Endogenous lateral electric fields around bovine corneal lesions are necessary for and can enhance normal rates of wound healing.

The strength of the electric fields in the vicinity of 1.5 mm circular lesions in the bovine cornea has been found to influence the rate of re-epithelization. A decrease in the field strength by submersion of the lesions or by treating the lesions with the Na+-channel blocker, benzamil, significantly retarded healing. An increase in the field strength of lesions treated with Na+-depleted Hanks' solution, by the addition of direct current, increased epithelization. Epithelization was fastest in wounds with field strengths raised to - 80 mV/mm, more than twice the normal field strength present in wounds maintained in Hanks' solution alone. Epithelization decreased, however, when the field strengths were increased to -120 mV/mm. A similar pattern was also observed when the field's polarity was reversed. By manipulating and monitoring the field strengths, we have been able to show for the first time that increased wound field strengths enhance corneal wound epithelization, and that field strengths with reversed polarity also enhance this epithelization.

Electric field strength and epithelization in the newt (Notophthalmus viridescens).

There is convincing evidence that endogenous electric fields are necessary for normal wound epithelization, but it is unclear whether normal epithelization rates can be accelerated increasing normal field strengths. Although we confirmed that normal lateral fields are required for normal Notophthalmus viridescens epithelization rates, significant increases in epithelization were not achieved by increasing normal field strengths with direct current. With increases of less than 50%, epithelization rates were slightly increased. When the field strengths were augmented by 50%, epithelization was significantly retarded. This pattern was also observed when we applied direct current to wounds whose fields were nullified with benzamil. Epithelization was more rapid in wounds with field strengths raised to 20, 40, and 60 mV/mm than in benzamil-treated wounds without field augmentation. Epithelization was most rapid at 40 mV/mm, the normal value. When fields were augmented to 80 and 100 mV/mm, the epithelization rate diminished significantly. We also augmented field strengths by increasing the Na+ concentration in the medium surrounding the digits. Wounds healed more slowly in 10 mM Na+ than in 1.5 mM Na+, the normal pond water concentration. When field strengths in 10 mM Na+ were diminished to levels found in contralateral digits at 1.5 mM Na+ by applying direct current, the epithelization rate was restored to normal. We conclude that newt wound epithelization rates are nearly maximal at normal wound field strengths. Field strengths significantly higher than normal (50% more) result in significantly diminished rates of epithelization.

Electrical fields in the vicinity of epithelial wounds in the isolated bovine eye.

A lateral potential drop along the outer surface of the cornea could be measured at the edge of wounds made in the corneal epithelium of the isolated bovine eye when the cornea was covered by simulated tear film of modified Hanks' solution. These lateral fields (LFs) had an average magnitude of 42 +/- 1.4 mV mm-1 in the first 0.25 mm from the wound edge. The polarity of these surface LFs is more positive at the wound than in regions away from the wound. Very little if any lateral field could be measured at the edge of wounds along the inner surface of the corneal epithelium. The surface LFs depend on the cornea's transepithelial potential (TEP), which in this isolated bovine eye preparation we determined (with conventional microelectrode techniques) to have an average value of 24.7 +/- 2.2 mV, stroma-side positive. We found that this TEP drives a current from wounds in the epithelium with an average current density of 55 +/- 12 microA cm-2. We also have found that the average transcorneal potential (TCP) was 3.2 +/- 0.5 mV greater than the average TEP, confirming that the major contribution to the TCP comes from the corneal epithelium.

Intrinsic electric fields promote epithelization of wounds in the newt, Notophthalmus viridescens.

The lateral electric fields (LFs) in the vicinity of small wounds made in hindlimb digit tip skin of Notophthalmus viridescens have been measured and manipulated. Healing of these wounds was assessed by crystal violet staining and by histology. Paired experiments were conducted on the animals: the healing of one digit's wound was compared with healing of the contralateral digit's wound whose LF was changed from that of its contralateral wound. When currents were passed through the animal (into one wound and out of the contralateral wound) so that the LF of one wound was zero while the contralateral wound had an enhanced LF, the wounds with the enhanced LF healed more rapidly than the wounds with the zero LF. When digits on one side were treated with 30 microM benzamil in an artificial pond water so that their wound LFs were reduced to approximately zero, and the contralateral wounds were kept in artificial pond water without benzamil so that they had normal wound LFs, there was significantly less epithelization of the benzamil-treated wounds than of the control wounds. This effect on wound healing was reversed by adding currents that restored the normal wound LFs, but not by adding currents that reversed the wound LFs to the opposite polarity. When currents were added to reverse the wound LFs on one side of the animal, leaving the contralateral wounds free of added currents, the wounds with the reversed LFs healed more poorly than the contralateral wounds with normal LFs. These results are consistent with the hypothesis that the intrinsic LFs in the vicinity of wounds promote epithelization of these wounds.

