A wide variety of visual functions show increases attributable to binocularity, and the question pursued here was whether a second eye enhances the visually stimulated skin blanching reaction of the larval salamander. Dermal melanin spots (produced by the aggregations of melanosomes within dermal melanophores and which contract or expand to lighten or darken the skin) were measured in eyeless (controls), one-eyed and two-eyed Ambystoma punctatum larvae after chronic adaptation of the subjects to a white background (i. e., stimulus conditions for maximum blanching). The eyeless subjects showed no blanching (thus remained dark) in white cups, and they exhibited melanin spots 7 or 8 times the size those of the other two groups. All one- eyed or two-eyed subjects exhibited blanching reactions; planometric comparison revealed a significantly larger melanin spot area for one-eyed than for two-eyed animals; i. e., the binocular condition permitted greater contraction of the pigment spots than did the monocular condition. Analytical data compared favorably with independently ascertained pigmentation indices. The results indicate that a second eye quantitatively elevates the blanching maximum of a larval salamander.
One such aspect occurs in the skin of the larval salamander. Most wild Ambystoma larvae can redistribute their skin melanin so as to adapt apparent coloration to changes in the immediate photic backgrounds [1, 6]. Earlier in this century, Laurens [5] demonstrated that these camouflage-mediating modulations of pigment are optically evoked. His observations have been reproduced in more recent years, and the camouflage reactions have been exploited for proving the functional recovery of xenoplastically grafted eyes [7, 8]. In the present investigation, we focused on the dermal melanophores that directly mediate blanching in Ambystoma larvae and analysed for differences in monocular versus binocular subjects.
The parameters and apparatus used for lighting are described elsewhere [7] but a brief summary of essentials will be presented here for the reader's convenience. A fluorescent light chamber was used to expose animals to approximately 1300 lux (i. e., some thousand fold above the threshold for blanching); the chamber was on the same circuit and duty cycle as the room lights and employed the same type of lamp. Blanching was the principal endpoint (for reasons presented with results). Except during photography or when being tested for the skin darkening reaction, animals were kept in white Styrofoam cups whose luminance in the light chamber was 360 (+/- 10 s.d.) nits, as measured on the cup wall, at the animal's resting level, with a Tektronix J6523 luminance probe. The lights came on at 6:30 A.M. To be sure that animals would be fully reacted, critical data were collected after 3:00 P.M.
Animals of a prospective group were anesthetized en masse in 1:5000 MS 222 (dissolved in 5% Holtfreter's solution) and when unconscious were randomly sampled into three subgroups: a) bilaterally enucleated --eyeless; b) left eye removed -- one-eyed; c) normal -- two-eyed. All subjects in a group were simultaneously removed from the anesthetic (to 5 % Holtfreter's solution). Ancillary experiments were performed to control for wounding per se: in a separate group of anesthetized animals the periorbital skin was circumferentially incised, but the eye was left in the orbit; the dermal pigment spots of these subjects were measured with an eyepiece micrometer preoperatively and again postoperatively. These wounds produced neither acute nor chronic effects on pigment spot sizes.
Using an automatic Zeiss Photomax camera-stereoscopic microscope system, we took photographs, at 20 X, three months following enucleation, of the dermal pigment spots in the occipital and cervical dermatomes; Kodak 35 mm plus X film was used; representatives of each group were recorded on serial frames of the same roll of film. Animals were not anesthetized for photography. Photographic images of dermal pigment spots, enlarged an additional 13 X, were projected onto the digitizing pad of a Zeiss Videoplan image analyzing computer. The outline of each dermal pigment spot in the frame was scribed using an electronic stylus. The Videoplan system was programmed to compute the area per spot in arbitrary planometric units. Statistical analyses of the data were performed with Videoplan software.
The Hogben-Slome [4] pigmentation index (where 1 is brightest and 5 darkest) was individually ascertained by inspecting the first cervical dermatome of each subject at the time of photography; these data were analyzed with Speakeasy software on a VAX 8650 computer.
The bright phase of the reaction was chosen over the dark phase as the principal endpoint because the photic environment could be controlled using eyeless animals (which blanch in total darkness but intensely darken when illuminated against a bright background). Moreover, as darkening progresses, the perimeter of the pigment spot becomes increasingly variegated, and tracing its outline, although still possible, becomes less precise than with the puncta presented during blanching. Finally, it was impossible to maintain control of stimulus conditions for the dark reaction (black photographic pan) during photographic sessions.
The qualitative aspects of the results are represented by the exhibits in Figure 1: three serial frames from the same roll of film, representing a two-eyed, a one-eyed and an eyeless subject, respectively; the animals had spent the previous three months in adjacent wells of the same cup rack and their entire life less than a meter apart (including during photography). Notice the subtle difference between the two-eyed and one-eyed subject, in contrast to the appreciably darker eyeless subject.
Three unretouched, serial frames showing from left to right: two-eyed, one-eyed and eyeless A. punctatum larvae from the same volley. The animals were maintained in individual bright white cups but spent their lives within a meter of each other, were of identical pigmentation patterns preoperatively, and pigment cells appeared precisely as exhibited in here for the previous three months. This photograph was taken (left to right) between 3:37-3:39 PM, e.s.t; i.e., about 9 hrs into the "on" phase of the light cycle for the day. Primary magnification 20 X.
