Abstract
The persistent influx of ions through nanopores created upon cellular exposure to nanosecond pulse electric fields (nsPEF) could be used to modulate neuronal function. One ion, calcium (Ca(2+)), is important to action potential firing and regulates many ion channels. However, uncontrolled hyper-excitability of neurons leads to Ca(2+) overload and neurodegeneration. Thus, to prevent unintended consequences of nsPEF-induced neural stimulation, knowledge of optimum exposure parameters is required. We determined the relationship between nsPEF exposure parameters (pulse width and amplitude) and nanopore formation in two cell types: rodent neuroblastoma (NG108) and mouse primary hippocampal neurons (PHN). We identified thresholds for nanoporation using Annexin V and FM1-43, to detect changes in membrane asymmetry, and through Ca(2+) influx using Calcium Green. The ED50 for a single 600 ns pulse, necessary to cause uptake of extracellular Ca(2+), was 1.76 kV/cm for NG108 and 0.84 kV/cm for PHN. At 16.2 kV/cm, the ED50 for pulse width was 95 ns for both cell lines. Cadmium, a nonspecific Ca(2+) channel blocker, failed to prevent Ca(2+) uptake suggesting that observed influx is likely due to nanoporation. These data demonstrate that moderate amplitude single nsPEF exposures result in rapid Ca(2+) influx that may be capable of controllably modulating neurological function.
Highlights
High voltage nanosecond pulsed electric fields have been shown to be effective stimuli to induce apoptosis in cells, activate platelet aggregation, and as a skin cancer therapy.[1,2,3] Theoretical models have predicted that nanosecond pulse electric fields (nsPEF) could be used to manipulate neurological signals responsible for motor movement and pain.[4,5] One experimental study performed by Pakhomov et al has validated the potential for nsPEF to interfere with motor signals from the brain.[6]
Probit analysis is optimal for dealing with this variability as it predicts the ED50 point based on binary data. By applying this statistical technique, we found that both NG108 and primary hippocampal neurons (PHN) display an ED50 at roughly 95 ns
The overarching goal of this work was to establish the threshold for nanopore formation in both NG108 and PHN cells
Summary
High voltage nanosecond pulsed electric fields (nsPEFs) have been shown to be effective stimuli to induce apoptosis in cells, activate platelet aggregation, and as a skin cancer therapy.[1,2,3] Theoretical models have predicted that nsPEF could be used to manipulate neurological signals responsible for motor movement and pain.[4,5] One experimental study performed by Pakhomov et al has validated the potential for nsPEF to interfere with motor signals from the brain.[6]. When exposed cells are under a whole cell patch clamp, nanopore activity appears to have unique electrical characteristics.[13] These characteristics include inward (depolarization) by allowing movement of otherwise impermeable cations, such as sodium (Naþ) and calcium (Ca2þ), across the plasma membrane. This induced depolarization fully recovers over several minutes.[10]
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