Hello everyone,
my postdoc is doing some LTP experiments in CA1 neurons from mouse brain slices (25-35 days-old) and, while everyting worked OK in some trial runs, with very stable and reliable LTP (using a paired induction protocol combining postsynaptic depolarization to 0 mV and HFS). he's now seeing a large EPSC rup-up at around 15 min after delivering the induction protocol. I've never seen this in the literature using such protocol, and he also didn't see it in the first recordings he did to test the protocol and the solutions. Has anyone ever experienced this? What could be the cause?
Thanks!
Hi Paulo,
I think lots of labs have observed this - it is not usually spelled out why but sometimes in methods sections you can see mention of 'prestimulation' periods. We first got a great tip from someone in the Kristen Harris lab a few years ago...
I assume that you normally let the slices sit in the recording chamber for at least a half an hour (after the initial recovery period) before you start to place the electrodes and then once you have all electrodes in place you begin the experiment. We now start to place the electrodes in the slice earlier and stimulate at the baseline frequency during at least the last 10 minutes of the 'settling time' (pick an intermediate stimulation intensity - maybe that gives about 1 mV fEPSP). Then after this stimulation period, determine the exact IO curve, you may need to adjust the exact electrode placement to have nice fEPSPs etc. at this stage. Then select your stimulation intensity for the experiment and begin the experiment. Plan to discard the first 10 minutes of the baseline anyways (i.e. record for 40 minutes when you want a 30 minute baseline) so that you can still discard the first ten minutes. These steps should improve the stability of the baseline - at least it did for us.
We also often see CA3-CA1 synapses 'waking up' when we perform patch-clamp recordings from CA1 neurons - there will be no response to the first couple of stimuli and then the EPSCs will suddenly appear - I really don't know why this. It is very difficult to study because although it is not so rare it also doesn't always happen...means that maintaining a constant stimulation rate is very important.
Boa tarde Paulo. are you guys making a cut between CA3 and CA1? it is possible when you don't cut that you are stimulating CA3 neurons too and then you get a feedback from CA3. ideally you should completely isolate CA1 by cutting CA3, subiculum and even DG.
Otherwise I agree with Christine, some times you see this and if you are recording fields, just keep a baseline recording for as long as you get a stabilization of the response. And discard the data before stabilization. Some times this changes in EPSP size are due to lack of stability of your perfusion system. I usually run my perfusion for at least 30 min before starting experiments, to be certain that the flow is constant and I recycle my solution. For whole cell, it is a little bit tricky because you don't have the time for a long baseline otherwise you washout important molecules for LTP. So in that case, for whole cell recordings I would just discard this data.
One more small point - if you are recycling your solutions (or even when not) you can also get interesting artifacts from changing osmolarity during the day...bubbling/heating increase evaporation a lot and we were shocked when we realized that even a change of about 5-10% noticeably changes the fEPSP amplitudes...so we now have a syringe pump to add dH2O at a steady rate to compensate the evaporation during our experiments. This also greatly improves the stability of fEPSP recordings...
Christine E Gee That is a good point! I will check my osmolarity. For the artifacts I have two breaks in my perfusion system (perfusion drips). One at entry level and the other one at exit level to break the circuit. It works fine!
Thank you for all the replies and suggestions. We are not performing fEPSP recordings, this is whole cell...And for the LTP protocols that we use it is described that HFS should be delivered within 5 min of break in, to avoid "LTP washout". Evaporation and osmolarity changes are indeed a thing, but not in the time of a whole cell recording. When this run-up happens it is there within 15 min of HFS. The funny thing is that we tested this protocol before using the transgenic mice and got text book LTP in 3/3 recordings... We are now trying with more limited simulation using pipettes filled with ACSF, since the concentric bipolars, in my opinion, stimulate a too large area in mouse slices. The papers we are trying to replicate make no mention of cutting CA1 from CA3, although I'm aware that this is routinely done for fEPSP experiments.