Retinopretectal and accessory optic projections of normal mice and the OKN-defective mutant mice beige, beige-J, and pearl.

Retinal projections to the pretectal and terminal accessory optic nuclei were studied in normal wild-type mice and mutant mice with abnormal optokinetic nystagmus (OKN, Mangini, Vanable, Williams, and Pinto: J. Comp. Neurol. 241:191-209, '85). The mutants used were pearl, which exhibits an inverted OKN in response to stimulation of only the temporal retina, and beige and beige-J, which show inverted OKN in response to stimulation of only the temporal retina and, in addition, exhibit eye movements with a vertical component in response to horizontally moving, full-field stimuli. These projections were studied following intraocular injections of 3H-proline or horseradish peroxidase (HRP) with, respectively, light microscopic autoradiography or HRP histochemistry. In wild-type mice, strong contralateral retinal projections covered the entire nucleus of the optic tract, the anterior and posterior divisions of the olivary pretectal nucleus, and the posterior pretectal nucleus. Similar heavy contralateral projections were distributed over the dorsal and medial terminal nuclei of the accessory optic system. Also, terminals sparsely covered the entire neuropil of the contralateral lateral terminal nucleus in some but not all wild-type mice. The most prominent accessory optic input was to the medial terminal nucleus and was provided by the inferior fasciculus of the accessory optic tract. A typical mammalian superior fasciculus of the accessory optic system with anterior, middle, and posterior components was present. Ipsilateral label was found in anterior and posterior olivary pretectal nuclei in all of the wild-type animals, but was found inconsistently in the ipsilateral terminal accessory optic nuclei. The pattern of contralateral retinal projection to the nucleus of the optic tract and posterior pretectal nucleus in mutants was indistinguishable from that seen in the normal wild-type mice. However, retinal inputs to the ipsilateral anterior and posterior olivary pretectal nuclei were significantly reduced in pearl mutants and were exceedingly sparse in the beige and beige-J mutant mice, while the contralateral inputs to these nuclei were increased in a complementary fashion in the mutants. The labeling of the accessory optic input to the contralateral dorsal terminal nucleus appeared to be substantially reduced in all of the mutant mice. The size of the principal accessory optic fascicle, the inferior fasciculus, was significantly smaller in beige, beige-J, and pearl mice; this reduction was greater in the beige and beige-J than in the pearl mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of amiloride analogues on adult Notophthalmus viridescens limb stump currents.

We have previously investigated the relevance to limb regeneration of epidermally driven, Na+-dependent limb stump currents by blocking epidermal Na+ channels with amiloride, 3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide. In order to reduce Notophthalmus viridescens stump currents more effectively than with amiloride, we have examined six amiloride analogues. Of these, only benzamil, 3,5-diamino-6-chloro-N-[(benzylamino)aminomethylene]pyrazinecarboxamide, was more effective than amiloride. The concentration of benzamil that reduced stump currents to half their initial value was 0.034 microM, while this concentration for amiloride was 0.42 microM. We also found a contribution of calcium ions to these currents. When immersed in water whose Ca2+ concentration decreased stepwise from 1 to 0 mM, stump currents decreased significantly, although to a variable extent, depending on the batch of newts. With 30 microM benzamil and 0.5 mM calcium (in water that also contained 1.5 mM NaCl and 0.06 mM KCl) stump currents could be reduced to very low levels and, in many cases, changed to incurrents.

The optokinetic nystagmus and ocular pigmentation of hypopigmented mouse mutants.

