I am using current clamp to record the action potential of the stellate cell in entorhinal cortex . I am try to find the rheobase (minimum injecting current to let cell produce a complete single action potential)
My mentor said I only when rheobase is accurate, the threshold of the action potential could be accurate. But after I adding 4-AP (kind of potassium channel blocker) into the external solution. The shape of the action potential changed, and I couldn't determine the rheobase. The enclosed is a graph indicating different action potential induced by different levels of injecting current.
Is there anyone know what is the criteria of finding the rheobase???I am very confused!!! Thanks a lot!!!
Hi Wanyu,
The three action potentials pretty much look the same to me. It is clear that you indeed inject less current for the black trace, as you see the membrane charging slower initally.
Blocking potassium channels increases the excitability of a cell i.e. reduce the spiking threshold and rheobase. It should also change the shape of the action potential, since this is affected by potassium currents.
As you said, the criterion for the rheobase is that it is the minimum current at which the cell generates an action potential (after a long duration) All currents you inject lead to the generation of an action potential, so the rheobase is lower than 300 pA (around 100 pA guessing from the curvature of the black trace). What is the response for smaller injected currents?
Hope this helps a bit.
Dear Wanyu,
I would suggest you to make stimulation protocol (script) which increases stimulation current step by step (let's say by 5pA) from the basal (holding) current. Each step (its stimulation part) should last for more than 300ms. I would also set the recover delay after eact step (e.g., 1 second)
Than check which step was the first with APs. This step's current may be considered as rheobase.
For more precise measurement one can also use ramp increase of current instead of stepped.
Pay attention on the X-scale of your graph (AP length less than 1ms!).
Good luck!
Hello Wanyu,
I agree with Tigran on how to obtain the rheobase. In addition to what he said after running a first current clamp protocol with 5pA steps and determining the current injection at which you had your first action potential, you can then run another protocol between the previous step (i.e step before action potential) with smaller steps( e.g. 1pA steps) that gives you a more accurate estimate. Good luck!
Dear Wanyu
I fully agree with the previous proposition of step protocol to exactly determine the rheobase and the fact that blocking potassium currents affect spike shape, as well as resting membrane potential. In some experiment, it is also shown that 4AP could induce spontaneous spiking.
However, to come back to rheobase, as a first approximation, you could look for the slope change in the depolarizing phase of the spike to estimate the membrane potential range, where you will apply steps with small increments.
Dear Wanyu,
Rheobase is defined as the current amplitude of infinite duration needed to depolarize the membrane potential to threshold for action potential generation.
In real life, you can inject depolarizing current steps of increasing amplitude (with small increments) until the cell fires. The rheobase will depend on the length of the current step, the longer the step the smaller the current needed usually.
Stellate cells of the entorhinal cortex show early spiking (i.e. the delay is short) so a step of 500 ms will be enough.
4-AP will block different potassium currents according to the concentration: 1 mM will block also Kv4 channels. The action potential shape will change in any case and it is very likely that you will get bursting after bath application of 4AP, but that is OK and the rheobase in 4AP will be the current needed to get a single burst. You should expect this current to be of smaller amplitude than in control condition.
Best,
Maximiliano
hello,
As said by Max, the rheobase is the minimum current of infinite duration (meaning 500 ms or 1 s long) required to trigger an action potential.
best
fabien
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