I am using HEKA in current clamp mode to record from mouse DRG neurons. I record action potential in DRG neurons by injecting 80 ms current pulses from 100 - 1000 pA in 50 pA steps (current step protocol) followed by 1 min wash, then another current step protocol. In my system, the minimum current needed to elicit an action potential (AP threshold) is changing (increasing) in the second current step protocol. Any suggestions about why this might be?
Dear Sampurna,
There are quite a few factors that may be contributing to the action potential threshold. One of those factors may be the changing resting membrane potential baseline; if the resting potential is slighly depolorized then you will notice a decrease in the threshold, and you will notice an opposite effect if it is hyperpolarized. Other factor may be the change in the access resistance of the microelectrode, changes in the electrode resistance usually due to blockage of the tip can significantly effect the voltage and current control. Another factor may be due to calcium loading of cells following prolonged depolarizations that can subsequently lead to changes in action potential generations. If you need to test for action potential threshold, you dont need to use such long (80ms) pulses. For this, I usually use pulses at around 1ms. Also amplitude of current steps you are using may be too much- you do not need to apply 1000pA steps (non physiological levels). If you reduce your maximum current then you may also be able to reduce the amplitude of your current steps in between intervals. For examples, please see my following publications in isolated DRG neurons and pelvic ganglion neurons.
Best wishes,
Refik
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Dear Sampurna: The duration of 80 msec used for recording action potential (AP) appears to be a littler longer. Current amplitude used to elicit AP could be quite variable, depending on the magnitude of “leak”component under experimental condition.
Dear Sampurna,
There are quite a few factors that may be contributing to the action potential threshold. One of those factors may be the changing resting membrane potential baseline; if the resting potential is slighly depolorized then you will notice a decrease in the threshold, and you will notice an opposite effect if it is hyperpolarized. Other factor may be the change in the access resistance of the microelectrode, changes in the electrode resistance usually due to blockage of the tip can significantly effect the voltage and current control. Another factor may be due to calcium loading of cells following prolonged depolarizations that can subsequently lead to changes in action potential generations. If you need to test for action potential threshold, you dont need to use such long (80ms) pulses. For this, I usually use pulses at around 1ms. Also amplitude of current steps you are using may be too much- you do not need to apply 1000pA steps (non physiological levels). If you reduce your maximum current then you may also be able to reduce the amplitude of your current steps in between intervals. For examples, please see my following publications in isolated DRG neurons and pelvic ganglion neurons.
Best wishes,
Refik
Article Tertiapin-Q Blocks Recombinant and Native Large Conductance ...
Article Postnatal Maturational Changes in Rat Pelvic Autonomic Gangl...