We tested the optokinetic nystagmus (OKN) reflex of various hypopigmented mutant mice and ultrastructurally examined the pigmentation of various ocular structures in these mutants. Using electron microscopy we examined the pigmentation of the choroid and retinal pigment epithelium (RPE) and measured the numerical density, volume density, and distribution of RPE melanosomes of mice with the following phenotypes: (1) wild type, (2) mutants that have abnormal or no OKN in response to horizontally moving, full-field stimulation, and (3) other mutants that have normal OKN but reduced choroidal pigmentation. We also measured the OKN of all these mice in response to horizontally moving stimuli that were restricted to the nasal or to the temporal retina. We found that in the mutants with normal OKN the numerical density of melanosomes in the RPE was within the range found for wild type, while the numerical density was reduced for the mutants with abnormal OKN. For one mutant with normal RPE pigmentation and normal OKN, the choroidal pigmentation was nearly absent. For the genotypes with abnormal OKN the volume density of the RPE melanosomes and percent apical melanosomes were sometimes greater and sometimes less than normal. The OKN patterns of these mice fell into the following categories: (1) wild type; (2) field-restriction dependent OKN with small following movements but no OKN in response to full-field stimulation, normal OKN in response to stimulation of the nasal retina, and OKN of reversed direction in response to stimulation of the temporal retina; (3) oblique with slow oblique following movements and reduced numbers of OKNs with oblique quick phases in response to horizontally moving, full-field stimulation, nearly normal OKN in response to stimulation of the nasal retina, and OKN of reversed direction in response to stimulation of the temporal retina. The horizontal component of the oblique response to full-field stimulation was in the same direction for the two eyes, but the vertical component was in the opposite direction. (4) Slow, small amplitude, with no or very small following movements in response to full-field stimulation, following movements in response to stimulation of the nasal retina and reversed "following" movements in response to stimulation of the temporal retina but few or no quick phases of the OKN for any stimulus condition. These results show that a variety of abnormalities of the OKN occur for hypopigmentation mutants of the mouse.(ABSTRACT TRUNCATED AT 400 WORDS)

Stump currents in regenerating salamanders and newts.

We report here that a variety of salamanders and newts from differing habitats all drive a steady ionic electric current out of the forelimb stump tip after forelimb amputation. Several hours after amputation the density of this stump current ranges from about 10 to 100 microA/cm2 in most species, and declines with time. In most cases, the magnitude of the stump current is dependent on the concentration of Na+ in the external medium (an artificial pondwater), suggesting that the well-known Na+ -dependent transcutaneous voltage described in amphibia (particularly frogs) is the EMF for this stump current. These measurements add to those previously reported for the North American red spotted newt (Notophthalmus viridescens), and suggest that electrical changes following amputation of urodele limbs are widespread among members of this group.

Visually evoked eye movements in mouse mutants and inbred strains. A screening report.

The authors screened various inbred strains and mutants of the mouse, Mus musculus, for qualitatively abnormal or reduced numbers of optokinetic nystagmus (OKN) eye movements. Thirteen hypopigmentation mutants and ten neuromuscular mutants were found to have abnormal or markedly reduced OKN, but none of these mutants had retinal degeneration. Ten other hypopigmentation mutants and nine other neuromuscular mutants had relatively normal OKN, thus showing that neither hypopigmentation nor neuromuscular abnormality per se are well-correlated with abnormal OKN. These findings show that many mutants with visual defects are available from existing holdings. These visual mutants may serve as a resource for the study of the function and development of the mammalian visual system.

The glabrous epidermis of cavies contains a powerful battery.

Voltages across various glabrous (and gland-free) regions of cavy skin range from 30 to 100 mV, inside positive; across hairy ones, 0 to 10 mV. (moreover, hairy areas also tend to maintain lower transcutaneous voltages in man.) When an incision is made through the glabrous epidermis of the cavy, a microampere flows through each millimeter of the cut's edge. These wound currents generate lateral, intraepidermal voltage gradients or fields of about 100-200 mV/mm near the cut; fields which decline with distance from the cut with a space constant of 0.3-0.4 mm. It is deduced from these data that the epidermis near a cut drives up to 300 microA/cm2 across itself; moreover, these currents and potentials can be grossly, rapidly, and (to some extent) reversibly reduced by amiloride. It is concluded that the hair and gland-free skin of cavies has a battery comparable in power and character to that of frogs; but it is suggested that this mammalian battery may primarily subserve epidermal wound healing rather than salt uptake.

Characterization of abnormalities in the visual system of the mutant mouse pearl.

