I am recording fEPSP from sl-m of area CA1 with the stimulation electrode in the same layer around 1000 micron apart. When I applied APV + CNQX with or without bicuculline, I got an enormousely increased fiber volley. Would you please tell me if the enlarged fiber volley implies a great preservation of perforant pathway fibers there? Thanks a lot!
The size of the fiber volley reflects the number of axons that are activated to give rise to a synaptic response. What stimulation intensity are you using for your experiments. I always do an input-output curve, and only use slice that gives me a small fiber volley relative to the synaptic response. Slices that show a large fiber volley are usually unhealthy.
You could have at least a couple different things going on.
1) In the presence of bicuculline, you can get increased excitability and thus a larger fiber volley.
2) More likely, by blocking the excitatory response with APV + CNQX, you are unmasking the fiber volley from underneath the postsynaptic response. These can get tangled, especially when responses aren't as clean, like in a place like SLM.
A couple other caveats about PP-SLM recording. Your responses can also be contaminated by dentate gyrus pop. spikes because of your close proximity or via ring-around through CA3 and into Schaffer-Collateral. Some people place remove DG place a cut between CA3 and CA1 (see PubMed ID: 10085331, Figure 1 and Methods) . Also, make sure you check to make sure there isn't contamination of the response by the Schaffer Collaterals. Without moving your stimulating electrode, place your recording electrode into stratum radiatum. If you have a positive field potential then you're good. If you're field potential is negative, you're stimulating electrode is exciting Schaffer Collaterals as well. You'll have to move things around until the negative deflection in stratum radiatum goes away.
Hi Walter,
Thanks a lot for your answer.
My anatomical data showed far better preservation of PP fibers in HEC slices than in frontal slices. To verify this, I started to record fEPSP in sl-m. Whereas fiber volley got a several-fold increase in HEC when APV-CNQX with or without bicuculine were applied, there was just a very limited increase in frontal slices when the blockers applied. What I really want to know is if the huge increase in fiber volley can attribute to the great preservation of PP fibers in HEC slices.
Xiong
As you know, fiber volley is a measure of the compound action potential of the axons you are activating with your stimulating electrode at the site of the recording electrode. As you also know, fiber volley should increase with stimulation strength.
If I'm understanding you correctly, you are seeing a much bigger change in the fiber volley after applying CNQX+APV+Picrotoxin when compared to before applying the blockers. Where I'm puzzled is that you see the large fiber volley increase after application of APV+CNQX+Picrotoxin only, i.e., after blocking postsynaptic activation. This greater change when synaptic blockers are added would indicate there is greater masking of the fiber volley under normal conditions.
Application of APV+CNQX+Picrotoxin is a great way to isolate fiber volley, especially, if you go a further step and then apply TTX to remove the shock artifact. Of course, you need to do this sequentially. So, if you are doing an input-output curve as Antoine suggested above, you would need to record your stimulator settings for each stimulation and then go back and record the responses at those stimulator settings in both APV+CNQX+Picrotoxin and then again after adding TTX. That way you have the whole response (no blockers), shock artifact + fiber volley (APV+CNQX+Picrotoxin), and shock artifact alone (APV+CNQX+Picrotoxin+TTX). Then you can isolate the synaptic response (Response - Rsponse in APV+CNQX+Picrotoxin) and fiber volley size (Response in APV+CNQX+Picrotoxin - Response in APV+CNQX+Picrotoxin+TTX) by subtracting the different responses.
When doing you input-output curves, if your maximum responses and maximum fiber volleys are larger, then I would say you have greater preservation of fibers. In theory, the response slope should only be different if there is some difference in postsynaptic response or presynaptic release probability, since you are controlling for fiber volley size in the way you plot your curve.
I hope this helps, but I may be missing the actual nature of your question.
Hi Walter,
Thank you so much for your suggestion in great detail. I shall say that your understanding is accurate at my question.
Actually I already did what you are suggesting. Attached, please check my PPT of fEPSP recording in HEC (A) verses frontal slices (B). I numbered traces as 1 to 4, which represent baseline, APV+CNQX, APV+CNQX+Bicuculine and plus TTX, sequentially and respectively. I also have to point out that the distance between stimulating and recording electrodes is more than 1200 micron (long axis of the mesh of the holding chamber) with a stimulation of 250 microA in (A), compared to a little longer than 650 micron (short axis) in distance and 350 microA in stimulation as in (B).
Do you think my data supports my conclusion that PP fibers are much better preserved in HEC slices than in frontal slices, as I can show in my anatomical data. Do you know any reference paper regarding fiber volley recording and pathway fiber preservation? Or, would you please tell me whose papers I should check about this issue? Thanks a gain.
Xiong
Xiong -
The short answer is that I'm not sure, but I'm suspicious that there is more going on in panel A. If what you have recorded is a true fiber volley, it's severely distorting your SLM postsynaptic response. The broad peak after the large trough in traces 2 & 3 sits right in the middle of your initial slope of the SLM EPSP in trace 1. What does your response look like, if you subtract 2 or 3 from 1?
I'm concerned that the second broad peak followed by a second noticeable trough indicate some kind of antidromic activation of the pyramidal neurons, which would distort possibly both the fiber volley and EPSP. If you look at the work of Heinemann (see, for example, PubMed ID: 7623286) and Schuman (see, for example, PubMed ID: 10601455), they make all sorts of cuts in the slice to prevent polysynaptic (shouldn't be an issue with APV+CNQX) and antidromic activation of CA1 pyramidal cells. I'm not sure if something like this could be a confound in your more intact preparation in A. It's interesting to note that in both of these papers, neither set of authors expressed the need to remove the fiber volley to reveal the true SLM response, which is what your traces clearly require from what you have shown here.
Sorry, that I cannot be more definitive. One thing you could do to test for antidromic stimulation, is to place a recording electrode in stratum pyramidale to see whether you elicit a population spike, or patch some pyramidal cells, and see if they spike in the presence of APV+CNQX. If they do, you know for sure that there's antidromic activation of pyramidal cells.
Best of luck!
Hi Walter,
Thank you very much for your inputs.
I think I need some time to digest your comments.
Xiong