Hi everyone,
I'm consistently having small fEPSP's of approximately -0.01 mv. I've been moving the position of my electrodes and increasing the stimulation intensity but the response doesn't change much. I'm looking at the CA1 region of the hippocampus and would like to do an LTP experiment.
I'm following a protocol that was done in our lab 10 years ago. However, I recently set up the rig after it had be unused for the last 10 years. I'm not sure how to rule out potential problems for the rig. This is a consistent problem that happens each time I record. Does anyone have suggestions for what to check for?
1- slice health. I use papouin’s recipe (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856250/)
2- how much Mg in your bath? Try lower it to 1 mM
2- bath temp? Try 32 degrees C.
Thank you for answering Fouad Lemtiri-Chlieh
Currently, I've been using 1.3 Mm of Mg in the bath but I can lower it to 1.0 to see if that would help.
BTW, I meant to write this by 4:00 am this morning, but I was too sleepy! :)
After I saw your recording, I am 99% sure that your trace is lacking the field response. You only have the fiber volley but no AMPA response. To be 100% sure, I need to see your time scale of that trace. So if am right, then just maybe, just maybe, you are not simulating in the right direction (i.e., from CA3 --> CA1). If it's true, then you have to exchange the rec. pipette position with the stim. electrode or change the orientation of your slice (by 180 degrees).
Hi! I agree with Fouad, Can be a problem of the quality of your slices. How old are your animals? If more than P20, I recommend to use a choline chloride solution for perfusion and slicing. Also if you are cutting 300uM try to get 400uM slices, they are normally better for fields.
Otherwise, how big is your pipette? I normally use 3-5 Mohms pipettes but if you are starting try to make pipettes, and break the tip very little. And see if you have an increased response.
Hi! it looks like you are recording only fiber volley response not AMPA. Do you have an image of your electrodes position?
Thank you everyone for the replies!
I'll take a better picture next time I have a brain slice on the rig that shows the time scale, and another picture that shows the position of the electrodes.
My animals are 12 weeks so I can use a choline chloride solution. My pipettes are usually between 1-10 Mohms but I can use 3-5 Mohms to see if that will make a difference.
Hi Don, did you resolve this issue? I just found it while looking for something else. I had the same issue and after going crazy for a wee or two I realized the problem was the amplifier parameters. Check that there are no filters that are hiding your signal. My waveforms looked exactly the same as yours so it might be the same! Feel free to message me and I'll explain better.
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