Mice of the mutant strain pearl (pe/pe) differ from the wild strain by a single gene mutation, which leads to a lightening of the coat color. We tested this strain to see if this mutant gene also expressed itself in one or more visual abnormalities. Pearl mice were found to lack totally the optokinetic nystagmus reflex that was present in every normal mouse that we examined. This lack of optokinetic nystagmus was not due to oculomotor defects, since postrotatory nystagmus was normal. As described for other pigmentation mutants, we found that pearl mutants had a reduced ipsilateral projection to the lateral geniculate nucleus, superior colliculus, and visual cortex. We recorded from single cells in the superior colliculus and found response properties and light sensitivities to be normal over the luminance range at which optokinetic nystagmus was tested. However, at very dim backgrounds (scotopic levels), the incremental sensitivities of these cells in pearl mice were about 100 times lower than those of normal mice. This reduction in sensitivity was restricted to scotopic backgrounds and was not due to abnormalities in either the time course of dark adaptation or the receptive field sizes of single cells. In recordings of the electroretinographic response, both the waveforms and the normalized magnitudes of the A and B waves of pearl were indistinguishable from those of normal mice, which seems to indicate that the cause of pearl's sensitivity defect is located central to the main electrical events in the photoreceptors. The normality of many aspects of the visual system of pearl mice contrasts sharply with the complete absence of optokinetic nystagmus, with the reduced ipsilateral projection, and with the reduced dark sensitivity of the cells in the superior colliculus.

Trochlear nerve regeneration in Xenopus laevis larvae.

The trochlear nerve of Xenopus laevis larvae was sectioned in the orbit, and the nerve distal to the section was removed, so that it could not serve to guide the return of the regenerating nerve to the superior oblique muscle (SOM). In a second series, in addition to removing the distal nerve segment, the SOM was removed or damaged. The regeneration pattern of the trochlear nerve fibers was recorded in situ at 1 to 5-day intervals with the aid of methylene blue staining. The early growth or regenerating fibers was multidirectional; only some fibers encountered the SOM by day 6 or 7; others reached nerves, inappropriate muscle, and other tissues. Ultimately, the fibers that reached the SOM persisted and were reinforced, while those that reached other locations were withdrawn. By 20 days or so, the regenerated trochlear nerve usually had an appearance that was close to that of an uncut nerve. In the second series, in which the SOM was removed or damaged, the multidirectional character of the growth persisted through 20 days after section. these data suggest that: 1) Trochlear nerve regeneration is not guided by cues that closely control fiber growth along a direct path to the SOM, and 2) The SOM acts as a trap for regenerating trochlear nerve fibers, promoting the maintenance and the fasciculation of the trochlear nerve fibers that reach it, and the degeneration of the fibers that do not.

Reduction of sodium dependent stump currents disturbs urodele limb regeneration.

We have asked the question whether the natural electric currents which leave urodele limb stumps are in any way needed for their regeneration. As an initial test, we have greatly reduced such currents in the tiger salamander, Ambystoma tigrinum, by applying 0.5 mM amiloride to the stump skin or by immersion of the animals in sodium depleted media. We have also reduced such currents in the red spotted newt, Notophthalmus viridescens, by such immersion. Limb regeneration in half of the amiloride-treated animals was either entirely blocked or grossly deficient, while the others regenerated normally. Limb regeneration in sodium depleted media was consistently inhibited for some weeks but then recovered. These results are consistent with the hypothesis that stump currents are in some way needed for normal regeneration.

Role of subdermal current shunts in the failure of frogs to regenerate.

Large, uniform, skin-driven currents (20-40 muamp/cm2) leave the ends of limb stumps of post-metamorphic frogs (Rana pipiens) from about the first through the tenth day after amputation. However, right after amputation, while currents of comparable density may leave the periphery of the cut surface, current densities are greatly depressed in the center of this surface. We suggest that this depression is brought about by shunting through the subdermal lymph space (characteristic of anurans but not urodeles); continues in covert form after formation of a wound epithelium; and helps explain the ability of small, imposed currents to initiate frog limb regeneration.

Bioelectricity and regeneration: large currents leave the stumps of regenerating newt limbs.

Electrical currents near regenerating newt limbs were measured with a recently developed vibrating probe. Steady currents with local surface densities of 10 to 100 muA/cm2 or more leave the end of the stump during the first 5-10 days after amputation and are balanced by currents with densities of only 1-3 muA/cm2 that enter the intact skin around the stump. They are immediately dependent upon the entry of sodium ions into this skin and are therefore inferred to be skin-driven. The outward currents are comparable in direction, density, duration, and position to artificially imposed currents previously found sufficient to induce significant regeneration of amputated adult frog limbs. This comparison suggests that the endogenous stump currents play some causal role in initiating regeneration.