Low intensity transcranial electric stimulation: Safety, ethical, legal regulatory and application guidelines.

Low intensity transcranial electrical stimulation (TES) in humans, encompassing transcranial direct current (tDCS), transcutaneous spinal Direct Current Stimulation (tsDCS), transcranial alternating current (tACS), and transcranial random noise (tRNS) stimulation or their combinations, appears to be safe. No serious adverse events (SAEs) have been reported so far in over 18,000 sessions administered to healthy subjects, neurological and psychiatric patients, as summarized here. Moderate adverse events (AEs), as defined by the necessity to intervene, are rare, and include skin burns with tDCS due to suboptimal electrode-skin contact. Very rarely mania or hypomania was induced in patients with depression (11 documented cases), yet a causal relationship is difficult to prove because of the low incidence rate and limited numbers of subjects in controlled trials. Mild AEs (MAEs) include headache and fatigue following stimulation as well as prickling and burning sensations occurring during tDCS at peak-to-baseline intensities of 1-2mA and during tACS at higher peak-to-peak intensities above 2mA. The prevalence of published AEs is different in studies specifically assessing AEs vs. those not assessing them, being higher in the former. AEs are frequently reported by individuals receiving placebo stimulation. The profile of AEs in terms of frequency, magnitude and type is comparable in healthy and clinical populations, and this is also the case for more vulnerable populations, such as children, elderly persons, or pregnant women. Combined interventions (e.g., co-application of drugs, electrophysiological measurements, neuroimaging) were not associated with further safety issues. Safety is established for low-intensity 'conventional' TES defined as <4mA, up to 60min duration per day. Animal studies and modeling evidence indicate that brain injury could occur at predicted current densities in the brain of 6.3-13A/m2 that are over an order of magnitude above those produced by tDCS in humans. Using AC stimulation fewer AEs were reported compared to DC. In specific paradigms with amplitudes of up to 10mA, frequencies in the kHz range appear to be safe. In this paper we provide structured interviews and recommend their use in future controlled studies, in particular when trying to extend the parameters applied. We also discuss recent regulatory issues, reporting practices and ethical issues. These recommendations achieved consensus in a meeting, which took place in Göttingen, Germany, on September 6-7, 2016 and were refined thereafter by email correspondence.

The expression and roles of Nde1 and Ndel1 in the adult mammalian central nervous system.

Mental and neurological illnesses affect one in four people. While genetic linkage analyses have shown an association of nuclear distribution factor E (NDE1, or NudE) and its ohnolog NDE-like 1 (NDEL1, or Nudel) with mental disorders, the cellular mechanisms remain unclear. In the present study, we have demonstrated that Nde1 and Ndel1 are differentially localised in the subventricular zone (SVZ) of the forebrain and the subgranular zone (SGZ) of the hippocampus, two regions where neurogenesis actively occurs in the adult brain. Nde1, but not Ndel1, is localized to putative SVZ stem cells, and to actively dividing progenitors of the SGZ. The influence of these proteins on neural stem cell differentiation was investigated by overexpression in a hippocampal neural stem cell line, HCN-A94. Increasing Nde1 expression in this neural stem cell line led to increased neuronal differentiation while decreasing levels of astroglial differentiation. In primary cultured neurons and astrocytes, Nde1 and Ndel1 were found to have different but comparable subcellular localizations. In addition, we have shown for the first time that Nde1 is heterogeneously distributed in cortical astrocytes of human brains. Our data indicate that Nde1 and Ndel1 have distinct but overlapping distribution patterns in mouse brain and cultured nerve cells. They may function differently and therefore their dosage changes may contribute to some aspects of mental disorders.

Chronic wound state exacerbated by oxidative stress in Pax6+/- aniridia-related keratopathy.

