Embryonic fibroblast motility and orientation can be influenced by physiological electric fields

Epithelial layers in developing embryos are known to drive ion currents through themselves that will, in turn, generate small electric fields within the embryo. We hypothesized that the movement of migratory embryonic cells might be guided by such fields, and report here that embryonic quail somite fibroblast motility can be strongly influenced by small DC electric fields. These cells responded to such fields in three ways: (a) The cells migrated towards the cathodal end of the field by extending lamellipodia in that direction. The threshold field strength for this galvanotaxis was between 1 and 10 mV/mm when the cells were cultured in plasma. (b) The cells oriented their long axes perpendicular to the field lines. The threshold field strength for this response for a 90-min interval in the field was 150 mV/mm in F12 medium and between 50 and 100 mV/mm in plasma. (c) The cells elongated under the influence of field strengths of 400 mV/mm and greater. These fibroblasts were therefore able to detect a voltage gradient at least as low as 0.2 mV across their width. Electric fields of at least 10- fold larger in magnitude than this threshold field have been detected in vivo in at least one vertebrate thus far, so we believe that these field effects encompass a physiological range.     

The growth of PC-12 neurites is biased towards the anode of an applied electrical field

We have exposed cultures of PC12 cells to uniform DC electric fields following the addition of NGF. The success of these experiments relied upon the design of new chambers enabling fields to be applied to mammalian cell cultures. After 48 h of field application, the distribution of neurite outgrowths was biased towards the anode. More neurites faced the anode than would be expected if growth was uniform. The magnitude of this bias was strongly correlated with field strenght, with a threshold value of about 1 mV/mm. At field strengths above 30 mV/mm, the neurites growing towards the cathode were shorter than those growing towards the anode or perpendicular to the field. This response was not correlated with field strenght. This report confirms that mammalian neurons respond to electrical fields and supports the notion that neurites are influenced by endogenous electrical fields during development. As far as we are aware, this is the only report the documents a response towards the anode. 1994 John Wiley & Sons, Inc.     

Morphologic responses of osteoblast-like cells in monolayer culture to ELF electromagnetic fields

Osteoblast-like cells (MC 3T3-E1) were exposed for 24 h, immediately after plating, to a 60 Hz, 0.7 mT rms magnetic flux density, sufficient to induce an electric field of 0.5 mV/m rms, in order to investigate the influence of ELF field exposure on cell morphology. Using phase contrast images of the live cells, computerized image-analysis permitted rapid and objective quantification of cell length, width, area, perimeter, circularity and angular orientation. While the field-exposed cells were consistently smaller than sham treated cells, the morphologic alterations were not significantly different in the exposed cell population when cell orientation was not considered. When analyzed with respect to cell orientation, cells oriented parallel to the induced electric field (orthogonal to the applied magnetic field) demonstrated a significant decrease in cell length and an increase in roundness. These results confirm and extend previous studies on the morphologic adaptation of cells to low level ELF electromagnetic fields. The results suggest that the observed responses most likely depend on the induced electric field, with a field intensity threshold well below 1 mV/m. Further, these results provide important clues to the specific mechanism by which such low level fields may be capable of influencing cell behavior, and help to explain some of the difficulties in obtaining robust responses in in vitro EMF experiments.     

Weak electric fields serve as guidance cues that direct retinal ganglion cell axons in vitro

Growing axons are directed by an extracellular electric field in a process known as galvanotropism. The electric field is a predominant guidance cue directing retinal ganglion cell (RGC) axons to the future optic disc during embryonic development. Specifically, the axons of newborn RGCs grow along the extracellular voltage gradient that exists endogenously in the embryonic retina (Yamashita, 2013 [8]). To investigate the molecular mechanisms underlying galvanotropic behaviour, the quantification of the electric effect on axon orientation must be examined. In the present study, a culture system was built to apply a constant, uniform direct current (DC) electric field by supplying an electrical current to the culture medium, and this system also continuously recorded the voltage difference between the two points in the medium. A negative feedback circuit was designed to regulate the supplied current to maintain the voltage difference at the desired value. A chick embryo retinal strip was placed between the two points and cultured for 24 h in an electric field in the opposite direction to the endogenous field, and growing axons were fluorescently labelled for live cell imaging (calcein-AM). The strength of the exogenous field varied from 0.0005 mV/mm to 10.0 mV/mm. The results showed that RGC axons grew in the reverse direction towards the cathode at voltage gradients of ≥0.0005 mV/mm, and straightforward extensions were found in fields of ≥0.2–0.5 mV/mm, which were far weaker than the endogenous voltage gradient (15 mV/mm). These findings suggest that the endogenous electric field is sufficient to guide RGC axons in vivo.     

