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School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland
ABSTRACT I. INTRODUCTION II. HISTORICAL BACKGROUND A. The Common Origins of Electrophysiology and Bioelectricity 1. Injury potentials and action potentials 2. Injury potentials and nerve regeneration 3. Injury potentials and wound healing B. Electrical Signals Vary in Space and Time III. FUNDAMENTAL CONCEPTS IN ELECTROPHYSIOLOGY IV. ELECTRICAL FIELDS EXIST EXTRACELLULARLY AND INTRACELLULARLY A. Example 1: Embryos Generate a Dynamic Voltage Gradient Across Their Skin 1. Voltage gradients exist within the extracellular spaces underneath the skin 2. Disrupting the natural EFs in amphibians disrupts development 3. Endogenous currents and voltage gradients are present in chick embryo: disrupting these disrupts development 4. A voltage gradient exists across the neural tube and neuroblasts differentiate in this gradient B. Example 2: Wounded Epithelia Generate a Steady EF That Controls Wound Healing 1. Wound healing is regulated by the wound-induced EF in rat cornea 2. Proliferation of epithelial cells is regulated by a physiological EF in vivo 3. The axis of cell division is regulated by a physiological EF in vivo 4. Nerve growth is regulated by a physiological EF in vivo 5. Electrically driven wound healing: closing remarks C. Example 3: The Establishment of Left-Right Organ Asymmetry D. Example 4: Intracellular Gradients of Potential Segregate Charged Proteins Within the Cytoplasm V. HOW DO CELLS RESPOND TO PHYSIOLOGICAL ELECTRICAL FIELDS: PHENOMENOLOGY AND MECHANISMS A. Nerve Growth Is Enhanced and Directed by an Applied EF 1. Embryonic frog spinal neurons as a model system 2. Neuronal growth cone turning and induced receptor asymmetry 3. EF-induced asymmetries of second messengers and cytoskeletal molecules? 4. Membrane protein electrophoresis or electroosmosis? B. DC EFs May Be Pulsatile C. Directed Cell Migration in a Physiological EF: Whole Cell Electrotaxis 1. The cornea as a model system 2. Electrical control of lens epithelium 3. Electrical control of vascular endothelium 4. Directional migration of Dictyostelium discoideum in electric fields D. Electrical Control of Wound Healing and Tissue Regeneration E. Electrical Fields and Cancer? VI. CLINICAL UTILITY: ELECTRICAL CONTROL OF REGENERATION IN THE CENTRAL NERVOUS SYSTEM VII. CONCLUSIONS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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II. HISTORICAL BACKGROUND |
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In the mid-1700s, the ability to store and discharge static electricity from a Leyden jar was discovered and was used to demonstrate the effects of delivering strong electrical shocks to people. Vanable (198), quoting Hoff (79), recounts that L’Abbe Jean-Antoine Nollet caused 180 of the King’s guards to leap simultaneously by having them all hold hands and then connecting the man at the end of the line to the discharge from a Leyden jar. The King was greatly amused! This public party-trick was repeated with the whole population of a Carthusian monastery, which strung out a mile’s worth of humanity that leaped in concert on receiving the charge.
Around the same time, the importance of the electrical control of cell physiology was becoming apparent from the famous experiments of Galvani (Fig. 1A) (70, 159, 160). His epic work on frog nerve-muscle preparations included the use of lightning rods connected to nerves via wires, which caused leg muscles to twitch during a lightning storm. Similarly, static electricity generators creating sparks that activated nerve conduction also caused muscle twitching. Equally important was his observation during a public experiment in Bologna in 1794 that the cut end of a frog sciatic nerve from one leg stimulated contractions when it touched the muscles of the opposite leg. Collectively, these experiments provided definitive evidence for "animal electricity." In addition, with this last experiment, Galvani had demonstrated the existence of the injury potential (Fig. 1A).
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An injury potential is a steady, long-lasting direct-current (DC) voltage gradient induced within the extracellular and intracellular spaces by current flowing into and around an injured nerve. Its discovery predated the discovery of the better known action potential, which is a rapid, self-regenerating voltage change localized across the cell membrane. In 1831, Matteucci extended Galvani’s observations by measuring injury potentials directly at the cut end of nerves and muscles using a galvanometer (named after Galvani). Ingeniously, he used the injury potential of damaged frog muscle to demonstrate the existence of action potentials in nerve and muscle for the first time. By placing a cut nerve into an injured muscle (Fig. 1A), he showed that the latter activated the nerve and caused contraction of the muscle innervated by that nerve. He showed also that muscles made to contract in this way would activate contractions in a second frog muscle preparation, whose intact nerves were placed across the belly of the twitching muscles. The action potentials of the uninjured muscle fibers had stimulated action potentials in the intact nerves and intact muscles of the second preparation. In the intervening two centuries, the relative importance of injury potentials and action potentials has shifted markedly. Action potentials are central in neuroscience and electrophysiology, but injury potentials are frequently not recognized, are neglected, or are grossly misunderstood.