Heterozygosity for the transcription factor PAX6 causes eye disease in humans, characterized by corneal opacity. The molecular aetiology of such disease was investigated using a Pax6+/- mouse model. We found that the barrier function of uninjured Pax6+/- corneas was compromised and that Ca2+-PKC/PLC-ERK/p38 signalling pathways were abnormally activated, mimicking a 'wounded' epithelial state. Using proteomic analysis and direct assay for oxidized proteins, Pax6+/- corneas were found to be susceptible to oxidative stress and they exhibited a wound-healing delay which could be rescued by providing reducing agents such as glutathione. Pax6 protein was oxidized and excluded from the nucleus of stressed corneal epithelial cells, with concomitant loss of corneal epithelial markers and expression of fibroblast/myofibroblast markers. We suggest a chronic wound model for Pax6-related corneal diseases, in which oxidative stress underlies a positive feedback mechanism by depleting nuclear Pax6, delaying wound healing, and activating cell signalling pathways that lead to metaplasia of the corneal epithelium. The study mechanistically links a relatively minor dosage deficiency of a transcription factor with potentially catastrophic degenerative corneal disease.

Developmental effects of physiologically weak electric fields and heat: an overview.

This study summarizes the possible effects on prenatal development of physiologically weak electric fields induced in the body by exposure to extremely low frequency (ELF) electromagnetic fields and of elevated temperature levels that might result from exposure to radiofrequency (RF) radiation. Both topics have been discussed at recent international workshops organized by WHO in collaboration with other bodies. Mammalian development is characterized by a highly ordered sequence of cell proliferation and differentiation, migration, and programmed cell death. These processes, particularly proliferation and migration, are susceptible to a variety of environmental agents including raised maternal temperature. In addition, there is growing evidence that physiologically weak endogenous DC electric fields and ionic currents have a role in guiding developmental processes, including cell orientation and migration, by establishing electrical potential gradients. Disruption of these fields can adversely affect development in amphibian and bird embryos, which are experimentally accessible, and may well do so in mammalian embryos. The extent to which induced ELF electric fields might influence these and other processes that take place during prenatal development, childhood, and adolescence is less clear. Organogenesis, which takes place primarily during the embryonic period, is susceptible to raised maternal temperatures; a large number of studies have shown that RF exposure produces developmental effects that can be attributed to heat. The development of the central nervous system is particularly susceptible to raised temperatures; a reduction in brain size, which results in a smaller head, is one of the most sensitive markers of heat-induced developmental abnormalities and can be correlated with heat-induced behavioral deficits. However, some aspects of CNS development have been less well explored, particularly effects on corticogenesis. In addition, the persistence of CNS developmental sensitivity through to childhood and adolescence is not clear.

Re-orientation and faster, directed migration of lens epithelial cells in a physiological electric field.

The vertebrate lens drives current through itself in a pattern which concentrates current efflux at the lens equator. Lens epithelial cells (LECs) move into this region where they change shape and differentiate into lens fibre cells. The mechanisms underpinning these cell behaviors are unclear. We have attempted to mimic, in isolation, the effects which such electrical signals have on LEC behaviors, by culturing LECs in a physiological DC electric field (EF) similar to that in lens. Primary human (PHLECs), primary bovine (PBLECs) and a transformed human cell line (THLECs) all changed shape to lie perpendicular to the EF, the same orientation which LECs adopt with respect to the equatorial EF as they differentiate into lens fibre cells. Exposure to an EF also significantly increased the migration rate of all three LEC types. All three LECs also showed directed cell migration although, curiously, different cell types moved in different directions. PBLECs and THLECs showed voltage-dependent, anode-directed migration, with a response threshold between 100-150 mV mm(-1)and 25-50 mV mm(-1), respectively. Small sheets of THLECs also migrated anodally. By contrast PHLECs migrated cathodally with a response threshold below 100 mV mm(-1). Reversing the polarity reversed the migration direction for each cell type. These observations raise three possibilities: (1) that small electric field may be one of the cues regulating lens epithelial cell behaviors in vivo; (2) that altering the in vivo electric field by lens replacement may contribute to the aberrant migration of epithelial cells in conditions such as posterior capsule opacification and (3) that applying electric fields may be one way of controlling aberrant lens epithelial cell behaviors.

Neurotrophins enhance electric field-directed growth cone guidance and directed nerve branching.