Lateral electrophoresis and diffusion of Concanavalin A receptors in the membrane of embryonic muscle cell

A uniform electric field of 10 V/cm applied across the surface of embryonic toad Xenopus muscle cells results in the asymmetric accumulation of concanavalin A (Con A) receptors toward one side of the cells within 10 min, as visualized by postfield fluorescent Con A labeling. This field produces an extracellular voltage difference of 20 mV across these 20-microns wide cells. The effect is reversible in two respects: (a) Additional exposure of the cell to the same field of opposite polarity for 10 min completely reverses the asymmetric accumulation to the other side of the cell. (b) Relaxation occurs after the removal of the field and results in complete recovery of the uniform distribution in 30 min. Both the accumulation and the recovery movements are independent of cell metabolism, and appear to be electrophoretic and diffusional in nature. The threshold field required to induce a detectable accumulation by the present method is between 1.0 and 1.5 V/cm (corresponding to a voltage difference of 2-3 mV across a 20-microns wide cell). The electrophoretic mobility of the most mobile population of nonliganded Con A receptors is estimated to be about 2 x 10(-3) microns/s per V/cm, while their diffusion coefficient is in the range of 4-7 x 10(-10) cm2/s. Extensive accumulation of the Con A receptors by an electric field results in the formation of immobile aggregates. The Con A receptors appear to consist of a heterogeneous population of membrane components different in their charge properties, mobility, and capability in forming aggregates.     

Neural crest cell galvanotaxis: new data and a novel approach to the analysis of both galvanotaxis and chemotaxis

The galvanotaxis response of neural crest cells that had migrated out of the neural tube of a 56-hr-old quail embryo onto glass coverslips was observed using time-lapse video microscopy. These cells exhibit a track velocity of about 7 microns/min and actively translocate toward the negative pole of an imposed DC electric field. This nonrandom migration could be detected for fields as low as 7 mV/mm (0.4 mV/cell length). We find that this directional migration is independent of the speed of migration and have generated a rather simple mathematical equation that fits these data. We find that the number of cells that translocate at a given angle, phi, with respect to the field is given by the equation N(phi) = exp(a0 + a1cos phi), where a1 is linearly proportional to the electric field strength for fields less than 390 mV/mm with a constant of proportionality equal to KG, the galvanotaxis constant. We show that KG = (150 mV/mm)-1, and at this field strength the cellular response is approximately half maximal. This approach to cellular translocation data analysis is generalizable to other directed movements such as chemotaxis and allows the direct comparison of different types of directed movements This analysis requires that the response of every cell, rather than averages of cellular responses, is reported. Once an equation for N(phi) is derived, several characteristics of the cellular response can be determined. Specifically, we describe 1) the critical field strength (390 mV/mm) below which the cellular response exhibits a simple, linear dependence on field strength (for larger field strengths, an inhibitory constant can be used to fit the data, suggesting that larger field strengths influence a second cellular target that inhibits the first); and 2) the amount of information the cell must obtain in order to generate the observed asymmetry in the translocation distribution (for a field strength of 100 mV/mm, 0.3 bits of information is required).     