2. Injury potentials and nerve regeneration
Part of this review will discuss the role of endogenous and applied electrical fields (EFs) in stimulating and directing nerve growth and nerve regeneration. It is important and instructive to draw a clear historical link. Recent work in this area begins with experiments that are direct descendants of Galvani’s work on injury potentials. Injury currents, like those discovered by Galvani, have been measured entering the cut ends of Mauthner and Muller axons in embryonic lamprey spinal cord (Fig. 1B) (22). These currents are of the order of 100 µA/cm2, and because the resistivity of soft tissues is 
1,000 
· cm, they give rise to steady voltage gradients of 10 mV/mm. The hypothesis that the injury potential these currents establish in the distal ends of cut axons might impede regeneration has been tested. So has the proposal that applying a DC EF to offset and reverse the polarity of this injury potential would promote regeneration (24). Both have proven correct. In the intervening 20 years, this work has progressed to human clinical trials using applied DC EFs to treat human spinal cord injuries (see sect. VI).
3. Injury potentials and wound healing
The great German physiologist Emil Du-Bois Reymond, considered to be the founder of modern electrophysiology, repeated Matteucci’s experiments and measured directly the propagating action potential. However, he was also interested in injury potentials. In 1843, he used a galvanometer (built with >2 miles of wire) to measure 1 µA flowing out of a cut in his own finger. The flow of current is due to the short-circuiting of the transepithelial potential (TEP) difference that occurs at a skin lesion (Fig. 1, C–F).
A second aspect of this review discusses the role of endogenous and applied EFs in stimulating and directing wound healing. Again, a direct historical link is appropriate. Recent work in this area begins with two studies that are clear descendants of Du-Bois Reymond’s demonstration of injury currents in skin. First, the stumps of regenerating newt limbs drive large currents out the cut end (29). Currents of between 10 and 100 µA/cm2 create a steady voltage drop of 60 mV/mm within the first 125 µm of extracellular space, and this is essential for regeneration (2, 95, 133, 134). Second, human skin and that of guinea pigs and amphibians maintain a TEP across the epithelial layers. When the skin is cut, a large, steady EF arises immediately and persists for hours at the wound edge, as current pours out the lesion from underneath the wounded epithelium (Fig. 1, D and F) (8). These fields measure 140 mV/mm and play important roles in controlling several aspects of the cell biology of wound healing (see sect. VD).
Interestingly, although frog nerve and frog skin were key preparations in the discovery of steady DC injury potentials and have been central to teaching in electrophysiology, it is for the action potential and the TEP that these tissues are best known. Few of the many pupils and teachers that have used these preparations are familiar with injury potentials or their essential role in tissue development and repair.
In parallel with Galvani’s work, Volta was developing these ideas to create the first battery. Recognizing the parallel with animal electricity, Volta used batteries therapeutically to treat deafness. Others, however, were less rigorous scientifically in the promotion of electrical-based therapies. For more than a century there was "widespread and irrational use of galvanism and static electricity" (198). Static electricity generators were in common use and were promoted and sold because they created an allegedly beneficial "electric air bath" or a "negative breeze." The electric air bath involved charging the patient and using a grounded electrode to draw sparks from a chosen part of the body. The negative breeze allegedly was helpful in treating insomnia, migraine, and baldness (Fig. 2). With the electrode polarity reversed, a "positive breeze" was used to treat kidney disease. Vanable (198) gives a good account of this charlatanism and makes the insightful point that this period of highly dubious pseudo-science tainted perceptions and may be a major reason why animal electricity and bioelectricity have been held in relatively low scientific esteem.
