Hi Diego Fernández-Fernández

Interesting! Let's go through some steps for an initial troubleshooting:

If you use the routine HFS-induced LTP protocol and then record and assess your fEPSPs, what protocol do you use to induce LTP as in HFS (train of pulses/square pulses before and after HFS, frequencies and timing)?

Do you monitor your baseline stability for at least 10 min before stimulation and applying HFS?

Could you share an image of your typical pre/post-HFS responses and fEPSP to check the amplitude and slope of the PS?

Although it might be far away from the focus of your research and hypothesis, have you tried an inverse methods/different protocols like LFS to see how the facilitation or even depression goes on (even to induce STP or LTD)?

Do you use any agents (receptor antagonists) to block synaptic transmission of a specific neurotransmitter?

My own experience was mostly on awake/freely-moving or anesthetized rats and recording from DG/CA1. You may check our paper if it helps with the stimulating/recording protocols (published in Hippocampus in 2018):

Article Chronic ghrelin administration restores hippocampal long‐ter...

You checked the stimulator, electrodes and as you said it was not the reason.

Are you recording with a pipette type micro-electrode or a Teflon-coated tungsten electrode (I assume the later)? This is also important for the impedance you have to watch for.

However, as you know, recording from hippocampal organotypic slices in baths heavily depend on the concentration of your solutions. Check your bath solutions and concentrations and even the temperature, pH.

There are many other questions but first let me know if you got any ideas in mind after checking the above or if I can be of more help here.

Bahman

You may also share or check the following:

the recipe/solutions, how you prepare your slices (temp, age of the animals and even the thickness of your slices).

Importantly, Mg concentration in you bath.

Stim/rec electrode positioning and orientation or angel.

Is the amplitude of the fiber volley also changing?

If you incubate your slices at ~34 C for an hour after slicing then try just leaving them at room temperature. I find the former better for patch clamp and the latter for fEPSPs.

Thank you all for your kind responses!

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Bahman Sadeghi , the LTP protocol is not the reason because this baseline swing happens regardless LTP induction. Before LTP induction I have baseline with shifts. In the figure that I attached, you see a interruption of stimulation around minute 80. This is where I induced LTP in this experiment. As you can see, before LTP induction there is a huge swing of the fEPSP responses.

I actually apply baclofen in some experiments. Do you think this could be some residual baclofen?

I changed the stim boxes to a different brand, and the problem was still there. I didn't change the stim electrodes though. But these are brand new FHC concentric bipolar electrodes and seeing that I have this issue in both my chambers I think these are not faulty.

I am actually recording with a normal patch pipette (3 MOhm resistance), filled with ACSF and I am using a Multiclamp 700B for the recording. Do you think this might be the cause?

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Hi Rachael Ingram !! :) It is so nice to hear from you! Actually FV are changing too.. and I think the stim artefact too, so I assume the problem is that stimulation intensities might be changing over time? Very bad...

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Thanks Paul James Banks , actually the quality of the slices doesn't seem to be very bad, because they show nice fEPSP responses with a good FV/amplitude ratio. The problem is that once I get a response, there happens ramdom fluctuations of amplitude during time, and I cannot get a stable baseline.

Thank you very much for all your responses! Very helpful

To clarify I meant that the stability of baseline fEPSPs is better for me when slices are only incubated at room temperature between slicing and putting them into recording chamber.

Okay, that sounds interesting . Normally I incubated them in 32 ºC just after cutting. I will definitely try your suggestion. Thank you Paul James Banks

Hi Diego,

I think the issue could be due to some sort of physical instability.

I've found with electrode and slim box issues normally theres more of a pattern to the fluctuations and not random changes in FV/EPSP amplitude. And when I've had issues with slices its more of a constant rundown issue.

So I'd try looking at things like are your manipulators moving in any way, is your suction constant, is there any tension in the cables, does the table feel like its floating, does using a different perfusion pump help.

How do you perfuse the slices? Is there a drip system to prevent oscillations from the pump?

Thank you Rachael Ingram . It was indeed a technical problem with the stim electrode, which was faulty. I changed the stim electrode of this chamber and now the recordings are much more stable. Thank you all for your helpful answers.