The relationship between sensitivity to 60-Hz electric fields and induced transmembrane potentials in plants root cells

Summary Growth rates and cell diameters were determined from 12 species of plant roots exposed to a 60-Hertz (Hz) electric field of 360 Volts per meter (V/m) in an aqueous inorganic nutrient medium [conductivity: 0.07–0.09 Siemens per meter (S/m)]. The degree of growth depression ranged from zero to nearly 100 percent of control. Cell diameters ranged from 13.5 to 31.8 µm as an averaged value for procambial, cortical, and meristem cells. Sensitivity to the electric field as determined by root growth rate reduction increased with increasing cell size. Sensitivity also increased with increase in 60 Hz induced transmembrane potentials; the transmembrane potential threshold for growth reduction was about 6.0 mV and the potential for near-complete cessation of growth was about 10–11 mV.Two different hypothetical mechanisms of action by which applied electric fields induce biological effects at the cellular level were tested. The two mechanisms pertain to different possible modes of action of applied electric fields: one mechanism postulates the involvement of the transmembrane field, the other mechanism postulates the tangential electric field as the important factor for inducing biological effects. The data support the transmembrane and not the tangential field mechanism. It is concluded that the effects observed are consistent with a membrane related mechanism and that there is a narrow range (a few mV) between threshold and debilitating induced membrane potentials.     

Microtubule arrays in regeneratingMougeotia protoplasts may be oriented by electric fields

Summary Initially non-polar protoplasts of the green algaMougeotia will regenerate to re-establish their original cylindrical cell shape. The orientation of the growth axis of regenerating protoplasts held in agarose was independent of both the direction of incident white light and gravity. Protoplasts elongated parallel to applied DC electric fields of approx. 0.2 Vcm^–1 (1 mV/protoplast) and greater, with an increasing percentage oriented with increasing field strength. At the maximum field strength used (10 mV/cell), 53% of protoplasts were oriented within +- 10° of the 0/180° axis of the field. In untreated controls, the orientation of elongation was random. Protoplast survival was unaffected by field treatment. Some protoplasts (up to 37% in 10 mV/cell fields) formed outgrowths towards the cathode and occasionally towards the anode. Regenerating protoplasts in fields displayed the normal sequence of microtubule reorganization. This means that the positioning of the ordered symmetrical array of microtubules centred on two foci that appears within 3 to 4 h, and the subsequent organization of microtubules by 8 to 12 h into a band that intersects both foci and which is transverse to the axis of elongation (Galway and Hardham 1986), may be controlled by externally applied electric fields. In the region of this microtubule band, the applied field causes the plasma membrane to be stretched parallel to the field (Bryant and Wolfe 1987). We suggest that microtubules may become oriented perpendicular to the direction of field-induced membrane stretching, and that membrane stretching may be one of the orienting mechanisms for membrane-linked microtubules in elongating plant cells.Abbreviations PBS phosphate buffered saline - PMM protoplast maintenance medium - DMM dilute maintenance medium - MES 2(N-morpholino)ethanesulfonic acid - TRIS tris(hydroxymethyl)aminomethane - ANOVA analysis of variance     

Suppression of T-lymphocyte cytotoxicity following exposure to 60-Hz sinusoidal electric fields

A significant 25% inhibition (P less than .005) of allogeneic cytotoxicity of the target cell MPC-11 by the murine cytotoxic T-lymphocyte line CTLL-1 was observed when the 4-h cytotoxicity assay was conducted immediately following a 48-h pre-exposure of the effector lymphocytes to a 10-mV/cm (rms) 60-Hz sinusoidal electric field. At 1.0 mV/cm a significant 19% inhibition (P less than .0005) was seen. At 0.1 mV/cm a nonsignificant 7% inhibition of cytotoxicity was noted. When the 4-h cytotoxicity assay was conducted in the presence of the field using previously unexposed effector lymphocytes, cytotoxicity was not significantly reduced. Cell proliferation in the presence of interleukin-2 was unaffected by the field. These data suggest a dose response and threshold (between 0.1 and 1.0 mV/cm) for inhibition of cytotoxicity in clonal T-lymphocytes by exposure to a 60-Hz sinusoidal electric field. These results suggest mechanisms by which 60-Hz electric fields could affect the function of cells of the immune system.     

Neurites grow faster towards the cathode than the anode in a steady field.