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B. Electrical Signals Vary in Space and Time
Contemporary electrophysiologists are familiar with fast membrane conductance changes that take place within milliseconds, for example, as an action potential is propagated along a neuronal membrane. In developmental biology too there is widespread recognition of similarly fast electrical conductance changes, for example, across the egg membrane. In this case, a transient and massive membrane depolarization of the newly fertilized sea urchin egg acts as an electrical block to polyspermy (87). Both of these examples involve very fast conductance changes that occur within membrane-embedded channels and a flow of ions that changes the voltage across the membrane only. The electrical events to be covered in this review last much longer and are present across hundreds of microns, rather than being confined to the immediate vicinity of the cell membrane. They involve steady DC gradients of electrical potential, usually in the extracellular spaces, but sometimes within the cytoplasm of a single cell or a syncytium of cells. Such gradients are present for hours, days, or even weeks during both development and regeneration. In addition, they are regulated both spatially and developmentally. Their undisputed existence and their physiological relevance, both of which will be established below, indicate that they should be integrated into current thinking. One way to do this is to consider throughout this review the issue of chemotaxis. This is a widely accepted form of directed cell motility based on the ability of cells to respond with directional movement in a chemical gradient. Many types of molecules are presented to cells in the form of chemical gradients in the extracellular spaces. Both in developing and in regenerating systems many of these molecules carry a net charge. Because steady voltage gradients also are present within the extracellular spaces and vary in space and time, these coexistent chemical and electrical signals must interact. This raises several issues. For example, are chemical gradients established by preexisting electrical gradients? Do chemical and electrical guidance cues activate shared signaling pathways to achieve directional growth? Are there hierarchies of guidance cues that vary in space and time?
Some other questions to be tackled are outlined below, to prime the reader. As the review develops, the answers to these questions will need to be integrated with prevailing views on the control of directed cell motility by chemical gradients (177), or by substrate topography (147), or by whatever other dynamic guidance cues coexist within the extracellular spaces. Where are steady electrical fields found? What is their basis? What are their functions? What tissue or cellular effects do they have? What mechanisms underpin these effects? How do electrical cues interact with other guidance cues? And can electrical fields be mimicked or manipulated to control cell behaviors?
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III. FUNDAMENTAL CONCEPTS IN ELECTROPHYSIOLOGY |
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100 · cm. If there is a voltage difference between any two points in a conductive medium, current will flow. The voltage difference per unit distance is the electrical field, and in a biological context, this is most intuitively expressed in millivolts per millimeter. The relationship between current density and the electrical field is E = J · 
, where E is the electrical field, J is the current density (in A/cm2), and is the resistivity of the medium. Inevitably then the existence of an EF and the flow of current are interdependent and inseparable events. Importantly, the EF and the current density are vectors, with both magnitude and direction. It is this directional quality of an EF that makes it a candidate spatial organizer, because it can impose directional movement on chemicals in the extracellular environment, on receptor molecules, on cells, and on tissues.
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60 mV/mm within 125 µm of the lesion. By 6 h postlesion as the stump wound resistance increases, the EF drops to 26 mV/mm, distally negative (133, 134). Second in development, the tight junctions between epithelial cells of the flank skin break down, but only in the precise areas where limb buds will form. The TEP becomes short-circuited only at these sites from which currents of 1–10 µA/cm2 leave the flank. These "prophetic" currents precede limb bud emergence by several days and predict the sites from which limb buds will appear in amphibians, chicks, and mice (1, 21, 25, 170). Before discussing more detailed examples of electrical fields and their control of development and regeneration, it is crucial to put the magnitude of these electrical signals into context. To depolarize a neuron and fire an action potential using surface electrodes requires field stimulation of 1–2 V/mm. The common technique of electroporation for drug or gene delivery into cells uses extremely large pulses of DC EF stimulation, roughly 100–500 V/mm (143). The DC EFs that play physiological roles in development and regeneration (see examples in sect. IV, A–D) are three or four orders of magnitude less than this (1–100 mV/mm)! It is a mistake to think of them having similar magnitude, but this is a major misconception made by those who dismiss EF as being "nonphysiological."
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IV. ELECTRICAL FIELDS EXIST EXTRACELLULARLY AND INTRACELLULARLY |
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A. Example 1: Embryos Generate a Dynamic Voltage Gradient Across Their Skin
The skin of adult frogs is well known as a transporting epithelium that sustains a TEP of 100 mV, inside positive, across the multilayered epithelium (102). The ion transport properties of the apical and basolateral cell membranes differ and are polarized (Fig. 1D). The apical cell membrane contains specialized amiloride-sensitive sodium channels allowing Na+ to enter the cells, while the basolateral membranes contain the well-known "sodium pump," the ouabain-sensitive Na+-K+-ATPase that electrogenically pumps three Na+ from the cytoplasm out into the extracellular fluid in exchange for two K+ entering the cells. Net ion transport therefore occurs across the epithelium, with Na+ being transported from pond water into the animal. A high-resistance electrical "seal" exists between neighboring cells in most epithelial sheets including amphibian skin. This is formed by tight junctions, and these greatly reduce the electrical conductivity of the paracellular space (5). Viewed end-on, the apical surface of each epithelial cell appears to be encircled by strands of specialized tight junctional proteins. Strands on neighboring cells abut each other to form a "seal" that restricts the paracellular passage of solutes and water. The whole structure may be pictured as similar to a series of interconnecting hoops around the end of a barrel. The same basic elements of polarized channels, pumps, and tight junctions are found in embryonic frog skin, and these also establish a TEP from very early stages of development (131, 168).
