Hi people,
I performed some experiments in Ca1 pyramidal neurons to study sodium and potassium currents. I used classic I/V recordings (10 mV steps starting from -80mV with the cell clamped to -60). Of course i had sodium currents and also potassium, how i can analyse the trace to be sure to not over- (or under) estimate the current? Is sufficient to measure at the peak of the current?
Thanks in advance
Hi Luca,
I am not quite sure what you mean by "to not over- or under- estimate the current", but typically if you clamp at -60mV most of the voltage-gated sodium channels are inactivated o you definitively underestimate the sodium component in your recordings, regardless if you measure the peak current of somewhere else.
What are you trying to measure exactly? Give us more details and we can certainly help you designing a proper protocol.
in voltage camp experiment it is quite difficult if you have combined current. if you have clampex then go in clamp-fit and get it pick for all the voltage, take the reversal potential and calculate the conductance and other para meter. See the paper singh et al., neuroscience 2013 http://www.sciencedirect.com/science/article/pii/S030645221201192X
and kharatmal et al., neuroscience 2015 http://www.sciencedirect.com/science/article/pii/S0306452215007150
if you need any particular paroblem let me know
good luck
JNS
Luca - If you haven't manipulated the currents in any way, there's no way to disassociate your INa from the other currents. Both the papers linked above use specific solutions to block other currents. The reason is pretty straightforward - if there are *any* voltage activated potassium channels (or calcium for that matter) there are multiple degenerate ways that the partial currents could add to the current that you measured. Even with the information from voltage steps from different places, reversal potentials etc. you just don't have enough information to say what part of the current is carried by sodium and what isn't. It might appear possible by just isolating the transient part of the current, but there you're also thwarted by pesky capacitance and the design of the patch-clamp amplifier circuitry. Even in optimal patching conditions, say you have a 10MOhm seal on a 30pA cell, your RC time constant is still hitting the wall of NaT kinetics. Worse, the current you read is actually that provided by your amplifier - since the action potential will be propagating away from you at the same time as your poor amplifier is trying to regain space clamp over those distal projections, you'll be systematically overestimating the current and widening its time course. Your best bet will be to get your bath nice and spiked with K+/Ca++ channel blockers, reduce the external concentration of Na++ and raise it internally, and pray for a quality seal. Best of luck :)
I agree with Jonny... the situation is hopeless unless you block all potassium and calcium currents. Even then, its hopeless, because of bad space clamp. The only way to do it is to acutely dissociate your cells and then patch. That way you can have good voltage and space clamp on a single neuron, but it will lose some of its dendrites and axon, of course. Sorry to be so pessimistic....
What I want to really suggest that without isolating single type of current is difficult to analyse accurately and papers linked above showing the use specific solutions to block the other currents. if current is with space clamp u will not get anything.
Hi Luca,
As others mentioned above, basically You cannot dissociate or analyse sodium currents from the I-V relationship what you got.
When you want to get the amplitude of the specific current, you have to use proper blockers.
Hi Luca,
You should record Na and K currents separately. For Na currents try a Cs-based pipette solution and TEA (20 mM), Cd (100 uM) extracellular. For K currents you should use a K-based pipette solution and TTx (1 uM) and Cd (100 uM) extracellular.
If for the purposes of your experiments you can't use two different solution, you should use a K-based pipette solution and record you mixed Na/K currents in control ACSF (+ Cd). After the control recordings you apply TTx to block Na currents and obtain K currents. After that you apply TTX + TEA (20 mM) (you may need also Ba 2mM), to block K currents. To get the isolated Na current you will need to subtract the K currents you obtained by applying TTX from the control traces. To get the isolated K currents you can subtract the traces in TEA from the traces in TTX.
A disadvantage of the second method is that you will not be able to clamp properly the cell with a K-based solution and it will affect fast and large currents such and Na currents and A-currents.
Hope this helps,
Maximiliano
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