We explanted fragments of embryonic chick dorsal root ganglia on to polylysine coated glass and cultured them in a medium containing one unit of nerve growth factor plus enough methylcellulose to give viscosities from 0.01-3,000 poise. We allowed them to grow out in the absence of a field, and then selected explants with halos of neurites which were relatively dense, relatively symmetrical, and practically free of glial cells. These selected explants were then exposed to electrical fields of up to 140 mV/mm for some hours. In media with viscosities of one poise or less, the field some times dragged the central cell mass of an explant towards the anode. However, in cases where the central cell mass did not move, fields of 70-140 mV/mm induced that sector of each neurite halo which faced the cathode to grow out several times faster than the one facing the anode.     

Hippocampal growth cone responses to focally applied electric fields

A wide variety of cell types respond to electric fields in culture. Despite evidence for electric fields existing in the mammalian embryo, there are few studies testing the effects electric fields exert on neurons from the mammalian central nervous system (CNS). The present study demonstrates orientation responses to focally applied electric fields of embryonic rat hippocampal neurons isolated in culture. The most striking result from this study is that different growth cones of the same neuron can show differential responsiveness to focally applied electric fields: growth cones on the short straight processes that are destined to become dendrites, oriented toward the cathode, whereas growth cones on the longest process, the presumptive axon, did not orient. The present experiments bring a significant increase in resolution to the study of neuronal growth cone orientation by applied electric fields: a novel examination of the early events leading to orientation. Growth cones on dendrites displayed a spectrum of orientation responses: directed lamellipodial extension, directed filopodial extension and/or reorientation, cytoplasmic swelling of existing filopodia, consolidation of filopodia, and rapid elongation of the entire process. Individual growth cones displayed only one or two of these responses. Additionally, not all growth cones on these short processes sustained their initial orientation response: 35% adapted within 6 min. © 1993 John Wiley & Sons, Inc.     

Growth rate and mitotic index analysis of Vicia faba L. roots exposed to 60-Hz electric fields

Growth, mitotic index, and growth rate recovery were determined for Vicia faba L. roots exposed to 60-Hz electric fields of 200, 290, and 360 V/m in an aqueous inorganic nutrient medium (conductivity 0.07-0.09 S/m). Root growth rate decreased in proportion to the increasing strength; the electric field threshold for a growth rate effect was about 230 V/m. The induced transmembrane potential at the threshold exposure was about 4-7 mV. The mitotic index was not affected by an electric field exposure sufficient to reduce root growth rate to about 35% of control. Root growth rate recovery from 31-96% of control occurred in 4 days after cessation of the 360 V/m exposure. The results support the postulate that the site of action of the applied electric fields is the cell membrane.     

Protein orientation in time-dependent electric fields: orientation before destruction

Proteins often have nonzero electric dipole moments, making them interact with external electric fields and offering a means for controlling their orientation. One application that is known to benefit from orientation control is single-particle imaging with x-ray free-electron lasers, in which diffraction is recorded from proteins in the gas phase to determine their structures. To this point, theoretical investigations into this phenomenon have assumed that the field experienced by the proteins is constant or a perfect step function, whereas any real-world pulse will be smooth. Here, we explore the possibility of orienting gas-phase proteins using time-dependent electric fields. We performed ab initio simulations to estimate the field strength required to break protein bonds, with 45 V/nm as a breaking point value. We then simulated ubiquitin in time-dependent electric fields using classical molecular dynamics. The minimal field strength required for orientation within 10 ns was on the order of 0.5 V/nm. Although high fields can be destructive for the structure, the structures in our simulations were preserved until orientation was achieved regardless of field strength, a principle we denote “orientation before destruction.”     