1. Voltage gradients exist within the extracellular spaces underneath the skin
Crucially, the potential difference across the skin is different in different parts of the developing embryo (180). With the use of standard glass microelectrodes, stable gradients of voltage have been measured around the neural plate area in axolotl embryos during the period of neurulation. Skin potentials are higher rostrally than caudally (Fig. 4, A–C). This is particularly marked at the head end of the embryo, where EFs of 75–100 mV/mm have been measured in the extracellular space below the epithelium (180), whilst EFs of 30 mV/mm are present in the region rostral to the developing blastopore (173). In both cases the orientation of the steady voltage gradient is rostrocaudal, that is, along the long axis of the embryo (head to tail). The skin potential also is high at the midline of the neural plate as it begins to fold over to become the neural tube, decreases at the neural folds, and increases again further laterally on the flank of the embryo. This pattern of skin potential gives rise to standing voltage gradients on either side of the dorsal midline, with a mediolateral orientation (Fig. 4, A–C).
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1,000 · cm, current densities of 100 µA/cm2 give rise to voltage gradients of 10 mV/mm. Importantly, the anterior neural folds also are sites of current exit, and here current densities of 2 µA/cm2 have been measured. The sites of current leaks are regions of major tissue movements (173). Because tissue movements disrupt tight junctional seals transiently (47) and therefore reduce tissue resistivity locally (but not in distal areas), current flows parallel to the tightly sealed epithelium in areas of high resistivity (intact tight junctions) and exits the embryo in regions of low resistivity (where tight junctions have broken down) (Fig. 4D). Although these two techniques demonstrate clearly that electrical signals exist during neurulation in amphibians, the signals at the blastopore and at the neural folds may have different functions. This is because the blastopore current persists after closure of the neural tube, but the rostrocaudal and mediolateral voltage gradients at the buckling neural plate stage disappear as the neural folds fuse at the end of neurulation (Fig. 5). The functional significance of switching spatially localized DC EFs on and off during gastrulation and neurulation, which are major developmental milestones, has been tested.
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If the EFs in embryos play a significant role in development, disrupting the normal electrical milieu of embryos would cause developmental defects. This has been tested directly in amphibians using two experimental approaches. The first involved placing whole axolotl embryos in an externally applied EF of physiological magnitude for a period spanning either gastrulation or neurulation (18–22 h). The applied EF was designed to disrupt the pattern and magnitude of the endogenous EFs by altering the TEP in a predictable way (139).
With no EF exposure (normal TEP), abnormalities occurred in no more than 17% of embryos. In embryos with no specific orientation to the imposed EF, 62% developed abnormally in an EF of 75 mV/mm and 43% at 50 mV/mm. Because the main axes of the measured endogenous voltage gradients run rostrocaudally and mediolaterally within the neural plate (Fig. 4), experiments were made using embryos with these body axes aligned with the EF vector. When the EF was imposed during neurulation with a cathode at the rostral end of the embryos, 95% (19/20) developed abnormally. When the cathode was caudal, 93% were abnormal, and of those with the long (rostrocaudal) embryonic axis perpendicular to the applied EF, 75% (12/16) were abnormal. The types of defects seen were profound and included absence of the cranium, loss of one or both eyes, a misshapen head, abnormal or absent brachial development, and incomplete closure of the neural folds in focused areas. In some cases, cells from the neural plate migrated out from the embryo onto the dish and continued to develop autonomously. In another experiment an EF of identical duration and magnitude was applied to embryos undergoing gastrulation. The EF was switched off at the end of gastrulation, and the embryos were allowed to develop to stage 36. Importantly, these embryos developed completely normally.
Interestingly, the polarity of the applied EF predicted the polarity of the developmental abnormalities that were induced. The most striking disruptions were seen in areas facing a cathode, where the skin TEP had become hyperpolarized. When the rostral (head) end of the embryo faced the cathode, head defects predominated, and when the caudal (tail) end of the embryo faced the cathode, lower abdominal and tail defects predominated (139).
The disruption to the embryonic skin TEP by applying an EF externally was marked and was polarized in a predictable manner. The TEP, which is normally internally positive, depolarized and switched polarity to become increasingly negative internally in regions of skin at the anode in an EF of 25–100 mV/mm. At the other end of the embryo, the TEP of skin under the cathode hyperpolarized and became increasingly more positive internally. Because the TEP was altered in this striking manner by the externally applied EF, the endogenous gradients of extracellular voltage under the skin will be scrambled by this imposed applied EF.