Neurotrophins play major roles in the developing nervous system in controlling neuronal differentiation, neurite outgrowth, guidance and branching, synapse formation and maturation, and neuronal survival or death. There is increasing evidence that nervous system construction takes place in the presence of dc electric fields, which fluctuate dynamically in space and time during embryonic development. These have their origins in the neural tube itself, as well as in surrounding skin and gut. Early disruption of these endogenous electric fields causes failure of the nervous system to form, or else it forms aberrantly. Nerve growth, guidance, and branching are controlled tightly during pathway construction and in vitro dc electric fields have profound effects on each of these behaviours. We have used cultured neurones to ask whether neurotrophins and dc electric fields might interact in shaping neuronal growth, given their coexistence in vivo. Electric field-directed nerve growth generally was enhanced by the simultaneous presentation of several neurotrophins to the growth cone. Under certain circumstances, more nerves turned cathodally, they turned faster, further, and in lower field strengths. Intriguingly, other combinations of dc electric field and neurotrophins (low field strength and neurotrophin 3 (NT-3) switched the direction of growth cone turning. Additionally, cathodally directed nerve growth was faster and directed branching was much more common when electric fields and neurotrophins interacted with neuronal growth cones. Given such profound changes in growth cone behaviour in vitro, neurotrophins and endogenous electric fields are likely to interact in vivo.

A small, physiological electric field orients cell division.

We report on an observation that the orientation of cell division is directed by small, applied electric fields (EFs). Cultured human corneal epithelial cells were exposed to a direct-current EF of physiological magnitude. Cells divided while attached to the culture dish, and most did so with a cleavage plane perpendicular to the EF vector. There are many instances in which cell divisions in vivo occur in the presence of direct-current physiological EF, for example, during embryonic morphogenesis, neuronal and epithelial differentiation, wound healing, or tumor formation. Endogenous physiological EFs may play important roles in some or all of these processes by regulating the axis of cell division and, hence, the positioning of daughter cells.

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin.

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

Modelling corneal epithelial wound closure in the presence of physiological electric fields via a moving boundary formalism.

A new framework for the modelling of corneal epithelial wound healing is presented, which can include the presence of a physiological electric field. The difficulty inherent in the inclusion of this biological phenomenon motivates our use of a moving boundary formalism. A key conclusion is that the model predicts a linear relation between the wound healing speed and the physiological electric field strengths over a physiologically large range of electric field strength. Another key point is that this linear relationship between electric field strength and wound healing speed is robust to variations in critical parameters that are difficult to estimate. The linearity is also robust to different realizations of the modelling framework presented.

The direction of neurite growth in a weak DC electric field depends on the substratum: contributions of adhesivity and net surface charge.

We investigated the influence of the growth surface on the direction of Xenopus spinal neurite growth in the presence of a dc electric field of physiological magnitude. The direction of galvanotropism was determined by the substratum; neurites grew toward the negative electrode (cathode) on untreated Falcon tissue culture plastic or on laminin substrata, which are negatively charged, but neurites growing on polylysine, which is positively charged, turned toward the positive electrode (anode). Growth was oriented randomly on all substrata without an electric field. We tested the hypothesis that the charge of the growth surface was responsible for reversed galvanotropism on polylysine by growing neurons on tissue culture dishes with different net surface charges. Although neurites grew cathodally on both Plastek substrata, the frequency of anodal turning was greater on dishes with a net positive charge (Plastek C) than on those with a net negative charge (Plastek M). The charge of the growth surface therefore influenced the frequency of anodal galvanotropism but a reversal in surface charge was insufficient to reverse galvanotropism completely, possibly because of differences in the relative magnitude of the substratum charge densities. The influence of substratum adhesion on galvanotropism was considered by growing neurites on a range of polylysine concentrations. Growth cone to substratum adhesivity was measured using a blasting assay. Adhesivity and the frequency of anodal turning were graded over the range of polylysine concentrations (0 = 0.1 < 1 < 10 = 100 microg/ml). The direction of neurite growth in an electric field is therefore influenced by both substratum charge and growth cone-to-substratum adhesivity. These data are consistent with the idea that spatial or temporal variation in the expression of adhesion molecules in embryos may interact with naturally occurring electric fields to enhance growth cone pathfinding.

