I am currently trying to get a stable I/O curve in the CA3-CA1 circuit. I am working with 400µm horizontal slices from Bl6 mice that are 4-8 weeks old. I am cutting in normal ice cold ACSF and can store my slices differently afterward. I can store them at 33°C in an interface chamber or submerged at RT (or warmer, but I havent tryed that). I also tryed NMDG aCSF for cutting and 10 min recovery with NMDG aCSF after cutting. I let the slices rest for at least 1 hour after cutting in normal ACSF bevor I start the recording.
My problem is, that if I get a fEPSP that is bigger than my FV, it drops after a few stimuli and is mostly gone after abot 5 minutes. Does anybody have an idea what I am doing wrong? How should the slices recover? What ACSF solutions are you using?
Thanks
4-8 weeks old mice is very young, Normal ACSF work well, please get I/O curve,and use the 30%-50% stimulation intensity of max response intensity. normally,a good fEPSP is ~0.7-1.0 mV,max to 2~3 mv. if you according to the standard protocol,may the Perfusion system is the key problem to the stability of fEPSP.
Hey,
have a view of this question and the answers some people provided. It is in the same direction as your question!
https://www.researchgate.net/post/Why_do_I_have_an_unstable_fEPSP_baseline_in_Hippocampal_CA1
Good luck!
Diego
Hi,
In order to try to understand what is happening, can you post images of the first fEPSPs, after 2 min, and 5 min?
Note that you should stimulate at least every 20s
Hard to tell based on the info provided here, but if your response is dropping that rapidly (e.g. within 5 minutes), your first considerations might be:
(1) A drift problem (the recording or stimulating electrode moving within the slice)
(2) Solution is leaking from the barrel of your recording electrode if using high salt concentration (e.g. 3M NaCl). If you are using aCSF, this is probably not an issue.
(3) Check to make sure your bath temperature is stable, typically 30-32C.
It sounds to me like something I have experienced when the stim or recording electrode are moved too frequently or are mechanically damaging the tissue in the slice. You have to be very patient in optimizing the electrode placement. Sometimes you need to move a bit, then be sure to wait for three or more stimuli (acquired at no more than once per 20 sec as noted), then if necessary, adjust slightly and iterate. If you move around too much or too fast you can induce adenosine release that will cause the fEPSP or pop spikes to depress rapidly potentially down to nothing. That can also happen if your stim is too high to begin with. I would be interested in Alexander's view on this since he should have expertise regarding Adenosine suppression in slices.
What kind of stim strength are you using and what type of stimulating electrode? I like the concentric bipolar electrodes from FHC for this purpose. I am also curious, can you elicit pop spikes with the recording electrode moved to the pyramidal cell layer but without moving the stimulating electrode? I typically get ~5mV stable pop spikes at max and ~1mV fEPSPs for CA3-CA1 area at this age in mice, including using the NMDG protocol you mentioned.
Re: adenosine, yes, it is possible that the rapid rundown is caused by elevations in endogenous adenosine levels. The easiest way to check for that is to allow the rundown to stabilize, then see if you can reverse it with DPCPX (A1 antagonist) or caffeine (non-selective antagonist).
But I agree w/Jonathan that you should optimize the electrode placement---gradually lower the electrode while monitoring the size of the fEPSP until it is maximum.
I had similar rundown problems when I first started learning how to do hippocampal field recordings. As Alexander suggested, my rundown was rapidly reversed by the A1AR antagonist DPCPX, in fact, the response was even larger than expected suggesting the presence of basal adenosine tone. As a second note, I also had run-down related to the home-made holding slice chamber because my volumes were too low and the metabolites like adenosine could rapidly build up over the experiment. This was improved by refreshing the solution in the holding slice chamber, or eliminated altogether by using a commercial slice holding chamber with large volume (I prefer the Brain Slice Keeper-4 sold by Automate Scientific).
We may have a similar issue in the lab. We are preparing 300 µm slices (from much older mice, ~1 year), cutting in ice-cold choline aCSF, letting the slices recover for 1 hour at 37°C (and RT afterwards) in normal aCSF. Recordings are made at 32°C in an immersion chamber, in aCSF + picrotoxon, stimulating with either a concentric bipolar electrode or a glass pipette filled with aCSF, connected to a Grass SD9 or Digitimer DS3 (stimulation amplitude is usually in the range of 2-6 V or 20-40 µA, respectively). Most of the time, we're getting basically no CA3 --> CA1 fEPSP. However, when looking at high mag, the CA1 neurons look OK, and can be patched. Without moving the stimulating electrode that was giving no fEPSP, we're getting large eEPSCs.
So our slices are at least in part healthy, with patchable neurons, and the stimulation is efficient, as we're getting eEPSCs, so why aren't we getting even a small fEPSP ?