These experiments allow three conclusions. 1) Endogenous EFs with normal polarity and magnitude are essential for normal development of the nervous system and other tissues. 2) There was no generalized harmful effect of an applied EF since embryos exposed at gastrulation developed normally. 3) Embryos responded to scrambling of their endogenous EF only during the period when cells were undergoing neurulation. This suggests that neuronal cells gain and then lose the ability to respond to an applied EF during a specific window of developmental time.
The second approach to modifying endogenous EFs involved impaling Xenopus embryos with glass microelectrodes, similar to those used to measure the TEP. Current was passed through the electrodes so that the endogenous current leaving the blastopore was reduced or reversed (84). The effect of injecting current on the magnitude of the blastopore currents was measured directly using the vibrating microprobe. Injected current at 100 nA nulled the blastopore current, and 500 nA approximately reversed it. Eighty-seven percent of embryos (20/23) injected with this level of current for 9–11 h at stage 14–16 showed gross external developmental abnormalities. These included the formation of ventral pigmented bulges, failure of the anterior neural tube to close, reduced head development, retarded eye formation, the extrusion of cells from the blastopore into the dish, and failure of embryos to form functional cilia. Control embryos with long-term impalement of an electrode in the same region but no current, those with low current (10 nA, which did not affect the magnitude of the natural blastopore current), or those with reversed current that augmented the blastopore current, showed a much lower rate of abnormality.
Therefore, although two markedly different methods have been used to disrupt the endogenous EFs of amphibian embryos, they nevertheless induced a surprisingly uniform and striking array of developmental defects. Brain and tail structures were especially vulnerable and, significantly, these are regions of endogenous outward currents and of measurable steady DC voltage gradients in the embryo. The high incidence of neural tube defects also may be significant. Mutations in a number of genes such as sonic hedgehog cause neural tube defects (41), so it would be instructive to determine the expression levels of these genes in normal embryos and in embryos where the endogenous EFs have been disrupted. Conversely, it would be useful to determine whether disruption of endogenous EF affects expression patterns of key developmental genes.
One interpretation of the experiments outlined above is that steady voltage gradients within embryos provide a gross template for the development of pattern during ontogeny and that developmental abnormalities resulted from experimentally scrambling electrical cues necessary for patterning and cell migration within the embryo.
3. Endogenous currents and voltage gradients are present in chick embryo: disrupting these disrupts development
Importantly, currents and voltage gradients that are analogous to the blastopore and neural fold currents in amphibians have been measured in chick embryos (81). In stage 15–22 chick embryos, ionic currents greater than 100 µA/cm2 leave the embryo via the posterior intestinal portal (PIP). This is the period of tail gut reduction; when there is extensive cell death at the caudal end of the embryo. The PIP is the opening into the hindgut from the yolk sac. These currents enter through the ectoderm of the embryo and upon flowing through the embryo generate a caudally negative voltage gradient of 20 mV/mm. If these voltage gradients play a necessary role in embryogenesis, then disrupting them should alter development. This has been tested by creating an alternative path for current flow out of the embryo. Hollow capillaries that formed an ectopic region of low resistance were implanted to create, in effect, a permanent, nonhealing wound (83). This procedure reduced the magnitude and altered the internal pattern of the natural EF. Conductive implants designed to shunt currents out of the embryo were placed under the dorsal skin at the midtrunk level (stage 11–15). These hollow capillary shunts were 100 µm outside diameter and 1 mm long and were filled with saline, in some cases gelled with 2% agarose to control against bulk fluid transfer between the embryo and its surroundings. They were placed perpendicular to the neural tube in a slit 250 µm long and inserted around 500 µm under the ectodermal epithelium parallel to the neural axis. Currents of 18 µA/cm2 left the conductive shunts. The net effect was to reduce the current leaving the PIP of these embryos by 30%. Ninety-two percent (25/27) of these embryos developed with gross abnormalities. The most common defect was in tail development, with the neural tube, notochord, and somites all either missing or truncated in the tail region. There were defects also in limb and head/brain development, but the frequency of defects increased in a rostrocaudal direction. Forty-four percent of embryos showed multiple developmental defects. Nonconductive, solid rod implants of the same dimensions were used in control embryos. No currents were measured escaping from these implants, nor did the implants influence the magnitude of the currents leaving the PIP. Only 11% (2/18) of control embryos with solid implants showed any developmental abnormalities; all the others developed completely normally, despite the continuous presence of the nonconducting implant. The abnormalities seen in experimental embryos were very similar to those produced in rumpless chicks, a naturally occurring mutation which can result in complete absence of all caudal structures (228). Vibrating probe measurements from rumpless chicks showed that currents leaving the PIP were 41% of the PIP current in normal embryos (83), suggesting that this electrical deficit contributes, at least in part, to the tail structure deletions.
