Difficulty recording mIPSCs

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beskph01
beskph01's picture
Difficulty recording mIPSCs

Hello all,

I've recently ran into trouble recording mIPSCs in a synaptically active in vitro neuron population. In all cases, I've been using whole-cell configuration and performing -70 VC recordings. For mEPSCs (in the presence of TTX and bicuculline), I use a 140 mM K-Gluconate based buffer and get beautiful recordings with mEPSC rates around 6-10 Hz. However, when recording mIPSCs (in the presence of TTX, CNQX, AP5) using either a 140 mM Cs-Cl based buffer or 140 mM KCl based buffer, I've recently started running into issues. Immediately after break-in, my holding current drops precipitously to around  -500 pA. This leads to a horribly messy recording in which I can't even pick out events amongst the noise. Previously (as of a few weeks ago), I was reliably able to record mIPSCs with a frequency of around 1-3 Hz. Now, even if I am able to occasionally get a somewhat stable seal, the noise is terrible. I've tried making up new intracellular and extracellular buffers 3 times now. Sterile filtered just prior to use as always. The osmolarity seems right, 290. The pH seems right. I've tried new electrodes. Same result. Tried a whole other rig. Same result. :( The neurons look horrible after the patch is on. Has anyone ever ran into this problem? Any tips from any expert patch-clampers out there on getting good mIPSC recordings? I haven't tried using Cs-MeSO4 and recording at 0 mV yet, only because I previously have gotten such nice recordings with Cs-Cl at -70 mV. At this point I'm stuck, and seriously hating mIPSCs!

cheers! 

c.hill
c.hill's picture
Hi beskph01,

Hi beskph01,

Have you tried re-chloriding your wire? Do you seal and break in using a syringe? If you do, have you tried using a brand new syringe? Do your neurones look ok when you start patching? If they're not looking great after you start patching then it strongly suggests that something is happening for them to look horrible. As you've made up new external and internal solutions, I'd be inclined to discount those as being the problem. Maybe when you're breaking in the membrane is disrupted too much, causing the neurones to shrivel/swell up.