planes, and horizontal slices seem to produce the best cell health. However, I prefer the orientation of frontal slices when targeting this cell type since dorsal hippocampus has been used for many of our past experiments (field recordings, etc) and since the PC layer is so much more prominent. Lastly, I am using a Cs-gluconate based internal solution. At most, I can get 3 cells per day. Some days though, I cannot get a single acceptable recording. I guess I'm just looking for any tips from veterans of patching that may improve my throughput in these experiments. It is well known that patching older animals is sometimes a challenge, but surely I can improve what I'm doing in some way. I'm happy to provide any further details should the need arise. Thank you in advance for your suggestions!
Patching with cesium-based solution is always difficult. When I've used straight cesium internal, I've had the worst time getting gigaohm seals. Clearly much worse than with potassium internal.
One thing that I've found helps is replacing some of the cesium with potassium -- say 2/3 cesium and 1/3 potassium. If you still get good space clamp with this (somewhat) diluted mixture, your data yield might go up considerably.
Good luck.
Hello Dr. Desai,
I am new to electrophysiology and i am trying to record Na+ current from avian nucleus magnocellularis neuron. But i cant make the giga seal.
I know it’s an old conversation but would you please provide me the protocol to prepare the internal you mentioned ( i couldn’t understand how i should change the ratio ).
My internal is : ( mM)
150 CsCl, 10 NaCl, 0.2 EGTA, 10 HEPES. Ph7.23 by CsOH.
I Don't use kCl here. Did you mean i should try making 100 mM CsCl and 50 mM kCl? In that case my extrenal kcl should be changed as well?
My external is 125 TEA-cl, 2.5Kcl, 26 NaHCo3, 1.25 NaH2PO4, 5 CsCL, 0.5 CaCl2, 1 MgCl2, 17 glucose, 1 CdCl, 0.5 Ni, 1 CdCl.
Thank you so much for the advice.
Hi Sristy Halder Yes, what I did was replace CsCl by an equal amount of KCl, so that the osmolarity remained constant. So, 100 mM CsCl and 50 mM KCl would be correct. And I made the solution in the usual way: add all ingredients, adjust pH (with HCl or NaOH), check osmolarity and adjust if needed (with sucrose or water). I didn't change the external solution, and I don't think there's any reason for you to do so either. The cesium (external and internal) and the TEA should do a good job of blocking most K + currents, and so the K+ reversal potential should not be relevant for your experiments. Best,
Niraj
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