Several aspects of these experiments are important. 1) They confirm that currents and endogenous voltage gradients are present during major episodes of chick development and are greatest in the tail region. 2) Reducing the PIP currents by shunting current out at an ectopic dorsal location has the greatest developmentally disruptive effect in the tail region. 3) The shunt placement is several millimeters away from the site of the main defects, indicating that current shunting did not have nonspecific and deleterious local effects. 4) Solid shunts had no effect. 5) A naturally occurring chick mutation may cause tail deformities because of aberrant electrical signals.
A further point of interest and one which requires further study is that the primitive streak of the chick embryo, which is analogous to the amphibian blastopore, also is a site of large outward currents of 100 µA/cm2 (94). A physiological role for these currents has not been explored.
4. A voltage gradient exists across the neural tube and neuroblasts differentiate in this gradient
The vertebrate neural tube forms when the lateral edges of the neural plate thicken, rise up, and fold over to fuse with each other at the dorsal midline (Fig. 5). A hollow tube called the neural tube that develops to become the brain and spinal cord forms from the folded ectoderm and then detaches to lie below the skin. The luminal surface of the neural tube therefore is equivalent to the outer surface of embryonic skin. Because spatial and temporal differences in the electrical properties of embryonic skin generate steady endogenous electrical signals (see above), the neural tube has been investigated to determine whether similar electrical signals are generated across its wall. Amphibian neural tube does establish a potential difference across its wall known as the transneural tube potential (TNTP; Fig. 6) (82, 179). In axolotl this may be as large as 90 mV, with the lumen negative with respect to the extracellular space at stage 28. Because the wall of the neural tube is roughly 50 µm wide, this large potential difference would create a steady voltage gradient across cells in the neural tube wall of a remarkable 1,800 mV/mm [90 mV/50 µm = 180 mV/100 µm = 1,800 mV/mm]. The neuroblasts (neuronal precursors) within the wall must migrate, differentiate, and sprout directed axonal projections whilst exposed to this high, continuous extracellular EF. The TNTP is largely the result of transporting Na+ out of the lumen, and this can be prevented pharmacologically by injecting benzamil or amiloride into the lumen. When this was done in axolotl embryos at stage 21–23 and the embryos were allowed to develop for 36–52 h, by which time uninjected embryos had developed to stage 34–36, the TNTP collapsed for several hours. In all of 28 embryos tested, collapse of the TNTP caused major abnormalities in cranial and central nervous system (CNS) development. The defects were characterized by a disaggregation of cells from structures that had already begun to form (26). The cells that had comprised the optic and otic primordia, brain, neural tube, and notochord disaggregated, but did not die, whilst new internal structures failed to form. In effect, the internal structure of most embryos had been reduced to a formless mass of apparently dedifferentiated cells, simply by collapsing the TNTP. Remarkably, the external form of some embryos with collapsed TNTP continued to develop, despite the complete absence of concomitant internal histogenesis (26). Making similar injections of a vehicle solution into the neural tube, or of the active agents amiloride, or benzamil beneath the embryonic skin immediately adjacent to the neural tube had no effect on the TNTP and did not disturb development. This shows that neither the injection, nor the drugs, had a generalized toxic effect on the embryos and that the disaggregation of the neural tube and other internal structures was a consequence of collapse of the TNTP.
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In short, vertebrate embryos possess steady voltage gradients, particularly in areas where major developmental events related to cell movement and cell division are occurring. Disrupting these electrical fields disrupts normal development.
B. Example 2: Wounded Epithelia Generate a Steady EF That Controls Wound Healing
The second example of a tissue in which a steady DC EF is found extracellularly is wounded epithelium. Skin and cornea are well-studied examples (8, 36). However, all epithelia that segregate ions to establish a TEP will generate a wound-induced EF for the reasons outlined below.