In arbitrary units, the mean area occupied by the pigment spots in the two-eyed animals was 4.09+/-2.0 s.d., as compared to 9.65+/-3.6 s.d. for one-eyed subjects and 67.92+/-23.8 s.d. for their eyeless siblings. These three means are significantly different beyond the 95% level (Table 2) using Student's t-test.
The medians were calculated, as well (so as to perform nonparametric statistical analyses): 3.89, 9.50 and 62.82 for two-eyed, one-eyed and eyeless groups, respectively; these values were significantly different beyond the 95% confidence limits when assessed with the Mann-Whitney U test.
The radii (r) of the spots computed algebraically (assuming the ideal spot to be circular) from the mean areas (using area = Pi r2) were: 1.14 units for two-eyed; 1.75 units for one-eyed and 4.65 units for eyeless.
The Hogben-Slome pigmentation indices are also shown in Table 1. Two-eyed subjects generated a mean index of 1.08; for one-eyed, the pigmentation index was 2.0; the eyeless Hogben-Slome index was 4.89.
Area was chosen as a parameter because it could be directly and accurately measured. But the virtue of area (amplification) exaggerates and thus creates a distorted impression of the differences. Pigment spot radius provides a useful, if indirect, means of comparing blanching maxima. The variations in the radii roughly approximate those in pigmentation indices. But, as indicated by the simple model in the next paragraph, pigment spot radius can also serve as a basis for comparing the relative change from fully expanded to maximally contracted.
Imagine the spot radius to be the hypotenuse of a right triangle on a Cartesian coordinate in which X represents the number of eyes removed and Y an unknown pigmentation value. With the Pythagorean theorem, we can quickly compute the value of Y for any X. For Eyeless, Y is 4.1979; the one-eyed Y is 1.4361; the two-eyed Y is 1.140. Using eyeless as the frame of reference, the coefficient of change relative to it is 0.34 for the one-eyed and 0.27 for the two-eyed spots; i.e., the relative decrease in the size of the melanin spot attributable to the second eye is some 7%, a subtle but readily quantifiable difference.
Parallel circumstances occur, one-eyed versus two-eyed Ambystoma larvae, during light-shock avoidance learning [9]. As was true of blanching, the differences were subtle and became evident only after quantitative analysis: of avoidances per trial were 0.24 and 0.30 for one-eyed versus two-eyed animals.
Nor are one-eyed and two-eyed differences in visual functions confined to salamanders. Watanabe et al [10] compared binocular and monocular discrimination learning in pigeons and found the former superior to the latter, especially as the difficulty of the task increased. In humans, an extensive review of the psychophysical literature led Blake and Fox [2] to conclude "...binocular performance is superior (to monocular performance) for all task categories..." In a brief but seminal report on the subject in question, Campbell and Green [3] showed visual acuity to be of greater sensitivity in binocular versus monocular viewing. At high contrast, the gain from the second eye was 7%. The similarity of the latter value to the 7% generated in the present investigation may be coincidental. But the improvement of phenomenologically unlike functions of the visual system -- and among widely differing species -- may be the manifestation of a principle of physiological optics transcending both phylogeny and modality.
2. Blake, R. and R. Fox The psychophysical inquiry into binocular summation. Percep and Psychophysics 14:161-185, 1973.
3. Campbell, F.W. and D. G. Green [1965] Monocular versus binocular visual acuity. Nature 208:191-192.
4. Hogben, L. T. and D. Slome The pigmentary effector system. VI. The dual character of endocrine coordination in amphibian colour change. Proc Roy Soc B 109:10-53, 1931.
5. Laurens, H. The reactions of normal and eyeless amphibian larvae to light. J Exp Zool 16:194-210, 1914.
6. Noble, G. K. The Biology of Amphibia. Dover Press, New York, 1954.
7. Pietsch, P. and C. W. Schneider Vision and the skin camouflage reactions of Ambystoma larvae: the effects of eye transplants and brain lesions. Brain Res 340: 37-60, 1985.
8. Pietsch, P. and C. W. Schneider Transplanted eyes of foreign donors can reinstate the optically activated skin camouflage reactions in bilaterally enucleated salamanders (Ambystoma). Brain Behav Evol 32:364-370, 1988.
9. Schneider, C. W. and P. Pietsch The effects of addition and subtraction of eyes on learning in salamander larvae. Brain Res 8:271-280, 1968.
10. Watanabe, S., W. Hodos and B. B. Bessette Two eyes are better than one: superior binocular discrimination learning in pigeons. Physiol Behav 32:847-850, 1984.
| Subjects | cells no. | Area/Cell* | Radius** | Hogben-Slome Pigmentation Index*** |
|
|---|---|---|---|---|---|
| median | mean (+/-s.d.) | mean (+/-s.d.) | |||
| Two-eyed | 150 | 3.89 | 4.09 (2.0) | 1.14 | 1.08 (0.2) |
| One-eyed | 75 | 9.50 | 9.65 (3.6) | 1.75 | 2.00 (0.0) |
| Eyeless | 175 | 62.92 | 67.92 (24) | 4.65 | 4.89 (0.2) |
| Comparison | degrees of freedom | t-value* | U-value** | significance level |
|---|---|---|---|---|
| Two-eyed versus One-eyed | 97 | 12.5 | 913 | 95% |
| One-eyed versus Eyeless | 191 | 31.6 | 255 | 95% |
Comments:pietsch@indiana.edu