New Insights into Biophysical Mechanisms of the nsPEF-Induced Neuronal Response

Abstract

Simulation studies of neuromuscular incapacitation using high-intensity electric pulses (EPs) were previously reported. These studies hypothesized that a reversible action potential (AP) block can be achieved based on energy deposition causing neuronal electroporation. However, theoretical concepts were presented without elaboration of specific details on possible biological mechanisms. Recently, we discovered that nanosecond pulsed electric fields (nsPEF) could initiate phosphatidylinositol-4,5-bisphosphate (PIP2) depletion in non-excitable cells. PIP2 is the precursor for important second messengers and is a key modulator of the neuronal ion channels involved in AP generation. By using primary hippocampal neurons (PHN) and the PLCδ-PH-EGFP optical probe of PIP2 hydrolysis, we demonstrated that electric field (EF) exposure induced PIP2 depletion in the PHN, and defined EF exposure parameters necessary to safely elicit reversible effects without neuronal damage. Results show that five days after neuronal dissociation (D5), the pre-exposure level of the cytoplasmic PLCδ-PH-EGFP fluorescence is significantly higher in D5 neurons than in D1 neurons, likely due to higher levels of tonic inositol1,4,5-trisphosphate (IP3). Such biological sensitization caused D5 neurons to respond intensely following a single 7.5 kV/cm 600 ns EP, while the D1 neurons did not respond. Despite age of development, the stronger 15 kV/cm 600 ns or longer 7.5 kV/cm 12 µs EPs initiated profound PIP2 depletion in all neurons studied, outlining direct impact on the neuronal plasma membrane during electroporation. Accordingly, D1 neurons exposed to such EPs had significant post-exposure propidium iodide (PI) uptake. In the more sensitive D5 neurons, PIP2 recovery was achieved within 10 min after all 600 ns EPs exposures, but 12 µs EPs caused irreversible PIP2 depletion. Thus, nsPEF-induced PIP2 depletion in neurons could be the primary biological mechanism responsible for both stimulation and latent inhibition of neuronal tissues.