2-ns Electrostimulation of Ca²⁺ Influx into Chromaffin Cells: Rapid Modulation by Field Reversal

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca²⁺ levels due to Ca²⁺ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca²⁺ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca²⁺ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.