4. Applied electric fields had no effect on the location of the origin of neurites on the cell body.5. Spherical myoblasts cultured in applied electric fields (36-170 mV/mm) elongated with a bipolar axis of growth which was perpendicular to the electric field. The response was graded and disappeared at field strengths below 36 mV/mm. 6. It is suggested that in vivo, the direction of neural outgrowth from the neural tube and the strict spatial organization of somites might be under the control, in part, of endogenous electric fields. Possible sources of these are discussed.
Calcium ions enter the prospective growth pole of polarizing Pelvetia eggs faster than the opposite pole and leave this antipode faster than the growth pole. The calcium current is greatest when first measured at 6 hours after fertilization and decreases as the time of final commitment to growth in a particular direction approaches.
Intracellular free-calcium concentration ([Ca2+]i) was measured in lamprey spinal axons using the fluorescent calcium indicator fura 2. We used both a photomultiplier tube and a video-image processing system to measure the temporal and spatial distributions of [Ca2+]i in the proximal segments of transected axons. Within 3 min following transection, a spatially graded increase in the [Ca2+]i was apparent in the last few millimeters of the axons. Superimposed on the initial gradient was a moving front of calcium that progressed up the axon, reaching 1.6 mm from the cut end in 3 hr. The [Ca2+]i behind the moving front exceeded 10 microM. This movement of Ca2+ was greatly reduced by an externally applied electrical field with the cathode distal to the lesion and was increased by an applied field of the opposite polarity. When axons were transected in Ca2(+)-free medium, no increases in [Ca2+]i occurred. One d after transection, [Ca2+]i was at or below the precut levels, except in the distal 250 microns, where it remained slightly elevated. Therefore, axons can survive the high levels of [Ca2+]i that occur after transection and can reestablish normal [Ca2+]i levels within 24 hr. Measurements of both the diffusion coefficient and the fluorescence polarization of fura 2 indicate that the dye is not significantly bound to axoplasmic components.
Xenopus neural crest cells migrated toward the cathode in an applied electrical field of 10 mV/mm or greater. This behavior was observed in relatively isolated cells, as well as in groups of neural crest cells; however, the velocity of directed migration usually declined when a cell made close contact with other cells. Melanocytes with a full complement of evenly distributed melanosomes did not migrate of their own accord, but could be distorted and pulled by unpigmented neural crest cells. Incompletely differentiated melanocytes and melanocytes with aggregated melanosomes displayed the same behavior as undifferentiated neural crest cells, that is, migration toward the cathode. An electrical field of 10 mV/mm corresponded to a voltage drop of <1 mV across the diameter of each cell; the outer epithelium of Xenopus embryos drives an endogenous transembryonic current that may produce voltage gradients of nearly this magnitude within high-resistance regions of the embryo. We, therefore, propose that electrical current produced by the skin battery present in these embryos may act as a vector to guide neural crest migration.
An extracellular vibrating electrode was used to map the current pattern around Xenopus laevis oocytes. Current was found to enter the animal hemisphere and leave the vegetal hemisphere; in fully grown oocytes from which the follicle cells had been removed, the maximal current density was about 1 uA/cm2. This current decreased to nearly zero in response to progesterone and several other maturation-producing agents. In the case of progesterone, the decline began within a few minutes of the addition of the hormone and proceeded with a half-time of about 20 min.An analysis of the effects on the current of the removal or addition of various ions and drugs led to the inference that the major current-carrying ion was chloride and that the chloride permeability was controlled by calcium. An ubiquitous feature of nonmammalian animal eggs is the presence of an animal-vegetal axis. The nucleus of the egg is generally displaced toward the animal pole and this pole is the site of polar body formation. In many amphibian eggs, the axis is readily distinguished by the pigment accumulation at the animal pole and the yolk mass at the vegetal pole. Superimposed on this visible polarity is a developmental polarity: the animal pole gives rise to the ectodermal parts of the embryo and the vegetal pole, to the endodermal. (For a recent review, see refs. 1 and 2.) When the full-grown anuran oocyte is exposed to progesterone, a sequence of events is triggered, leading to the reinitiation of meiosis and formation of a fertilizable egg. This process is also a polar one. The basal portion of the germinal vesicle is the first to break down as the entire germinal vesicle moves toward the animal pole (3).Our knowledge of the physical forces that might produce such asymmetries and direct polar movements remains limited. The oocytes are randomly oriented in the ovary; thus gravity is not the causal agent. Strong evidence has now accumulated in the case of fucoid algae eggs that electrical currents and ion gradients are involved in transducing environmental asymmetries into cellular polarity (4-6). Electrical currents associated with growth and localization processes have also been shown to exist in insect oocytes (7), pollen tubes (8), and regenerating Acetabularia (9). There are also a few older surface potential measurements from which the existence of polar currents in animal eggs can be inferred (10). It is against this background that this investigation of animal-vegetal currents in Xenopus oocytes and eggs was begun.METHODS AND MATERIALS Sexually mature Xenopus laevls females were obtained from the South African Snake Farm, Fish Hoek, South Africa. To obtain oocytes, females were anesthetized by chilling in an ice bath, portions of their ovaries were removed, and the oocytes were then manually dissected from their follicles. In some cases, the remaining follicle cells were removed by a 5-min treatment with Pronase (50 ;tg per ml in Steinberg's solution).A vibrating platinum electrode was used to measure extracellular currents. T...
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 strength, 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 strength. 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 that documents a response towards the anode.
We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities . Current leaves the first cleavage furrow after new, unpigmented membrane is inserted . This outward current may be carried by K+ efflux . No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane ; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane . This suggests a heterogeneous ion channel distribution within the old membrane . Experiments suggest that the inward current may be carried by Na' influx, Ca" influx, and CI-efflux . No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage .The cleavage process in amphibian eggs includes the addition of new membrane at the cleavage furrow that forms blastomeres with two distinct membrane domains . Each blastomere retains some of the original egg surface with pigment granules underlying the membrane and each gains new, unpigmented membrane . In addition to being morphologically distinct, these membrane domains appear to have different electrical properties . Woodward (45) and de Laat and co-workers (7,8) have suggested that the membrane hyperpolarization observed during first cleavage is due to the addition of new, unpigmented membrane that has a high potassium permeability . We investigated the electrical properties of these two membranes in Xenopus laevis embryos, using the extracellular vibrating probe that is used to measure steady ion currents which cells drive through themselves .Ion currents have been shown to be correlated with growth and pattern formation in a number of developing systems (5,19,22,23,28,44) . In a previous study of immature Xenopus oocytes, Robinson (31) detected an animal-vegetal current which falls to nearly zero in response to a variety of maturation-promoting factors. No steady transmembrane currents have been detected in the mature, unfertilized egg or in precleavage fertilized eggs aside from a ring-shaped wave of inward current which spreads over the egg for 4 min following activation (D. Kline and R. Nuccitelli, manuscript in preparation) . Here we report on electrical measurements made beginning 15 min after fertilization and on through second cleavage . Large steady currents are found after new membrane
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