Hello everybody. We are trying to write a script to analyze current-clamp electrophysiological traces. I write asking for help because we stumbled on an issue that we couldn't really solve. Depolarizing current injections trigger numerous action potentials. From literature, manuals, and guides for analysis programs, it appears that the AP amplitude must be measured from the V threshold. However, the first V threshold is used as a reference to calculate also the amplitude of ALL the following APs. The issue is that, depending on the cell, the APs after the first (especially the last ones) will be triggered at higher V thresholds. This causes a series of issues in the measurement, mainly due to the fact that:

• the amplitude of the APs after the first will be overestimated
• the measurement of the AP width, taken either at the Vthreshold or at the peak half-amplitude, will be wrong. For reference, please see the picture included. It shows a sweep from an example cell where the half-amplitude (calculated considering the Vthreshold of the first AP) is represented by the orange line. As you can see, it is not measuring the AP.

The question is: How do we measure amplitudes, voltage thresholds, and spike widths for action potentials following the initial AP? Or do we just calculate the measurements for only the first AP.Thank you for your help!