Input-output relationship in galvanotactic response of Dictyostelium cells

Under a direct current electric field, Dictyostelium cells exhibit migration towards the cathode. To determine the input-output relationship of the cell's galvanotactic response, we developed an experimental instrument in which electric signals applied to the cells are highly reproducible and the motile response are analyzed quantitatively. With no electric field, the cells moved randomly in all directions. Upon applying an electric field, cell migration speeds became about 1.3 times faster than those in the absence of an electric field. Such kinetic effects of electric fields on the migration were observed for cells stimulated between 0.25 and 10 V/cm of the field strength. The directions of cell migrations were biased toward the cathode in a positive manner with field strength, showing galvanotactic response in a dose-dependent manner. Quantitative analysis of the relationship between field strengths and directional movements revealed that the biased movements of the cells depend on the square of electric field strength, which can be described by one simple phenomenological equation. The threshold strength for the galvanotaxis was between 0.25 and 1 V/cm. Galvanotactic efficiency reached to half-maximum at 2.6 V/cm, which corresponds to an approximate 8 mV voltage difference between the cathode and anode direction of 10 microm wide, round cells. Based on these results, possible mechanisms of galvanotaxis in Dictyostelium cells were discussed. This development of experimental system, together with its good microscopic accessibility for intracellular signaling molecules, makes Dictyostelium cells attractive as a model organism for elucidating stochastic processes in the signaling systems responsible for cell motility and its regulations.     

Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment

The transmembrane potential on a cell exposed to an electric field is a critical parameter for successful cell permeabilization. In this study, the effect of cell shape and orientation on the induced transmembrane potential was analyzed. The transmembrane potential was calculated on prolate and oblate spheroidal cells for various orientations with respect to the electric field direction, both numerically and analytically. Changing the orientation of the cells decreases the induced transmembrane potential from its maximum value when the longest axis of the cell is parallel to the electric field, to its minimum value when the longest axis of the cell is perpendicular to the electric field. The dependency on orientation is more pronounced for elongated cells while it is negligible for spherical cells. The part of the cell membrane where a threshold transmembrane potential is exceeded represents the area of electropermeabilization, i.e. the membrane area through which the transport of molecules is established. Therefore the surface exposed to the transmembrane potential above the threshold value was calculated. The biological relevance of these theoretical results was confirmed with experimental results of the electropermeabilization of plated Chinese hamster ovary cells, which are elongated. Theoretical and experimental results show that permeabilization is not only a function of electric field intensity and cell size but also of cell shape and orientation.     

Pulsed electric current induces the differentiation of human keratinocytes

Although normal human keratinocytes are known to migrate toward the cathode in a direct current (DC) electric field, other effects of the electric stimulation on keratinocyte activities are still unclear. We have investigated the keratinocyte differentiation under monodirectional pulsed electric stimulation which reduces the electrothermal and electrochemical hazards of a DC application. When cultured keratinocytes were exposed to the electric field of 3 V (ca. 100 mV/mm) or 5 V (ca. 166 mV/mm) at a frequency of 4,800 Hz for 5 min a day for 5 days, cell growth under the 5-V stimulation was significantly suppressed as compared with the control culture. Expression of mRNAs encoding keratinocyte differentiation markers such as keratin 10, involucrin, transglutaminase 1, and filaggrin was significantly increased in response to the 5-V stimulation, while the 3-V stimulation induced no significant change. After the 5-V stimulation, enhanced immunofluorescent stainings of involucrin and filaggrin were observed. These results indicate that monodirectional pulsed electric stimulation induces the keratinocyte differentiation with growth arrest.     

Weak Sinusoidal Electric Fields Entrain Spontaneous Ca Transients in the Dendritic Tufts of CA1 Pyramidal Cells in Rat Hippocampal Slice Preparations

