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Abstract. A transplanted eye of a different species can reinstate the optically evoked camouflage reaction in a salamander (Ambystoma) larva whose own eyes had previously been removed. Usually complete,the recovery was equal in quality, frequency and magnitude to bilaterally enucleated controls with one of their own eyes reimplanted (autografts). The observations confirm Stone's thesis that an eye can work in host of a foreign species.During the 1930's, L. S. Stone and his associates at Yale xenografted the eyes of various urodele amphibians and reported that the host would eventually see. Depending on the species,the grafted eye reportedly could endow the recipient with better visual acuity than the animal exhibited preoperatively (see especially Stone, 1930; Stone and Ellison, 1940; additional references and review in Jacobson, 1970).
Stone, of course, did not lack in critics. Indeed, even his most ardent admirers had to admit that his criteria for vision (behaviorally based) were not rigorous. A review of the literature indicated to us that the quality of the evidence had not substantially improved during the years since Stone's reports. We undertook the present investigation to see if Stone's fundamental thesis could be substantiated in tests involving reflex reactions rather than behavior, per se, and for this purpose turned the visually evoked, neuro-endocrine skin camouflage reactions of Ambystoma larvae (Pietsch and Tokarski, 1992). The latter reaction was discovered early in this century (Laurens, 1914) and we have more recently employed it to assess vision in ectopically transplanted eyes (Pietsch and Schneider, 1985).
In nature, salamander larvae would stand little chance of escaping the swift and vicious predators that share their native waters except for the marvelously attuned adaptive reactions of their skin pigmentation. (For general treatment of amphibian pigmentation see Bagnara and Hadley, 1973). When placed against a brightly reflecting background an Ambystoma larva with at least one functional eye will blanch within about thirty minutes. When transferred to a dark receptacle the same animal's will gradually darken. If its eyes are removed, however, the animal, if illuminated, will invariably darken whether the container is black, white, transparent or of a wide variety of colors; i.e., in the eyeless condition, dermal pigmentation patterns are independent of the photic background. (The animal will blanch in total darkness.) The direct effectors of the changes are a class of dermal melanophores that redistribute melanosomes to and from peripherally extended cytoplasmic processes (dendrites) and the cell soma with the net cumulative effect of altering the animal's surface reflectance. Readily visible under the stereoscopic microscope, these cells can be non-invasively monitored during prolonged intervals and can be rated according to a conventionally employed pigmentation index (Hogben and Slome, 1931). Since the changes occur gradually over some minutes they can readily be photographed.
In the present investigation, we sought to find out first if a xenoplastic eye could revive the camouflage reactions; if so, with what frequency and how well vis-a-vis recipients of an autografted eye?
Recipients of both autoplastic or xenoplastic transplants recovered the camouflage reaction as early as 4 and as late as 8 weeks postoperatively or else never showed a camouflage reaction again. The qualitative aspects of these trends are illustrated by Figures 3-8 (the quantitative features findings will be described below): Figure 3 shows an A. opacum larva with the eye of an A. tigrinum , an animal whose camouflage reactions had recovered by 5 weeks postoperatively; the subject of Figure 4, by way of contrast, is an A. opacum larva with an autoplastically grafted eye, also at 5 weeks, but whose camouflage reactions never recovered during the remaining several months of its life.
Figure 5 is a view at higher magnification of a camouflage-competent A. tigrinum recipient of an A. opacum eye. Compare its pigment spots with those of the One-Eyed subject. A. tigrinum larva in Figure 6. Figure 7 is a close up of an Eyeless A. tigrinum presented here as a frame of reference for the subject in Figure 8: an A. tigrinum that, although it received an autoplastic eye transplant, never recovered the camouflage reactions. The point is that success and failure was not confined to the recipients of foreign eyes but attended autotransplants as well. Animals that recovered the bright reaction were tested for the dark phase of the response by placing them in brown cups for 24 hours whereupon they darkened to the same extent as concomitantly tested One-Eyed and Unoperated subjects of the volley. Figure 9 shows a record photograph of a brown-tested pair: an Unoperated A. opacum and a sibling that served as the recipient of an A. tigrinum eye. The partially darkened animals of the brown tests were re-tested in white cups for the bright reaction. The transplant recipients regained the bright color as rapidly as the One-Eye and Unoperated subjects. Figure 10 illustrates re-testing in white cups; this is the same pair as in Figure 9.
During both brown-testing and bright re-testing, animals judged not to have recovered the camouflage were indistinguishable from the Eyeless subjects (see Fig. 11).
