Hello Everyone,
Recently, I've started experiencing an issue that I haven't encountered over the past couple years of doing whole-cell current clamp recording. I typically record from cells for about 30-40 minutes at a time, and this has not really presented an issue in the past. However, I've noticed over the past few months that my access resistance is dramatically increasing at a certain point during the recording session, and I'm not sure what's causing it. Here are some details pertinent to these experiments:
- I use the following internal pipette solution: 130 mM K-Gluconate, 10 mM KCl, 4 mM Mg-ATP, 0.3 mM Na-GTP, 10 mM HEPES, 10 mM Na-phosphocreatine, and 0.5% Biocytin (for post-hoc streptavidin labeling). Usually have an osmolarity of ~295 mOsm
- The external solution (aCSF) is constantly perfused through the recording chamber at a temperature between 32-34 degrees C. The osmolarity is usually somewhere around 305 mOsm.
- Pipette resistance is typically ~2.5 MOhms.
- I typically inject a slightly negative current (between -20 and -45 pA) to maintain a constant RMP around -68 mV. This varies based on the true RMP of the neuron, but on average, the current injection tends to fall somewhere in that range.
- I inject a -80 pA test pulse to perform bridge balance and capacitance compensation. This is done about a minute after break-in, and the test pulse is immediately turned off once compensation is complete.
- Because the issue only began recently, I thought it may be the batch of internal solution I was using, so I made a fresh batch but had the same problem.
I typically attribute this to membrane closure, but I feel that's unlikely to be causing this issue just by how frequently I've observed it recently. Just curious if anyone has any tips for how to alleviate this problem. Thank you all in advance for your help!
Matt Russo great details! Nothing seem to be wrong with your protocol. Nonetheless, there could be numerous variables and thus multiple questions arise to find the problem:
1. But when exactly do you face that problem with resistance? Does it happen exactly at breaking-in? Do you have a sudden hyperpolarization then?
Did you test on cell recording to see any spontaneous activity or simple spiking once current injected?
Your pH and osmolarity seem OK though. I already assume that carbogen aeration of aCSF, its concentrations and bubbling should be OK too.
2. But is there any changes in internal/external solution ionic concentrations (low potassium ...)?
3. Have you checked loose seal or on-cell recording too to compare the seal resistance?
4. A simple issue that may cause big problems: aCSF amount in the bath! Is your slice submerged completely? Lack of enough aCSF in the bath that leaves the slice uncovered would lead to higher resistances (though not necessarily mean the access resistance). Submerging too much would inversely lead to lower resistance making it impossible to record anything on the other hand.
Hi Bahman Sadeghi , thanks for your reply! I'll try to answer your questions as thoroughly as possible and try some of your recommendations.
1. The problem doesn't seem to occur consistently at the same time for every recording, but I'd say on average it's probably about 20 minutes into the recording. It hasn't happened right after break-in. I usually notice it after a bit of recording. I also haven't seen any sudden hyperpolarizations upon break-in either, the cells typically sit somewhere in a healthy range (between -63 and -70 mV).
I haven't tested any on cell recording to see spontaneous activity (other than the membrane test that I use to determine the formation of a GOhm seal), so I can try that in my next session.
You are correct in assuming the aeration with carbogen, so no issues there.
2. I must admit, I don't think I completely understand your second question, but I'll try to address as best I can. There shouldn't be any changes in the internal/external solution ionic concentrations over time during recording sessions. I keep the internal on ice during recordings, and the external is continuously perfused over the slice chamber throughout the entirety of recording. Additionally, I correct for the junction potential using the MultiClamp 700B commander auto correction (just in case that is of interest).
3. I have not tried loose-seal or cell-attached configurations outside of the membrane test to determine the formation of the GOhm seal (the seal resistance is at least 1.2 GOhm before I even attempt break-in, but I've seen it get as high as 2.5 GOhm). I always allow seals to stabilize before attempting to break in, which usually doesn't take long.
4. The aCSF level is an interesting concern. I believe the slice is totally submerged, but I did recently experiment with lowering the level of the bath slightly to try and reduce capacitance across the pipette walls. I don't know that it's made a huge difference for capacitance, so perhaps going back to the current level of solution would be a good idea.
Again, thanks for your suggestions!
Great Matt Russo !
But about question 2, which I used telegraphic speech! what I meant was that there should not be any changes in the ionic concentration of either of your solutions! Potassium ion in particular could be a tricky one to control in you may not even notice that it has been changed until you get hyperpolarization or unexpected errors! But it appears that you do not have such problem with ion concentrations or any abrupt or unnoticed changes there.
You may want to test some of those suggestions. Give them a shot! then come and tell the result. If no changes occurred, maybe others or I can be of help.
- Badges
- Science topic
- Similar topics
- Filing