Human corneal epithelial cells reorient and migrate cathodally in a small applied electric field.

PURPOSE: To test whether human corneal epithelial cells (HCECs) respond to small applied electric fields (EFs) in a similar manner to bovine corneal epithelial cells (BCECs), the orientation and directed migration in small EFs of both primary cultures and of a human corneal epithelial cell line were quantified. METHODS: Primary cultures of human corneal epithelial cells (PHCECs) and transformed human corneal epithelial cells (THCECs) were exposed to EFs (100 mV/mm-250 mV/mm) in different media. Cell migration was traced using an image analyser. RESULTS: PHCECs and THCECs reoriented and migrated towards the cathode (negative pole) when cultured in small direct current (dc) EFs. Both the reorientation and directional migration were voltage- and serum-dependent, as shown previously for bovine cells. PHCECs and THCECs showed significant perpendicular orientation in EFs at 150 mV/mm in medium with serum, while at the same voltage, no significant orientation was found in serum free medium. PHCECs started to show perpendicular reorientation around 30 min after onset of EF at 150 mV/mm. They showed significant directional migration at 150 mV/mm, with directedness of 0.35 +/- 0.07 and a migration rate of 9.1 +/- 0.7 microns/h (n = 90), both significantly higher than that of cells in serum free medium. Addition of EGF-induced significant reorientation and directional migration of THCECs at 100 mV/mm. Additionally, as for BCECs, which remained viable and responsive to electric fields for at least 75 h at 150 mV/mm, THCECs also remained viable and showed responsiveness during long periods of exposure to EFs (at least 20 h). CONCLUSIONS: Cultured human primary CECs and a human corneal epithelial cell line both responded to small EFs with perpendicular reorientation and cathodally-directed migration. Cell responses were qualitatively similar to those reported previously for bovine CECs. The endogenous EFs generated by wounded cornea may play an important role in promoting cell shape changes and directed migration of CECs during the healing process.

Physiological electrical fields modify cell behaviour.

Steady direct current (dc) electric fields exist in many biological systems over many hours. At these times cells are dividing, differentiating, moving to final locations and extending motile processes. Each of these events may be influenced by physiological electric fields in tissue culture and when electric fields are disrupted in vivo, major developmental abnormalities arise. The likelihood of physiological electric fields playing a role in cell behaviours and some potential mechanisms are outlined.

Integrated interactions between chondroitin sulphate proteoglycans and weak dc electric fields regulate nerve growth cone guidance in vitro.

During development and regenerative growth, neuronal pathways are defined in part by several endogenous cues that collectively determine directed growth. The interactions between such cues largely are unknown. To address potential interactions, we have examined in vitro the combined effect on nerve growth of two endogenous growth cone guidance cues: chondroitin sulphate proteoglycans and weak dc electric fields. Addition to the culture medium of a chondroitin 6-sulphate/keratan sulphate containing PG (BNC-PG) markedly enhanced the cathodal re-orientation of embryonic Xenopus neurites in an electric field, whereas a proteoglycan containing chondroitin 4-sulphate (RC-PG) was inhibitory. These effects of BNC-PG and RC-PG were reproduced by their chondroitin sulphate glycosaminoglycan side chains alone. Chondroitin 6-sulphate or chondroitin 4-sulphate, respectively, enhanced and inhibited cathodally-directed nerve re-orientation. This was dependent on the integrity of the glycosaminoglycan chain structure; when digested into their disaccharide subunits both molecules became inactive. Keratan sulphate, a minor component of BNC-PG, was found to be inhibitory, whereas dermatan sulphate, an epimer of chondroitin 4-sulphate, had no effect. We conclude that in vitro specific interactions between these two nerve guidance cues do occur and that the specificity of the response is critically dependent on the charge pattern of the proteoglycans chondroitin sulphate side chains. The expression of a host of proteoglycans with differing glycosaminoglycan side chains varies in both time and place in the developing nervous system, thus the scope is vast for spatial and temporal modulation of nerve guidance by interacting cues.

Directed migration of corneal epithelial sheets in physiological electric fields.