The stratified mammalian cornea supports a transcorneal potential difference (TCPD) of around +30 to +40 mV, internally positive (Fig. 7A). The outer cells of the corneal epithelium are connected by tight junctions, as in amphibian skin, and these form the major electrical resistive barrier. Intact mammalian corneal epithelium transports Na+ and K+ inwards from tear fluid to extracellular fluid. Cl– is transported in the opposite direction, out of the extracellular fluid into the tear fluid (34, 99). This separation of charge establishes the TCPD. Wounding the epithelial sheet creates a hole that breaches the high electrical resistance established by the tight junctions, and this short-circuits the epithelium, locally. The TCPD therefore drops to zero at the wound (Fig. 7B). However, because normal ion transport continues in unwounded epithelial cells behind the wound edge, the TCPD remains at normal values around 500–1,000 µm back from the wound. It is this gradient of electrical potential, 0 mV at the short-circuited lesion, +40 mV 500–1,000 µm back in unwounded tissue, that establishes a steady, laterally oriented EF with the cathode at the wound (Fig. 7B). So, in contrast to the TCPD generated across the intact epithelium, which has an apical to basal orientation, the wound-induced EF has a vector orthogonal to this. It runs laterally under the basal surfaces of the epithelial cells and returns laterally within the tear film across the apical surface of the epithelium (Fig. 7B). Mammalian skin also supports a TEP. When the skin is cut, a wound-induced EF arises immediately for the same reasons as in cornea (compare Fig. 7, C and D). Importantly, the wound-induced EF persists until the migrating epithelium reseals the wound and reestablishes a uniformly high electrical resistance, at which point the wound-induced EF drops to zero.
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0.3 mm, which means that at 330 µm back from the wound edge the voltage gradient would have decayed to 1/e, or 37%, of its maximum value at the wound edge. Direct measurements in skin showed voltage gradients of 140 mV/mm in the first 250 µm, 40 mV/mm between 300 and 500 µm, and around 10 mV/mm at 500–1,000 µm from the wound edge (8).
These findings have several important implications. 1) All cell behaviors within 500 µm of a wound edge in skin and cornea (and probably any ion-transporting epithelium; gut, for example) inevitably take place within a standing gradient of voltage. These include epithelial cell migration, epithelial cell division, nerve sprouting, leukocyte infiltration, and endothelial cell remodeling with associated angiogenesis; in short, the whole gamut of cellular responses to injury! 2) Because of the exponential drop in voltage gradient with distance from a wound, any cell behaviors governed by the endogenous EF would be regulated differentially with distance from the wound. 3) Increasing or decreasing the TEP would inevitably increase and decrease the voltage gradient profile at the wound.
In light of these issues, we have been studying the cell physiology at experimental wounds made in rat cornea in vivo. We have shown that a diverse array of inter-related cell behaviors is controlled by the endogenous EF at corneal wounds. These include directed migration into the wound of epithelial cells, the proliferation of epithelial cells, the axis of division of epithelial cells, the proportion of nerves sprouting at the wound, and the directional growth of nerve sprouts towards the wound edge (184, 185, 130).
1. Wound healing is regulated by the wound-induced EF in rat cornea
Increasing or decreasing the TCPD pharmacologically has the inevitable consequence of increasing or reducing the wound-induced EF. We chose to modulate the TCPD using six different chemicals that act by different cellular mechanisms but whose only common effect was to change the TCPD (Fig. 8 and Ref. 185). In rat corneas treated with PGE2 (0.1 mM), which increases Cl– efflux, or with aminophylline (10 mM), which inhibits phosphodiesterase breakdown of cAMP and enhances Cl– efflux, the TCPD increased three- to fourfold. Wounds treated with these drugs healed 2.5 times faster within the first 10 h than control wounds (184). In contrast, wounds treated with the Na+-K+-ATPase inhibitor ouabain (10 mM), which reduced the TCPD fivefold to 18% of normal, showed markedly slower wound healing (Fig. 9). The rate of wound healing was directly proportional to the size of the TCPD and therefore to the size of the wound-induced EF (185).
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2. Proliferation of epithelial cells is regulated by a physiological EF in vivo
One element of the modulated wound healing response in cornea is due to EF-regulated proliferation of epithelial cells. We tested also whether epithelial cell proliferation is regulated electrically. Cell division is rare in the central area of unwounded corneal epithelium. In contrast, most cell division takes place within a peripheral ring of stem cells called the limbus. To provide cells for epithelial turnover, limbal stem cells differentiate and migrate within the basal layer of corneal epithelium and then move up through the epithelium to the surface layer (43). Wounding the corneal epithelium stimulates epithelial cells near the lesion to divide. Enhancing the endogenous wound-induced EF with PGE2 or aminophylline induced a 40% increase in cell divisions within 600 µm of the wound edge, and suppressing the EF with ouabain caused a 27% suppression of mitoses (Fig. 10) (184). Manipulating the EF therefore clearly regulated the cell cycle and altered the frequency of cell division. Because this will also modulate the population pressure of cells within the epithelium, this could contribute to the rate of wound healing.
