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. 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?
If it acts on the postsynaptic GPCR, GDP-betaS, a G-protein inhibitor, in your intracellular solution will block the effects of the compound.
If you can record "Miniature"-EPSCs, check the frequency and amplitudes of mEPSCs before and after the application. In general, change in the frequency is derived from presynaptic mechanisms, and change in the amplitude is postsynaptic.
Glutamate (or AMPA) iontophoresis in the presence of TTX will also help.
These methods are common, and the combination of these experiments will provide a strong evidence for the postsynaptic mechanisms.
This paper will help you (PMID: 22586220), although the main focus of the paper is not EPSCs.
Hope it will help you.
Hi am also looking for the basis mechanism for D4 regulation of dopamine release synaptic or post synaptic effect.
I'd suggest you to do Paired pulse ratio test before and after application, in the absence of TTX. Alternatively, as suggested by Akihiro you can also look for mEPSC frequency as it is attributed to change in release probability.
Hi
I would use TTX to block presynaptic spiking, and depending on the current you are interested in, you may also have to block other inhibitory and/or excitatory currents that could potentially modify the current of interest via presynaptic mechanisms. Once you have your drug cocktail, you could record mini EPSCs or mini IPSCs, as needed. However, if the postsynaptic current is large you should get a steady (i.e., "tonic") shift in either inward or outward current, depending on the channel. The sustained current is unambiguously postsynaptic, whereas the interpretation of minis can give you a headache! You also have to play careful attention to the reversal potential for your system, so you can set the appropriate holding potential for the voltage clamp.
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