PURPOSE: To characterize the effects of small applied electric fields (EFs) (100 to 250 mV/ mm) on cultured bovine corneal epithelial cell (CEC) sheets and to determine how EFs interact with other environmental cues in directing CEC sheet migration. METHODS: Primary cultures of bovine CECs were exposed to EFs in medium with or without serum, epithelial growth factor, basic fibroblast growth factor, or transforming growth factor-beta 1. Cell sheet migration was traced using an image analyzer. RESULTS: Cell sheets migrated toward the cathode (negative pole). The directional migration was voltage dependent, and, at low field strength (up to 200 mV/mm), it required serum in the medium. Sheets showed no migration responses up to 200 mV/mm in serum-free medium, whereas those in medium with serum showed evident migration toward the cathode, at an average rate of approximately 15 microns/h (n = 15 approximately 20) at 150 mV/mm. When serum was present, the threshold was below 100 mV/mm, very close to the measured wound field strength (approximately 42 mV/mm). After supplementing serum-free medium with individual growth factors or with combinations of epithelial growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1, significant restoration of cathode-directed migration occurred at 150 mV/ mm. Lamellipodia were abundant at the leading edges of migrating sheets, extending the area of sheets covered. The extension of cell membranes toward the cathode was more prominent in cell sheets than in single cells. CONCLUSIONS: The endogenous EFs generated by wounded cornea could play an important role by interacting with other environmental factors to promote changes in shape and in directed migration of CEC sheets.

Lectins implicate specific carbohydrate domains in electric field stimulated nerve growth and guidance.

Both endogenous lectins and DC electric fields may control aspects of early nerve growth and nerve guidance. To test whether such endogenous cues interact, lectins of varying sugar affinity and valency were studied for effects on electric field induced growth and reorientation of cultured Xenopus neurites. Concanavalin A (Con A), succinylated concanavalin A (S-Con A), and wheat germ agglutinin all completely inhibited field-induced cathodal reorientation. Lentil and pea lectins, which share the same sugar affinity as Con A/S-Con A, were only partially effective in inhibiting reorientation. Because S-Con A does not alter lateral mobility of membrane receptors, the previously accepted notion that Con A inhibited field-induced reorientation by preventing receptors from translocating and becoming redistributed asymmetrically in the membrane may be oversimplified. There are likely to be additional steric interactions that Con A and S-Con A share that inactivate asymmetrically redistributed receptors and prevent reorientation. Additionally, nerves growing in an applied field branch more commonly toward the cathode. Con A and S-Con A alone prevented this development of asymmetric branching. All the lectins tested prevented the normal field-induced increase in nerve growth rate, while all, except peanut agglutinin, prevented the usual faster growth cathodally than anodally. We suggest that lectin interactions with electric field effects in vitro may involve modulation of neuronal nicotinic acetylcholine receptors, neurotrophin receptors, or voltage-dependent calcium channels. Similar interactions between endogenous lectins and endogenous electric fields are to be expected.

Orientation and directed migration of cultured corneal epithelial cells in small electric fields are serum dependent.

Reorientation and migration of cultured bovine corneal epithelial cells (CECs) in an electric field were studied. Electric field application was designed to model the laterally directed, steady direct current electric fields which arise in an injured corneal epithelium. Single cells cultured in media containing 10% foetal bovine serum showed significant galvanotropism, reorienting to lie perpendicular to electric field vector with a threshold field strength of less than 100 mV/mm. Cells cultured in serum-free medium showed no reorientation until 250 mV/mm. Addition of EGF, bFGF or TGF-beta 1 singly or in combination to serum free medium significantly restored the reorientation response at low field strengths. Both the mean translocation rate and directedness of cell migration were serum dependent. Cultured in medium with serum or serum plus added EGF, single cells showed obvious cathodal migration at 100 mV/mm. Increasing electric field strength enhanced the cathodal directedness of single cell migration. Supplementing serum free medium with growth factors restored the cathodal directed migration of single cells and highest directedness was found for the combination of EGF and TGF-beta 1. Corneal epithelial sheets also migrated towards the cathode in electric fields. Serum or individual growth factors stimulated CEC motility (randomly directed). Applied fields did not further augment migration rates but added a vector to stimulated migration. Electric fields which are present in wounded cornea interact with other environmental factors and may impinge on CECs migration during wound healing. Therapies which combine the application of growth factors and electric fields may be useful clinically.

