I am quite confused on how action potentials (intracellular AP) and PSP's (either EPSP or IPSP by having GABA antagonists or AMPA and NMDA antagonists) are recorded using the same patch clamp technique. Are there any differences in use of recording solutions or cell-attached vs. whole cell mode etc... Does it mean people use TTX while they measure PSP's; where as to measure AP's they simply don't have TTX in solution. Please point me to direction where I could read and understand the basics of how to record different potentials and what they actually mean.
Hi Srinivasa,
The recording solution for Cell attached mode and Whole cell mode is different. There are multiple reasons for that and one of that is to avoid dialysis when you go whole cell. When you go whole cell the milieu inside the cell and your pipette solution becomes one and you want to make sure that the ionic contents of your pipette solution is as close as possible to the concentration of ions inside the cell. If not, the ions will move one way or the other depending on the concentration difference and that is not ideal for the cell and also for your recording. Pipette solutions for whole cell mode also has ATP, GTP and other phospho-nucleotides to mimic the cellular contents and they are also very important in maintaining the internal cellular environment. This follows the other reason, which is, the price of the phospho-nucleotides are really high and you don't want to waste them by adding in your pipette solution meant to record in cell-attached mode. Since, you are not breaking in the cell you don't need any of these and having ionic concentrations that loosely mimics the concentration of ions inside the cell will do it. Another thing to keep in mind is to have the osmolality of the pipette solution for whole cell mode similar to what's inside the cell.
As far as measuring PSP's go I am not sure how much I can help since I don't record them but I think adding TTX is probably not the way to go. Here is my reasoning. Since PSP's are a result of release of neurotransmitter from a presynaptic cell in response to an action potential (AP), adding TTX in the bath solution will block those action potential and as a result the relase of neurotransmitter will be blocked as well. Therefore, you cannot record PSP's in that case and in this experimental paradigm I think you can only record miniEPSP (not quite sure though). My other reasoning is that since PSP's can be either excitatory or inhibitory depending what channels it opens and if the stimulus is excitatory which also can depolarize the membrane beyond its threshold, adding TTX will block all that and you won't be measuring those PSP's. I know I am not giving you an answer but just something to think about. I am sure there are people in researchgate that will have more direct answer to this question.
Good Luck!
Hi, with patch clamp whole cell approach you can record APs and PSPs which happen in between AP. But by penetrating the cell membrane you alter the cell and its excitability too. The best way to record spiking is a patch-clamp in cell attach mode, but in this case you don't see PSPs. Also there is a perforated patch but it is tricky. If you are interested in postsynaptic events use voltage clamp mode. Yo can clamp the neuronal with holding potential far from AP threshold and record tha PSC (post synaptic currents). People use TTX to record miniature PSCs (mPSCs) they are postsynaptic events evoked by spontaneous thermodynamic release of neurotransmitter, not associated with AP generation.
Good luck
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