Impact of Nanosecond Pulsed Electric Fields on Primary Hippocampal Neurons Caleb C. Roth 1 , Jason A. Payne 2 , Marjorie A. Kuipers 2 , Gary L. Thompson 3 , Gerald J. Wilmink 2 , Bennett L. Ibey 2 1 General Dynamics Information Technology, Fort Sam Houston, TX, USA 2 Radio Frequency Bioeffects Branch, Human Effectiveness Directorate, Air Force Research Laboratory, Fort Sam Houston TX, USA 3 National Research Council, Fort Sam Houston, TX USA ABSTRACT Cellular exposure to nanosecond pulsed electric fields (nsPEF) are believed to cause immediate creation of nanopores in the plasma membrane. These nanopores enable passage of small ions, but remain impermeable to larger molecules like propidium iodide. Previous work has shown that nanopores are stable for minutes after exposure, suggesting that formation of nanopores in excitable cells could lead to prolonged action potential inhibition. Previously, we measured the formation of nanopores in neuroblastoma cells by measuring the influx of extracellular calcium by preloading cells with Calcium Green-AM. In this work, we explored the impact of changing the width of a single nsPEF, at constant amplitude, on uptake of extracellular calcium ions by primary hippocampal neurons (PHN). Calcium Green was again used to measure the influx of extracellular calcium and FM1-43 was used to monitor changes in membrane conformation. The observed thresholds for nanopore formation in PHN by nsPEF were comparable to those measured in neuroblastoma. This work is the first study of nsPEF effects on PHN and strongly suggests that neurological inhibition by nanosecond electrical pulses is highly likely at doses well below irreversible damage. Keywords: Nanopores, nanosecond electrical pulses, membrane damage, primary hippocampal neurons, calcium green, FM1-43 1. INTRODUCTION Nanosecond pulsed electric fields (nsPEF) are high voltage square wave pulses with duration (τ) under 1 µs. Originally engineered for plasma formation, various biological applications of nsPEF are being pursued including electromuscular incapacitation, cancer therapy, gene transfection, and pain suppression. 1-7 When applied directly to mammalian cells, multifarious effects have been observed including nuclear granulation, cellular swelling, bleb formation, and apoptosis. 1,2 Recent studies have shown that when nsPEF are applied directly to cells small pores, termed nanopores, are preferentially formed in the plasma membrane. 8 In contrast to classical electroporation, these nanopores are believed to have a diameter of only a few nanometers and therefore do not readily allow large molecules such as propidium iodide to enter the cell. 8,9 Therefore, unlike longer duration pulses, nsPEF enable the manipulation of cellular function without a high degree of mortality. Further distinguishing nsPEF induced- nanopores, many unique properties have been identified using a variety of techniques including patch clamp, fluorescent microscopy, direct ion measurement in bulk solution, and flow cytometry. 10,11,12 Specifically, the direction of ion flow appears predominantly inward, they open and close regularly at a slower rate than protein ion channels, and they have a lifetime at room temperature over many minutes. 8 Interestingly, nsPEFs may provide a unique technique for controlling the activity of neurons within deep tissue by causing either stimulation (at low dose) or inhibition (at high dose). Previous work by Rogers has shown that low voltage nsPEFs can trigger action potentials (AP) leading to contractions in isolated frog muscle. 13 It was believed that nsPEF can cause AP generation by activating the release of acetylcholine into the synaptic cleft. 6 Jiang and Cooper demonstrated that a single, 12 ns pulse at 403 V/cm was capable of activating skin nociceptors. They demonstrated this same effect at 100 pulses delivered at 4000 Hz with very low voltages (16.7 V/cm) without any Photonic Therapeutics and Diagnostics VIII, edited by Nikiforos Kollias, et al., Proc. of SPIE Vol. 8207, 820763 © 2012 SPIE · CCC code: 1605-7422/12/$18 · doi: 10.1117/12.911802 Proc. of SPIE Vol. 8207 820763-1 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/27/2012 Terms of Use: http://spiedl.org/terms