Exploring Sub-Microsecond Plasma Membrane Potential Shifts and Bioeffects Under Low-Energy Electric Pulse Stimulation

Low-energy micro- and millisecond electric pulses (EPs) charge and depolarize the cellular plasma membrane (PM) below the electroporation threshold. Conversely, individual nanosecond EPs (NSEPs) are too brief to initiate PM depolarization by charging the cells. It is hypothesized that a single NSEP induces cell depolarization via PM electroporation upon application of high-power EPs. However, low-energy NSEP bursts with a very high pulse repetition frequency may prevent electroporation. This method, a temporal summation of NSEPs, results in subsequent PM charging and depolarization. To visualize PM voltage changes during low-energy EPs, we employed optical measurements using FluoVolt™ (an organic fluorescent reporter of membrane potential [MP]) and a custom-made streak imaging system. Ultra-fast streak kymographs depicting MP changes were obtained after exposure to a ∼0.2 kV/cm single 200 µs EP and 5 MHz trains of 1000 and 2000 NSEPs with 100 ns duration. Immediately following exposure, a small FluoVolt™ response (up to ∼7% fluorescence change) was observed in the PM areas facing electrodes. The response duration directly correlated with the pulse width (PW) or duration of the NSEPs burst interval. The single 200 µs EP was more effective at charging the PM than an equivalent-energy 5 MHz burst of 2000 NSEPs of 100 ns duration. Furthermore, similar amplitudes of PM fluorescence changes between ∼0.2 kV/cm bursts of 1000 and 2000 NSEPs suggest that increasing either PW or applied voltage is necessary to enhance the extent of PM depolarization. Nonetheless, the modest depolarization effect reported herein was sufficient to open voltage-gated Ca²⁺ channels in neurons. These findings indicate that a 5 MHz burst of low-energy NSEPs and single µs EPs effectively induce PM depolarization and Ca²⁺ responses without causing any cellular damage.