So I've adoped a project examing LTP (stimulating Schaffer collaterals and recording in stratum radiatum of CA1) and am able to get apparent LTP following high frequency stimulation (four trains of 100 Hz at 20 s intervals) however the shape of my fEPSPs look odd following the tetanus. Attached is a picture of the fEPSP before and after tetanus. It looks like my stimulus artifact is merged with the fEPSP, for lack of a better way to describe it, and I'm not sure if this is normal or if I'm doing something wrong. Under these circumstances, how should I measure the peak and slope relative to the stimulus artifact and baseline which is not clearly distinguishable in the fEPSPs post tetanus?
Also, are there any good resources which discuss more of the technical aspects of recording field potentials for LTP?
EDIT: My reply from below posted here for more visibility:
I tested another slice and actually found that while I get this spike contamination with the 3 h recording protocol, I do not observe it with the baseline protocol, even after tetanus, so I'm thinking this isn't a consequence of the tetanus but rather the protocols I'm using for the 20 minute baseline and the 3 h recording were not set up the same way. As I understand it, the only difference between the baseline protocol and the 3 h recording protocol should be the number of sweeps and everything else should be exactly the same, correct?
Thanks for any and all help!
Tom
That looks like spike contamination after LTP. Try to move your recording and stimulating electrodes further apart so you can see an increase in the slope of the EPSP without merging into your stim artifact.
Also, I would do the LTP starting with a smaller baseline (~25% of max response) so spike contamination is less likely to occur.
The control recording looks fine. Did you determine the threshold for the fEPSP. One advice is to set the stimulus intensity at 1.2 times threshold in order not to get a back propagating spike like the one you seem to have after LTP induction. In your case, you cannot measure any slope (which is what you should do with extracellular recordings).
I tested another slice and actually found that while I get this spike contamination with the 3 h recording protocol, I do not observe it with the baseline protocol, even after tetanus, so I'm thinking this isn't a consequence of the tetanus but rather the protocols I'm using for the 20 minute baseline and the 3 h recording were not set up the same way. As I understand it, the only difference between the baseline protocol and the 3 h recording protocol should be the number of sweeps and everything else should be exactly the same, correct?
I agree with Olivier. It looks like your potentiated response is contaminated with action potentials. You could change the filter parameters of your amplifier to determine whether you have a multiunitary or population spikes after LTP.
On the other hand, the "disappearance" of the stimulation artifact might be due to the polarization of your recording electrode with your tetanic stimulation. If this is the case, you should see the same artifact modification in the absence of a slice. You could also try reducing the duration of your stimulus. How long is your electrical stimulation pulse? Normally you should be able to get a response with 0.1ms long pulses and the adequate stimulation current.
Hi Gregorio,
Thanks for your response. I am suspecting that it might be the duration of the stimulation pulse as I only observe this when I run the 3 hour recording protocol, but not when I run the 20 min baseline recording protocol. I think that the protocols are producing the stimulation pulse differently.
Thomas, that would be messed up. The stimulation parameters (usually 100 us pulse duration, at whatever voltage or current you set) during the baseline have to be the same AFTER the induction protocol. That is the only way to compare the same number of fibers activated before the induction with after the induction.
Make sure this is the case. Some people increase duration during LTP induction but it is imperative to bring it back to pre induction levels for the 3 hour post induction recording protocol.
Thanks for your response, Olivier. This project is new to me and so I've never configured a protocol before but will be examining them later today to see if I can figure out how they are producing the stimulus. I imagine the easiest thing to do would be just to make a copy of the baseline protocol and extend the recording time rather than trying to alter the stimulus in the 3 h protocol so hopefully this will not be difficult. Thanks again for your help!
While you're learning how to write protocol files, also look up how to configure sequencing keys. Knowing how to write/troubleshoot protocol files and sequence keys will give you a lot of flexibility in designing and running experiments. Good luck!
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