I'm assessing LTP by stimulating the CA3 schaffer collaterals and recording for CA1 and I'm wondering what is an acceptable amount of variability in the peak and slope between each sweep from a single slice. As of now my slope varies by as little as 0.15 mV/ms to as large as 0.4 mV/ms (calculated by subtracting the smallest from the largest slope value during baseline). Are there any standard criteria to determine an acceptable amount of variability in a slice?
Thanks for any and all help!
Will marks from Dr. Hauser's lab is currently doing electrophys in hippocampal slices I believe. He might be someone to ask.
You should stimulate the slice at around 30-40% of the I/O curve. I use to normalise my fEPSP slope or amplitude all along the baseline before inducing LTP in the CA1. I use to look at the overall shape of the baseline. It should be stable from start to finish and then proceed with the LTP induction. If you are recording fEPSPs, the recorded potentials are not that quantitative compared to patch clamp. Also excitability can vary with time within the slice between slices and between days. Considering a fEPSP lasts around 20ms you might have a problem given your numbers. Have you verified your slope and amplitude cursors placement for the quantification sometimes miss-placement can bias your analysis. I don`t really know if that would help.
I have never quantified that (variability sweep-to-sweep), but as long as the first 5 min of baseline are within ~15% of the last 5 min of baseline then I think you are alright.
The lower the percent change between early and late baseline period, the better obviously.
I would use a baseline of twenty minutes whereby the slope of the field potential could not differ by more than 5% of the mean. I also agree with Charles that it should be a percentage of the peak response. In my case I kept the baseline at 20% of my peak response. The problem with doing that, at times, was that finding the peak response every now and then made the response unstable due to the large input. You will need to be careful when you do that.
I have less than 5 percent variation from start to finish of my baseline
To obtain stable baseline response depends on many factors. May be your slice is not on the rigid positioning (enough tight net under slice?) and its micromovement may elicit different contact of the tissue and electrodes...
Between slice runs can vary more than 5%, due to dissection health, vibratome slicing protocol and slice number within sequence (discard first few ans last few), temperature of Acsf, age of the pefrusate etc... within a single slice once recording fEPSp measures started should vary less than 5% and baselines should be carried out for minimum of 30minutes prior to tetanization. Also need to be careful when running baseline that the number of sweeps recorded per minute does not exceed 6 (1/10s; and 4/min or 1/15s is preferable), as there is a little known phenomena known as low frequency potentiation, which can appear based upon other variables ie bath temperature Acsf composition etc... Can check out figures and details from Cliff Abraham's lab. Paper listed below can be downloaded from my research gate or my website "Eric L Hargreaves PageONeuroplasticity". Other papers from that lab around that era, should also be very consistent on methods and recordings etc. Particularly slope measurement and duration of slope measurement segment and whether segment is closer to inital roll off or slid closer to the peak. most canned packages should have rules of thumb for measurements, or reference a number of papers that utilized the system and its measurments Its pretty amazing what can be done with the same sweeps in different hands and thats why its important to document document document.
A DOUBLE DISSOCIATION WITHIN THE HIPPOCAMPUS OF DOPAMINE D1/D5 RECEPTOR AND b-ADRENERGIC RECEPTOR CONTRIBUTIONS TO THE PERSISTENCE OF LONG-TERM POTENTIATION.
J. L. SWANSON-PARK,*† C. M. COUSSENS,† S. E. MASON-PARKER,† C. R. RAYMOND,†E. L. HARGREAVES,† M. DRAGUNOW,‡ A. S. COHEN†§ and W. C. ABRAHAM† Neuroscience, 1999, Vol92, p485.
Can also checkout fEPSP as % of max I/O value and its impact upon degree of potentiation. Where you are on the I/O curve also impacts the variability of measures. the closer you are to max the less variable are the measures. The trade off of course the closer you are to max the less LTP you will observe (which is more complex than just playing with percentages versus absolute differences). Can check out Ambrose Au's thesis in Stan Leung's lab. Brain Research Bulletin in 1994 (when BRB was a reputable journal).
Long-term potentiation as a function of test pulse intensity: A study using input/output profilesOriginal Research Article
Brain research Bulletin, 1994 Vol33, Pages 453-460
L.Stan Leung, Ambrose S. Au
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