How to determine whether a drug's effect is presynaptic or postsynaptic?

Rookie question here: In slice electrophysiology, if you’re recording from a postsynaptic neuron and you perfuse a drug, how do you determine whether the effect of the drug is directly on the postsynaptic neuron from which you’re recording or rather is due activity elicited in presynaptic neurons? As a real example, I have a compound that I think acts directly on a GPCR in a spinal neuron population, and I know that the GPCR is Gq coupled, so it should lead to some calcium increases intracellularly. I want to test this by recording from the spinal neuron and looking at the EPSC in the neuron. However, I suspect that the same GPCR might also be on the presynaptic afferents that terminate on the spinal neuron of interest. How do I exclude the presynaptic component and identify the portion of the effect that is through direct interaction with the GPCR on the postsynaptic neuron? Does adding TTX to the bath solution, to block sodium channels, allow you to say that whatever effect you record from the postsynaptic neuron is not due to presynaptic activity?

@MGradwell @YongHo

Cross-posted at ResearchGate:

The straightforward way to do this would be to record mEPSCs pre and post-drug. Then analyze your responses for changes in frequency or amplitude. Changes in frequency are usually reflective of a presynaptic effect, while amplitude changes are more postsynaptic. If you have changes in both then it’s likely that your drug has a mixed pre/post-synaptic influence.

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Thanks @ljmartin. Yes, that’s generally what I’ve seen in papers but I was wondering if there was a more definitive way. I think what you suggested should suffice.

This is pretty well accepted among electrophysiologists. If there’s another – perhaps more clever – way of doing this then I’d also like to know.

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Awesome. Thank you @ljmartin . Yeah, i don’t know that there is, but I’m new to spinal ephys so that’s why I asked. Thanks also for joining the forum.

You check for Paired-pulse ratio when you record from evoked EPSCs etc… decreased or increased ratio will give you a clue about the pre- or postsynaptic nature of the events.

Also, postsynaptic application of drugs through your recording pipette is another way to know if your response is localized specifically at the posysynaptic site.

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Thanks @soroush.dg. Also a very good idea, although more technically challenging than the suggestion about mEPSCs. I’ll keep in mind. Thanks for joining the forum.