I would suggest to record this response at several different membrane potentials to estimate its reversal potential. Then compare the estimate with calculated reversal potential of IPSCs under your experimental conditions.

I guess you see the sum of many IPSCs here.

What happens if you decrease the illumination intensity?

As Maksim already mentioned, check the responses at different holding potentials and estimate/calculate the reversal potential.

Can you block the IPSCs?

Another alternative approach is to use a long-lasting ramp voltage, to assess the value of reversal potential and then to identify whether it is a stretch-activated non-selective current, TRP-related current, or chloride current. SN

Dear Emma,

I think it is looking very good and that is a normal response when you simultaneously evoke responses from many many inhibitory synapses by stimulating a large number of inhibitory synapses. As already mentioned above you are observing summation of many evoked IPSCs occuring in response to your stimulation. It is looking very good, and f you know the mean of spontaneous miniture (individual) IPSC amplitude size, then you can calculate the number of synapses you are activating during light stimulation, by simply dividing evoked response amplitude by spontaneous responses (evoked/spontaneous). There are also a number of other criteria you may consider calculating from your results such as quantal release etc. Also if you can measure the size or area and location of your stimulation (such as soma, distal or proximal dendrites) you may be able to produce some exciting results in relation to density of of inhibitory synapses in specific locations.

best wishes,

Refik

That looks way too slow to be a GABA-A IPSC, as it's lasting almost 1.5 seconds. It could be a slow synaptic current, but that is really slow even for those. Are you sure that you don't have ChR expressed in the neuron you're recording from? It looks suspiciously like a ChR mediated direct current. I would also advise trying picrotoxin or gabazine if you do think it is a direct IPSC.

I agree, decrease intensity, duration of the pulse (2-4ms) and Gabazine to confirm the GABAergic nature of the light evoked current. Best of luck!

Ammari Rachida Aaron G Roseberry Refik Kanjhan Sheng-Nan Wu Stefan Weigel Maksim V Storozhuk Hi, thanks for everyone's suggestions, I have measured the estimated reversal potential for this. It is around -40mV and decreasing light intensity would make this current smaller. One thing that also appear after multiple sweeps is that after multiple sweeps, the IPSCs shape look thinner (more normal looking). We speculated that this might be due to the internal taking time to fill the whole cells. And since these synapses theoretically locate on the dendrite, the currents are more affected. How does this sound?

Hi Emma,

You need to keep in mind that ChR2 will give you a huge and rather slow current wherever it is expressed, so your IPSCs will probably be affected due to an enormous presynaptic Ca2+ entry leading to a LOT of vesicle fusion. This fact paired with dendritic current filtering, summation and maybe some asynchronous presynaptic release (your timescale is also wide and slow!) will give you this unorthodox kinetics. Regarding the IPSC shape after several stimulations, it makes sense if you think that after some stimuli you deplete the presynaptic terminal leading to a reduced vesicle release when your AP arrives at the presynaptic side. It happens because the inter stimulus interval is shorter than the time needed to replenish the readily releasable pool. You can test this by giving the preparation more time to recover between stimuli - it should preserve the IPSC shape.

If you try to stimulate the afferent fibers electrically, would you see a similar kinetics?

Good luck,

Andre

-40 mV is reasonably close to reversal potential for Cl ions (it would be expected for Cl out 150 mM and Cl in 30 mM).However, a component of the response mediated by other than GABAA receptors, also cannot be excluded (and, actually may be worth studying). In this regard, effect of (e.g.) bicuculine on the responses would be of interest.

Hi Emma,

I would also like to add to above comments that as you apply multiple (trains of stimulation) or prolonged stimulations, the cell will be loaded with Cl- ions as the currents are quite large. Over time such accumulation of internal Cl- will also effect the reversal potential of responses, resulting in some sort of accomodation. Therefore you will need to take into consideration when you interpreting responses from subsequent stimulations.

best wishes,

Refik