I am planing some experiments to study difference in basal synaptic transmission accross different animal conditions. I am thinking about producing a fiber volley vs fEPSP slope curve, but I haven't done this before.
Can anyone advise me for this exercise?
Regarding the x-axis of this putative curve, should I give fixed increasing steps for the stimulation intensity (for me this option seems easier) or should I try to find representative FV values (e.g. 0.05, 0.1, 0.15, 0.2, etc...; for me this option seems harder, since the FV size changes among preparations).
It would be great if you can help me.
Best regards,
Diego Fernández
Diego,
It is easiest to plot the fiber volley amplitude against the fEPSP slope for each individual slice placement, rather than trying to match fiber volleys. This will mean that you will have different values for each slice (placement). Then you can plot a sigmoidal function (FV vs. fEPSP) and use a predicted value from that curve for each slice to add to group data. For example, FV1/2, which would be the fiber volley that evokes a 1/2 maximal fEPSP response. This is a value that will normalize across slices within groups and can then be averaged and compared across groups.
You can see an example of the different I/O that you could generate in this paper
Article Kalirin-7 is necessary for normal NMDA receptor-dependent sy...
Dear Diego,
The proper way to do it is to increase progressively the stimulus intensity, and to plot the fiber volley amplitude versus slope of fEPSP
Since you never get the same fiber volley across slices/animals/conditions you just bin the fiber volley say from 0 to 0.5; 0.51 to 1.0; 1.01 to 1.51 (artificial units); etc.
You calculate the average and SEM in each bin for the volley amplitude and slope of fEPSP. This makes a point with double error bars (one horizontal for fiber volley amplitude and vertical for slope of fEPSP.
The difficult part is to get a nice fiber volley, not contaminated by the stimulation artefact. These are key experimental tips:
1. always ensure that your recording electrode and the stimulus electrodes are aligned along the path of the axons (on beam).
2. use twisted wire (i.e. 50 microns NiCr twisted wire) for stimulation, or better glass theta tube with both barrels filled with ACSF (gives the smallest artefact) - my preferred procedure.
3. you have to carefully adjust the distance between the stim and the recording electrode with trial and errors. The closer you are, the more contamination you get from the artefact. The further you are, the more difficult it is to be on beam.
Hope that helps!
cheers
I agree with Christophe Bernard recommendations. I also would add that as always the necessity of keeping the electrical noise as low as possible is important. In brain slice work I found keeping a low bath level also helpful in reducing stimulus artifact size.
Thank you everybody very much for your help!
Thanks Christophe for such useful tips for performing this curve.
Unfortunately, during my recordings I am not able to see the fiber volley regularly. Sometimes it appears, but normally it is just "mixed" with the background noise. So I don't feel very confident to do this measurement.
I found another possibility in a paper from Taqatqeh et al. (Taqatqeh J neurosci 2009 29). What they have done is to get a maximal saturated response with stimulus of 200 µs of duration, and after that, using the same stimulation intensity, they perform a curve for fEPSP slope responses against different durations for the stimulation ranging from 40 to 200 µs. What do you think about this procedure?
Thank you!