Neurons might interact via electric fields and this notion has been referred to as ephaptic interaction. It has been shown that various types of ion channels are distributed along the dendrites and are capable of supporting generation of dendritic spikes. We hypothesized that generation of dendritic spikes play important roles in the ephaptic interactions either by amplifying the impact of electric fields or by providing current source to generate electric fields. To test if dendritic activities can be modulated by electric fields, we developed a method to monitor local Ca-transients in the dendrites of a neuronal population in acute rat hippocampal slices by applying spinning-disk confocal microscopy and multi-cell dye loading technique. In a condition in which the dendrites of CA1 pyramidal neurons show spontaneous Ca-transients due to added 50 μM 4-aminopyridine to the bathing medium and adjusted extracellular potassium concentration, we examined the impact of sinusoidal electric fields on the Ca-transients. We have found that spontaneously occurring fast-Ca-transients in the tufts of the apical dendrites of CA1 pyramidal neurons can be blocked by applying 1 μM tetrodotoxin, and that the timing of the transients become entrained to sub-threshold 1-4 Hz electric fields with an intensity as weak as 0.84 mV/mm applied parallel to the somato-dendritic axis of the neurons. The extent of entrainment increases with intensity below 5 mV/mm, but does not increase further over the range of 5-20 mV/mm. These results suggest that population of pyramidal cells might be able to detect electric fields with biologically relevant intensity by modulating the timing of dendritic spikes.     

Airway epithelial wounds in rhesus monkey generate ionic currents that guide cell migration to promote healing

Damage to the respiratory epithelium is one of the most critical steps to many life-threatening diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. The mechanisms underlying repair of the damaged epithelium have not yet been fully elucidated. Here we provide experimental evidence suggesting a novel mechanism for wound repair: endogenous electric currents. It is known that the airway epithelium maintains a voltage difference referred to as the transepithelial potential. Using a noninvasive vibrating probe, we demonstrate that wounds in the epithelium of trachea from rhesus monkeys generate significant outward electric currents. A small slit wound produced an outward current (1.59 μA/cm(2)), which could be enhanced (nearly doubled) by the ion transport stimulator aminophylline. In addition, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) with CFTR(Inh)-172 significantly reduced wound currents (0.17 μA/cm(2)), implicating an important role of ion transporters in wound induced electric potentials. Time-lapse video microscopy showed that applied electric fields (EFs) induced robust directional migration of primary tracheobronchial epithelial cells from rhesus monkeys, towards the cathode, with a threshold of <23 mV/mm. Reversal of the field polarity induced cell migration towards the new cathode. We further demonstrate that application of an EF promoted wound healing in a monolayer wound healing assay. Our results suggest that endogenous electric currents at sites of tracheal epithelial injury may direct cell migration, which could benefit restitution of damaged airway mucosa. Manipulation of ion transport may lead to novel therapeutic approaches to repair damaged respiratory epithelium.     

Membrane potential perturbations induced in tissue cells by pulsed electric fields

Pulsed electric fields directly influence the electrophysiology of tissue cells by transiently perturbing their transmembrane potential. To determine the magnitude and time course of this interaction, electrotonic cable theory was used to calculate the membrane potential perturbations induced in tissue cells by a spatially uniform, pulsed electric field. Analytic solutions were obtained that predict shifts in membrane potential along the length of cells as a function of time in response to an electrical pulse. For elongated tissue cells, or groups of tissue cells that are coupled electrotonically by gap junctions, significant hyperpolarizations and depolarizations can result from millisecond applications of electric fields with strengths on the order of 10–100 mV/cm. The results illustrate the importance of considering cellular cable parameters in assessing the effects of transient electric fields on biological systems, as well as in predicting the efficacy of pulsed electric fields in medical treatments. © 1995 Wiley-Liss, Inc.     

The electromechanics of DNA in a synthetic nanopore

We have explored the electromechanical properties of DNA on a nanometer-length scale using an electric field to force single molecules through synthetic nanopores in ultrathin silicon nitride membranes. At low electric fields, E < 200 mV/10 nm, we observed that single-stranded DNA can permeate pores with a diameter >/=1.0 nm, whereas double-stranded DNA only permeates pores with a diameter >/=3 nm. For pores <3.0 nm diameter, we find a threshold for permeation of double-stranded DNA that depends on the electric field and pH. For a 2 nm diameter pore, the electric field threshold is approximately 3.1 V/10 nm at pH = 8.5; the threshold decreases as pH becomes more acidic or the diameter increases. Molecular dynamics indicates that the field threshold originates from a stretching transition in DNA that occurs under the force gradient in a nanopore. Lowering pH destabilizes the double helix, facilitating DNA translocation at lower fields.