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Cultured corneal epithelial cells divide along a cleavage plane that forms perpendicular to the EF vector (220), in other words the mitotic spindle aligns parallel to the EF before cytokinesis. The reasons for this are completely unknown. Importantly, however, the same striking phenomenon occurs in vivo (184). In untreated corneal wounds, which generate their own endogenous EF, the mitotic spindles lie roughly parallel to the EF vector, with cleavage occurring perpendicular to this (Fig. 10A). Enhancing the wound-generated EF with PGE2 or aminophylline roughly doubled the proportion of dividing cells whose cleavage planes were perpendicular to the EF vector. In contrast, reducing the wound-generated EF to <20% of its endogenous value with ouabain (Fig. 10, B and C) abolished wound-oriented cell divisions.
If the endogenous EF in rat cornea is causal in directing the axis of cell division, then its effects should be highest at the wound edge and decline back from here, because the EF declines exponentially away from the wound. This is the case. Orientation of the mitotic spindle was strongest in the first 200 µm and roughly halved 200–400 µm back from the wound. By 600 µm back from the edge, the angle of the mitotic spindle was not different from those seen in the limbus (1,700 µm away); both were randomly oriented with respect to the wound-generated EF vector (184). Importantly, 600 µm corresponds to the measured distance that the EF penetrates into the tissue (see Fig. 7D). Oriented division therefore dropped to zero as a function of distance back from the wound edge, and there was no oriented division in the distant limbus where the EF would be zero.
Enhancing the EF with PGE2 or aminophylline increased the orientation of cell division with significant orientation now occurring further from the wound edge, at 600 µm. Collapsing the EF with ouabain abolished oriented cell division, even within 200 µm of the wound edge. Clearly, the naturally occurring EF controls the orientation of cell divisions in vivo.
One clue to potential mechanisms indicates that phospholipid second messenger signals may transduce the EF into oriented cell division. This is because the aminoglycoside antibiotic neomycin, which had no effects on the TCPD (EF), but which inhibits phospholipase C, abolished oriented cell divisions in vivo. Interestingly, neomycin also prevents EF-induced orientation of embryonic myoblasts and of neuronal growth cones (58, 127).
It may be significant that a local environmental guidance cue (an EF) directs the plane of cell division. In the developing CNS for example, crucial decisions regarding the fate of neuroblasts and, consequently, the fundamental architecture of the brain are made by fixing the axis of neuroblast division. Symmetrical cleavage of progenitor cells in the ventricular zone with an axis that retains both daughters in the proliferative pool leads to reentry into the cell cycle and an exponential expansion of the ventricular zone population. In contrast, asymmetrical cell division with a cleavage plane parallel to the ventricular boundary releases one daughter cell from the cell cycle, and this cell differentiates and migrates away. Control over these events is exerted by a host of asymmetrically distributed protein molecules such as numb, miranda, prospero, and bazooka (74) and is determined in part by where the rotating mitotic spindle comes to rest (77). Whether the distribution of the determinative protein markers or the dynamics of spindle rotation and arrest are regulated by an endogenous or an applied EF remains to be determined.
4. Nerve growth is regulated by a physiological EF in vivo
Nerves sprout in response to wounds in skin (64, 117) and in cornea (10, 175). In cornea, with its rich sensory innervation, this is a biphasic process. Early collateral sprouts appear within only a few hours, mostly from intact fibers near the wound. In rabbit cornea these early collateral sprouts show a striking orientation with many parallel nerve bundles growing directly towards the wound edge (175). The early sprouts are transient and over the following 7 days or so, they retract and are replaced by regenerating neurites (10). The cues guiding growth cones of early sprouts directly towards the wound edge have not been explored. Electrical guidance of nerve growth cones has been proposed since the time of Cajal, a century ago, and there is much evidence for this robust phenomenon in tissue culture (see below). Because a corneal wound generates its own EF, we have tested the hypothesis that the wound-generated EF is causal in directing nerve sprouts to grow directly towards the wound edge. These experiments have provided clear evidence of a physiological role for electrical guidance of nerve growth in vivo (185). A 4-mm-long nasal to temporal slit wound was made in rat cornea. Early nerve sprouts are evident by 16 h, but they are not yet oriented with respect to the wound edge. Between 16 and 24 h, many more nerve sprouts appear, and most are perpendicular to the wound edge (Fig. 11). When the wound-generated EF was enhanced with PGE2, aminophylline, AgNO3, or ascorbic acid (Fig. 8), neurite growth towards the wound was enhanced. More sprouts appeared, sprouts appeared earl
