Dear all,
I would like to seek your opinion regarding analysis of EPSC when drugs or treatment compounds are wash-in or washout from the slices. For a recording period of 10 minutes upon acute application of treatment into the system, what type of data analysis can better mirror the effect of treatment ----
1. time-point whereby 10 minutes recording period is divided into 10 1-minute epoch, and all 10 epochs are then averaged out to get the results. Or maybe using the same method, I select every alternate minute as 1-minute epoch.
2. last 3-minute of recording, whereby I analyse only the last 3 minute of the 10 minute recording period. Or instead of the last 3 minute, is it common to do analysis by using the middle 3-minute?
3. total average of the 10 minutes recording.
Thank you.
Well,
It would depend on what kind of EPSC you record.
Are you talking about evoked EPSC (eEPSC obtained by a stimulation of a fiber tract) ?
Or are you recording from spontaneous EPSC (sEPSC) or from miniatures (spontaneous EPSC recorded in the presence of TTX: mEPSC) ?
For the former eEPSC, you may want to measure the amplitude before, during a after drug. The time course would give you a hint about the effect.
Remember that the amplitude of EPSC may quite variable (due to the quantal release), so you need at least 10 mins of stable baseline before applying drugs. Then depending of the time course of the effect you may measure it at the steady-state during x mins depending on the kinetics.
For the latter (sEPSC or mEPSC), it is more problematic because the EPSC frequency is stochastic and the amplitude would be highly variable. You need at at least 300 events before and as many after drugs. You may then bin them over 10- s for Visual inspection of frequency and amplitude but if you need statistical analysis of expected changes you should use non parametric tests such as Kolmogorov-Smirnov.
Hi Fabian,
I am working on sEPSC. Generally I record 10 mins of stable baseline before adding in drugs and record for another 10 min and follow by washout.
Ok,
sEPSC are a mixture of mEPSC and action potential driven EPSC (by spontaneous firing of pre-synaptic neurons).
Literature suggests that mEPSC and evoked EPSC may not due to the same vesicular pool and therefore may not be considered as equivalent in terms of regulation and functional role.
It would be best therefore to test your drugs in both conditions (on sEPSC then on mEPSC by adding TTX).
Generally :
1/ a change in frequency is interpreted as a pre-synaptic effect such as an alteration of the release machinery or activation of pre-synaptic channels.However, if the effects is only observed on sEPSc you may infer that your drug has altered the firing of the pre-synaptic neurons or the your drug specifically targets the ready-releasable pool of synaptic vesicles dedicated to action potential driven release.
2/ a change in amplitude is interpreted as a post-synaptic alteration of post-synaptic receptors (withdrawal or increase expression depending on the changes you observed).
3/ sometimes you get both : a decrease in frequency and a decrease in amplitude. In this case you should remind that sEPSC detection is prone to uncertainty due to the background noise. To say it differently, a decrease in amplitude may lead to a decraese in frequency because most EPSC would be now hidden in the noise.
As I wrote before
For sEPSC or mEPSC analysis, you need at at least 300 events before and as many after drugs. You may then bin them over 10- s for Visual inspection of frequency and amplitude but if you need statistical analysis of expected changes you should use non parametric tests such as Kolmogorov-Smirnov
Regards
Thank you very much Fabien for your advice! I shall try to test on mEPSC.
Regards