title: Nanosecond pulsed electric field thresholds for nanopore formation in neural cells.
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.
authors.affiliation: University of Texas Health Science Center, Department of Radiology, San Antonio, Texas 78229, USA. caleb.c.roth.ctr@mail.mil
authors.forename: Caleb C
authors.initials: CC
authors.lastname: Roth
authors.forename: Gleb P
authors.initials: GP
authors.lastname: Tolstykh
authors.forename: Jason A
authors.initials: JA
authors.lastname: Payne
authors.forename: Marjorie A
authors.initials: MA
authors.lastname: Kuipers
authors.forename: Gary L
authors.initials: GL
authors.lastname: Thompson
authors.forename: Mauris N
authors.initials: MN
authors.lastname: DeSilva
authors.forename: Bennett L
authors.initials: BL
authors.lastname: Ibey
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004558:Electric Stimulation; D004560:Electricity; D055664:Electrochemical Techniques; D005456:Fluorescent Dyes / Q000737:chemistry; D006624:Hippocampus / Q000166:cytology; D008959:Models, Neurological; D058608:Nanopores; D036103:Nanotechnology; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D009930:Organic Chemicals / Q000737:chemistry; D051381:Rats
chemical_list: D017304:Annexin A5; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C071336:calcium green; D002118:Calcium