The incidence and extent of recovery of xenografts versus autografts was judged in an experimental series involving A. punctatum as the host of either its own eye or that of an A. tigrinum larva. In this series all hosts had developed simultaneously through the same Harrison stages; the donor A. tigrinum had come from one clutch of eggs; operations were performed in groups of 12 daily over a period of a week; at 8 weeks postoperatively all surviving animals were examined, tested for camouflage reactions and rated for HS pigmentation index. The latter results are summarized in Table 1.
Of 22 A. punctatum larvae with an A. tigrinum eye, 18 were able to brighten in a white cup (attain an HS index of 2.5 or less); 4 reacted as the Eyeless animals in the series. Of 22 autograft recipients, 17 regained the camouflage reaction, but 5 failed to show recovery.
The HS indices of all 22 Xenograft and 22 Autograft subjects generated means of 2.09 (+/-1.0 s.d.) and 2.20 (+/-1.1 s.d.), respectively and a t-value of 0.36, which indicated no significant differences between the means. However, the latter data included indices from animals that had not recovered the camouflage reactions, thus falsely elevating the means. For a more faithful comparison, the HS pigmentation scores for non-responding transplantees were excluded and the analysis repeated; these t-values appear in the right-hand columns of Table 1.
The mean values for Xenograft, Autograft and One-Eye were similar, and their t-values indicate no significant difference at the 0.05 level. The means for the latter three types of subjects were somewhat greater than those for Unoperated, suggesting, perhaps, an extra natural eye may deliver a greater degree of brightening. (The latter point is currently being reexamined, but is not directly relevant to the present investigation.)0 Eyeless animals generated a mean HS index appreciably (4-fold) and significantly higher than those of the transplant group.
Animals of the latter series presented an opportunity to conduct a well-controlled, independent test of the premise that the camouflage reactions are causally related to the eye. Animals were formed into paired groups representing Xenograft or autograft recipients whose HS indices were similar to Unoperated and One-Eyed values. The test group also included pairs of Unoperated and One-Eyed animals. All animals were simultaneously anesthetized; one member of each pair was enucleated (bilaterally in the case of the previously Unoperated) but the other member, which would serve as the control, was not. The animals were removed from the anesthetic, placed in white cups and examined some hours later. All control pair members exhibited the normal blanching reaction whereas their enucleated partners had appreciably darkened; the melanophores of the latter were identical whether the enucleated animal had borne a xenogenically transplanted eye or its own natural eyes (cf. figs. 12 and 13).
Finally, experiments with A. tigrinum as the donor provided direct evidence of the general health of the xenogenically transplanted eye during the study. Because of seasonal differences in A. opacum and A tigrinum, the latter eyes were comparatively small at time of the operation, the donors being much younger than the hosts. However, A. tigrinum larvae eventually become much larger than A. opacum (or A. punctatum ). After grafting, A. tigrinum eyes grew appreciably in the host site, the extent of which may be judged by comparing Figure 3 with Figures 9 or 10, where the same subject can be seen at 5 and 10 weeks postoperatively.
The present results support Stone's general thesis (that the optic nerves can functionally regrow into a foreign brain). But they do not furnish direct evidence of whether vision, qua vision, recovered concurrent with the camouflage reactions. We are currently investigating this question and will defer the discussion of this issue until all the data are available. It is worthy of note at this juncture that several diverse optic pathways course through the brain of the salamander (see references in Pietsch and Schneider, 1985)
A discursive morphological investigation of the regenerated camouflage circuitry would be difficult, but nevertheless feasible with contemporary methods (e.g., HRP); such investigations would be instructive in terms of the relationship of the recovery of a neural function to the reconstruction of a particular morphological pattern. Also, a small but significant percentage of transplant recipients failed to recover the camouflage reactions even though the eye remained healthy and grew normally. A comparison between recovered and unrecovered animals could yield useful information about the anatomy of the camouflage network.
Herrick, C. J. Development of the optic nerve of Amblystoma. J. Comp. Neurol. 74:473-534 (1941).
Herrick, C. J. The brain of the tiger salamander (University of Chicago Press, Chicago, 1948).
Hogben, L. T. and Slome, D. The pigmentary effector system. VI. The dual character of endocrine coordination in amphibian colour change. Proc. Roy. Soc. B 109:10-53 (1931).
Jacobson, M Developmental neurobiology (Holt, Rinehart and Winston, New York, Chicago, London, