Embryonic cell motility can be guided by physiological electric fields

Migratory embryonic quail somitic fibroblasts display a striking sensitivity to small, steady electric fields. There are three components to their response. They begin to orient their long axes perpendicular to the field lines within 5 min of current application at the optimal field strength of 600 mV/mm. The threshold field for significant orientation in 90 min is 150 mV/mm (only 3 mV/cell width). The cells migrate toward the cathode with a similar low threshold. At field strengths greater than 400 mV/mm, the cells also elongate beginning about 1 h after field application. The importance of this embryonic cell galvanotaxis and orientation by electric fields lies in the possible utilization of this behavior both by the embryo in the guidance of embryonic cell migration in vivo and by the investigator to control cell morphology and directionality of movement in vitro in order to study mechanisms of motility.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Endogenous electric currents might guide rostral migration of neuroblasts

Mechanisms that guide directional migration of neuroblasts from the subventricular zone (SVZ) are not well understood. We report here that endogenous electric currents serve as a guidance cue for neuroblast migration. We identify the existence of naturally occurring electric currents (1.5±0.6 μA/cm², average field strength of ～3 mV/mm) along the rostral migration path in adult mouse brain. Electric fields of similar strength direct migration of neuroblasts from the SVZ in culture and in brain slices. The purinergic receptor P2Y1 mediates this migration. The results indicate that naturally occurring electric currents serve as a new guidance mechanism for rostral neuronal migration.     

Evaluation of the electrostatic field strength at the site of exocytosis in adrenal chromaffin cells.

Exocytosis in secretory cells consists of release from intracellular storage granules directly into the extracellular space via fusion of the granule membrane with the plasma membrane of the cell. It is considered here as comprising two distinct processes. One is the close apposition of granule and plasma membranes. The other arises from interactions between the two membranes during the process of apposition, leading to the formation of a fusion pore. In the following it is shown for the case of the adrenal medullary chromaffin cell that the fusion pore can be ascribed to electroporation of the granule membrane, triggered by the strong electric field existing at the site of exocytosis. Based on an electric surface charge model of the cytoplasmic side of the plasma membrane, resulting from the negatively charged phosphatidylserine groups, it is found that the electrostatic field strength at the site of exocytosis reaches values on the order of 10(8) V/m at small intermembrane distances of 3 nm and lower. The field strength increases with the size of the disc-shaped plasma membrane region generating the electric field, reaching an approximate limit for a radius of 10 nm, at a surface charge density of 5.4 x 10(-2) C/m2. According to previous experimental evaluations of threshold field strength, this field is sufficiently strong to cause membrane electroporation. This step is a precondition for the subsequent membrane fusion during the ongoing process of apposition, leading to secretion.     

Cyclic AMP response in cells exposed to electric fields of different frequencies and intensities

The action on intracellular cyclic AMP (cAMP) of therapeutically used 4000-Hz electric fields was investigated and compared with 50-Hz data. Cultured mouse fibroblasts were exposed for 5 minutes to 4000-Hz sine wave internal electric fields between 3 mV/m and 30 V/m applied within culture medium. A statistically significant decrease in cellular cAMP concentration relative to unexposed cells was observed for fields higher than 10 mV/m. The drop in cAMP was most pronounced at lower field strengths (71 % of controls at 30 mV/m) and tended to disappear at higher field strengths. An increase of cAMP content was observed with 50-Hz electric fields, as was also the case when 4000-Hz fields were modulated with certain low frequencies.     

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     

Measurement and analysis of static magnetic fields that block action potentials in cultured neurons

To characterize the properties of static magnetic fields on firing of action potentials (AP) by sensory neurons in cell culture, we developed a mathematical formalism based on the expression for the magnetic field of a single circular current loop. The calculated fields fit closely the field measurements taken with a Hall effect gaussmeter. The biological effect induced by different arrays of permanent magnets depended principally on the spatial variation of the fields, quantified by the value of the gradient of the field magnitude. Magnetic arrays of different sizes (macroarray: four center-charged neodymium magnets of ?14 mm diameter; microarray: four micromagnets of the same material but of ?0.4 mm diameter) allowed comparison of fields with similar gradients but different intensities at the cell position. These two arrays had a common gradient value of ?1 mT/mm and blocked >70% of AP. Alternatively, cells placed in a field strength of ?0.2 mT and a gradient of ?0.02 mT/mm produced by the macroarray resulted in no significant reduction of firing; a microarray field of the same strength but with a higher gradient of ?1.5 mT/mm caused ?80% AP blockade. The experimental threshold gradient and the calculated threshold field intensity for blockade of action potentials by these arrays were estimated to be ?0.02 mT/mm and ?0.02 mT, respectively. In conclusion, these findings suggest that spatial variation of the magnetic field is the principal cause of AP blockade in dorsal root ganglia in vitro. © 1995 Wiley-Liss, Inc.     

Assessment of exposure to intermediate frequency electric fields and contact currents from a plasma ball

While electric fields at intermediate frequencies are not widely utilized for industrial technologies, surprisingly, certain toys emit the highest electric fields found in our living environment. These toys, plasma balls, are devices that use high voltage to create ionized light discharges. In this study, we assessed exposure to electric fields and contact/induced current from a recreational plasma ball device. The electric field strength was measured as a function of distance from the device, and the contact/induced current was measured with a current clamp in different exposure situations with point or grasping contact. The characteristic spectra of the electric field and contact current were measured, and both the multiple frequency rule and weighting of the spectra were applied according to the International Commission on Non-Ionizing Radiation Protection (ICNIRP) 1998 and 2010 guidelines. The results indicate that the recommended reference levels for the general public are exceeded at distances <1.2 m, and that the contact currents in the hand may be twice higher than recommended by the general public guidelines.     

Effects of a low-voltage static electric field on energy metabolism in astrocytes

Mouse astrocytes (glial cells) in primary cultures were exposed to a low-voltage static DC electric field with no current flow and thus with no generation of magnetic fields. The electric field altered the rate of glycolysis, measured by 2-deoxyglucose accumulation. The magnitude and direction of this effect depended on the polarization of the field and the applied voltage. The maximum effect was an increase of ∼30%, which occurred with field across the cells at an intensity that can be calculated to be 0.3 mV/cm or less. Reversal of the polarization converted the stimulation to a small but statistically significant inhibition. Bioelectromagnetics 18:77–80, 1997. © 1997 Wiley-Liss, Inc.     

Electroporation of DC-3F Cells Is a Dual Process

Treatment of biological material by pulsed electric fields is a versatile technique in biotechnology and biomedicine used, for example, in delivering DNA into cells (transfection), ablation of tumors, and food processing. Field exposure is associated with a membrane permeability increase usually ascribed to electroporation, i.e., formation of aqueous membrane pores. Knowledge of the underlying processes at the membrane level is predominantly built on theoretical considerations and molecular dynamics (MD) simulations. However, experimental data needed to monitor these processes with sufficient temporal resolution are scarce. The whole-cell patch-clamp technique was employed to investigate the effect of millisecond pulsed electric fields on DC-3F cells. Cellular membrane permeabilization was monitored by a conductance increase. For the first time, to our knowledge, it could be established experimentally that electroporation consists of two clearly separate processes: a rapid membrane poration (transient electroporation) that occurs while the membrane is depolarized or hyperpolarized to voltages beyond so-called threshold potentials (here, +201 mV and −231 mV, respectively) and is reversible within ∼100 ms after the pulse, and a long-term, or persistent, permeabilization covering the whole voltage range. The latter prevailed after the pulse for at least 40 min, the postpulse time span tested experimentally. With mildly depolarizing or hyperpolarizing pulses just above threshold potentials, the two processes could be separated, since persistent (but not transient) permeabilization required repetitive pulse exposure. Conductance increased stepwise and gradually with depolarizing and hyperpolarizing pulses, respectively. Persistent permeabilization could also be elicited by single depolarizing/hyperpolarizing pulses of very high field strength. Experimental measurements of propidium iodide uptake provided evidence of a real membrane phenomenon, rather than a mere patch-clamp artifact. In short, the response of DC-3F cells to strong pulsed electric fields was separated into a transient electroporation and a persistent permeabilization. The latter dominates postpulse membrane properties but to date has not been addressed by electroporation theory or MD simulations.