Growth cone neurotransmitter receptor activation modulates electric field-guided nerve growth.

We have studied the interactions between two nerve guidance cues, which alone induce substantial growth cone turning: endogenous neurotransmitters and small dc electric fields. d-tubocurarine, a nicotinic AChR (acetylcholine receptor) antagonist, inhibited field-induced cathodal orientation of cultured neurites, whereas atropine, a muscarinic AChR blocker, and suramin, a P2-purinoceptor antagonist, markedly enhanced the guidance properties of the applied field. These experiments implicate the activation of growth cone nicotinic AChRs by self-released acetylcholine in the mechanism underpinning electric field-induced neurite orientation and raise the possibility that growth cones release neurotransmitter prior to target interaction in order to assist their own pathfinding. Additionally, they provide the first evidence that coactivation of several neurotransmitter receptors may interact to regulate directed nerve growth. Such interaction in vivo, where guidance signals coexist, would add further levels of control to neurite guidance.

Calcium channel subtypes and intracellular calcium stores modulate electric field-stimulated and -oriented nerve growth.

In culture, embryonic spinal neurites from Xenopus laevis show striking growth responses to steady dc electric fields, at a time when endogenous electric fields of similar size impinge on the developing nervous system. A high proportion of cultured neurites reorient, with both turning and branching directed cathodally. Neurite growth rates are increased and growth is differential (faster cathodally than anodally). Voltage-dependent calcium channels and calcium release from intracellular stores are shown to control these events. However, the pharmacological sensitivities of these phenomena differ, indicating different control mechanisms.

The effects of lyotropic anions on electric field-induced guidance of cultured frog nerves.

1. Dissociated Xenopus neurites turn cathodally in small applied electric fields. Increasing the external polycation concentration alters the direction and extent of field-induced orientation. A decrease in membrane surface charge may underlie these effects. 2. Lyotropic anions increase membrane surface charge and we have examined the effect of perchlorate (ClO4-), thiocyanate (SCN-) and sulphate (SO4(2-)) on galvanic nerve orientation. 3. Perchlorate and SCN- had no effect on field-induced cathodal turning, whereas incubation with SO4(2-) was inhibitory. In addition to its effects on surface charge, SO4(2-) increases production of the second messengers diacylglycerol and inositol trisphosphate. Interestingly, lithium (Li+), a blocker of polyphosphoinositide metabolism, had a similar effect to SO4(2-) on field-induced neurite orientation. 4. We conclude that increasing surface charge with lyotropic anions neither enhances galvanotropic orientation nor underlies the inhibitory effects of SO4(2-) and suggest that modulation of galvanotropism by SO4(2-) occurs owing to changes in the inositolphospholipid second messenger system.

Electric field-directed growth and branching of cultured frog nerves: effects of aminoglycosides and polycations.

The direction and rate of earliest nerve growth are critical determinants of neuronal architecture. One extrinsic cue that influences these parameters is a small direct current electric field, although the underlying mechanisms are unclear. We have studied the orientation, rate of growth, and branching behavior of embryonic Xenopus spinal neurites exposed to aminoglycoside antibiotics, to raised external cations, to applied direct current electric fields, and to combinations of these treatments. Field-induced cathodal turning and cathodal branching of neurites were blocked by the aminoglycosides, by raised extracellular calcium ([Ca2+]0) and by raised extracellular magnesium ([Mg2+]0). Neomycin together with high external Ca2+, by contrast, induced a reversal in the polarity of turning and branching, with neurites reorienting and branching more frequently anodally. Aminoglycosides decreased neurite growth rates, and for neomycin this was partially reversed by high external Ca2+. Raised [Ca2+]0 alone but not raised [Mg2+]0 altered growth rates in a field-strength dependent manner. Modulation of membrane surface charge may underlie altered galvanotropic orientation and branching. Such an effect is insufficient to explain the changes in growth rates, which may result from additional perturbations to Ca2+ influx and inositol phospholipid metabolism.