Hi Hong Lu,

That is a tricky problem. The recurrent collateral from CA3 are basically impossible to avoid recruiting 100% of the time, don't listen to people who tell you that if you place your stimulating electrode in the right place you can avoid it. If you are restricted to performing field potential recordings, then using DCG-IV is the only option I am aware of.

However, if you are performing patch-clamp recordings, you can wash on CNQX (or other AMPA antagonist) then patch a CA3 cell, depolarize it, and then record NMDA-EPSPs. The blockade of AMPA receptors will stop the activity in CA3 from getting supra-treshold, i.e. only the cell you are patching will get excited. In that case, you want to be stimulating outside of CA3, or otherwise you will risk directly activating CA3 axons.

DCG-IV washes out relatively fast. 20 minutes of wash should be more than adequate. But you should confirm that that is the case in your hands (i.e. different baths are different levels of efficiency at washing out drugs). If you get it to wash out quickly, then you can reuse the slice (at least in my opinion).

Hi William, thanks for your answer. Could you please explain how the blockade of AMPA receptors would led to only the cell I am patching getting excited?

Hi Hong Lu,

It is my understanding that the if you try to record EPSPs in CA3 cells by stimulating the mossy fibers, you will often record EPSP that is not due to mossy fiber input at all, but actually due to recurrent pathways within CA3. This is because the cell that you are recording from may only be weakly activated by mossy fibers, but other cells in CA3 may be strongly activated. These other cells in CA3 fire action potentials, and if their axons synapse on the cell you are recording from, then you will see an EPSP.

However, if you block AMPA receptors, then it is extremely unlikely that any mossy fiber input will be strong enough to make any CA3  spike. i.e. with AMPAergic input, there is no depolarization, and hence NMDA receptors will not open. However, if you are patching a cell, you can depolarize it, relieving the Mg2+ block from NMDA receptors. Then when the mossy fibers release glutamate, you will see a current through NMDA receptors. As no other CA3 cells will spike, you can be confident that they input you are recording is from the dentate.

Hope that makes it clear.

By depolarize the cell I am patching, do you mean holding it at a positive potential (such as +40mV)? If so, then the proper way to do this experiment would be to 1) incubate brain slice with ACSF + CNQX + bicuculline, 2) patch onto a CA3 pyramidal cell, then hold it at +40mV, 3) stimulate mossy fibers to activate release of glutamate, after which there will be an NMDA-mediated EPSC that can be recorded.

Does that sound right?

Hi Hong Lu,

+40 mV sounds good to me. In my experience, the cells wont like it quite as much as letting them sit at rest, but they should last long enough for most experiments.

And yes, they way you describe the experiment seems fine with me.

To get monosynaptic synaptic response, I located the stimulation electrode in the DG, at the border of the granular layer and moved the stimulation electrode along it until I got a monosynaptic response. For that you need to be able to move the stage of the microscope after you patched a CA3 neuron, so you can visualize the DG to move the stimulation electrode and set your stimulation to low intensity.

So what I ended up doing was patching onto CA3 pyramidal neurons and stimulating in stratum lucidum at low enough intensities so that I got failures half the time and successes the other half. That was my starting point for recordings. Then I incrementally increased the stimulation intensity, while monitoring whether the responses are monosynaptic or not. At the end of experiments, I added DCG-IV to see if evoked responses were mediated by mossy fibers. My stage is set up so that the patch pipette moves with it if it moves, so unfortunately I was unable to stimulate at a region that I could not visualize in the same field as the area I was patching, although I could try blind patch at a lower mag, and then stimulate in other regions, such as DG, for the future.

Sam, I was wondering if you had a tip for not losing cells everytime you move the stimulation electrode? Often when I moved my stimulation electrode while my CA3 cell was patched, I would lose it or the recordings would become unstable. If there a way to mitigate that? One way I can think of is perhaps stimulating, as you said, in DG, a further location. However, is there a way to not disturb the patched cell if the stimulation electrode was 100-200 µm away?

Hi Hong

Because my stimulation electrode was distant enough from the patch electrode, I didn't have this problem. Of course, it is important that your stimulation electrode doesn't touch the slice grid since any movement of the stimulation electrode will move the entire slice. I did patch from the CA1 pyramidal layer with a puff pipette in the stratum radiatum at 50-100 um distance between the pipettes and it was fine to move the puff pipette in the slice to find the best effect of the puff. I was using the diagonal approach of the micromanipulator to enter the tissue and moving in/out the pipette with a slow speed.

I forgot to mention in my previous answer that to avoid sectioning the mossy fiber during the slicing, you have to make a special cut. I will advice not to do horizontal or coronal slices. There are 3 ways:

- you can make the "magical cut" as explained in this article from P. Jonas lab (Bischofberger et al., Nat. Prot. 2006)

- you can prepare transversal slices (by isolating the whole hippocampi and cut them with a chopper)

-you can also try to cut sagittal slices but then only few slices from the dorsal hippocampus can be used.

If you preserve well the mossy fibers you might be able to have a longer distance between your stimulation and recording pipettes.

Good luck!