I am recording mEPSC (at -80mV, 2micromolar TTX) and mIPSC (at -60mV, 2uM TTX, 20uM APV, 50uM DNQX) in amygdala C57 mice (8 to 10 weeks).
there is a report which says a sodium resistant channel Nav1.5 is present in amygdala, but it is usually reported to be blocked by 1uM TTX, to be safe I am using 2uM TTX...
But, my supervisor insists that if I see large amplitudes (compared to the more frequent smaller events) and very fast rise time and two decay phases, initial sharp and then slower... then it is a spike (sodium channel mediated) and not a synaptic event... both for mEPSC and mIPSC, additionally if there are these large events one after another, then its a regenerative process where sodium channels are activating themselves in feedforward loop to cause these events...
I've added a file to show which traces are rejected under this criteria.
What parameters are usually used to select mEPSC and mIPSC events ? What are the criteria used to exclude an event?
I cannot find any papers mentioning this phenomenon of TTX- R Na current in mEPSC and mIPSC events ...Please help. IS there any EXPERIMENTAL way I can prove there are no sodium channel currents in presence of 2uM TTX?
Switch to current clamp and see if you get any action potentials, either spontaneously or with current injection--that will tell you for certain whether your TTX is blocking V-gated Na channels. The traces you uploaded certainly look like miniPSC's. Even if you have horrible space clamp issues, the chances of seeing Na spikes in voltage clamp, especially at -80 mV, but even at -60 mV is pretty slim, so you should be ok.
I agree with Aaron, just test it. I would comment on your supervisor opinion: to have biphasic mEPSCs is actually common and reveal mixed AMPA / NMDA receptors. NMDARs are blocked at -80mV, but the local depolarization done by an AMPA mEPSC is enough to remove the voltage block. So you can see biphasic events: a sharp AMPA current followed by a longer NMDA tail.
Dear Pranali,
Given you have nice clamp conditions? the shape, rise time and decay of synaptic potentials will depend on distance to the source of them and on the membrane resistance. So, if they are large and with fast rice time you are recording close to the source of synaptic potentials. Far away they would be slower and smaller.
That may explain the shape and amplitude of your synaptic potentials.
Regarding existence of a Na current probably you may block all other currents and create conditions to isolate the Na current the block with TTX and if something remain is it.
That is a long way but would give you a definitive answer.
Best regards
to settle the argument, at the end of your experiment, throw in some DNQX or Gabazine to block your minis (EPSCs and IPSCs respectively) and record for 3 to 5 minutes. If you see anything left, your supervisor has a point. If you don't, he has none :)
Fouad Lemtiri-Chlieh Did This experiment for IPSCs, within 2 minutes all the events disappeared....
There you go...Done! You win the argument for IPSCs!
Do the same for EPSCs, this time with DNQX & CPP.
Actually I didn't , counterargument to that was that since now no gaba or ampa are open, the local depolarization is not there to activate the sodium channels.
Their logic is that when you have mini IPSC or EPSC currents, that will cause a local depolarization which will activate the sodium currents and that current will get combined with the gaba or mapa current and increase the overall amplitude.
They want me to do an experiment where I reduce the extracellular sodium by 90% and see if the bigger amplitude events dissapear ..
I agree with all the others regarding solutions to show this is synaptically driven and not AP dependent. The caveat to reducing the sodium by 90% is that this will reduce all EPSCs by a significant proportion also, as the current at -80 mV is driven predominantly by sodium as well. Furthermore, at least 80-90% of spontaneous EPSCs are actually mEPSCs even when recorded in the absence of TTX, so the likelihood of these AP driven events being driven by spontaneous spiking is already pretty low, and only going to be driven lower by the further application of TTX.
If you really want to prove the point though, you could prove it further by blocking IPSCs with gabazine and record mEPSCs at their reversal potential (~0 mV ) as sodium currents should reverse at around +40 mV they should be present even if the EPSC is essentially shunting.
Hope my 2 cents helps or is at least vaguely useful.
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