I am recording AMPA:NMDA ratios, and I am having difficulty eliciting a single-peak AMPA response. I am often getting a polysynaptic/multi-peak AMPA current response. Is this a space clamp problem, or a matter of distance between the stimulator and recording electrode? Thanks
If you have AMPA currents from different dendritic compartments (which is very likely) it may be difficult to get clean single peaks. Best bet would be to record more and try to isolate them in post, or perhaps lower the injected charge (i.e. pulse duration) if you are going for channels close to your pipette.
Edit: yes, it would be a space clamp issue.
It may help to know what system you are working on (hippocampus, cerebellum, neuronal culture?), how you are stimulating (electrical stimulation, paired recording, optogenetics), and what your recording conditions are (electrode and bath solutions).
Multiple peaks could be, as Apostolos suggests, multiple synaptic inputs onto one cell, in which case moving the stimulating electrode or decreasing the stimulation intensity may help.
It could also be polysynaptic activity, as you suggested in your question. Just to make sure we mean the same thing, I use this term to mean the situation where you stimulate one neuron, which activates your intended target (neuron 1 -> recorded neuron), but also a second neuron that also synapses on your intended target (neuron 1 -> neuron 2 -> recorded neuron). In this case, your best recourse may be to reduce excitability, to try to prevent action potentials in neuron 2. For example, you could lower Ca or increase Mg in the bath, or use a receptor antagonist at low concentration (e.g. 250 nM NBQX).
Another possibility is that the later peak is delayed/asynchronous release. In this case, the later peaks would be the same size as spontaneous mEPSCs. You can reduce delayed release by applying EGTA-AM to your preparation, though it will also reduce the synchronous EPSC somewhat.
Another possibility is that the second peak is sodium channel activation. That could happen if the neuron you are recording from is escaping from voltage clamp, and the axon hillock is trying to fire an action potential. This is a problem for us, because our cells have huge AMPA currents (e.g. 10 nA). We deal with this issue by using recording pipettes with resistances below 2 M-Ohm, using series resistance compensation, and putting QX314 (Na channel antagonist) in the recording electrode. Usually, the sodium peak blends in with the AMPA current and isn't very distinct.
Hope one of these suggestions may be relevant!
-Matthew
Thank you for your suggestions, Apostolos Mikroulis and Matthew Xu-Friedman
I am working in slice in the cortex and am using electrical stimulation. And yes exactly, that's what I mean by polysynaptic activity. I will try both of your suggestions by isolating in post and adjusting the cation ratio in the bath.
Thanks again for your helpful insights! Much appreciated.
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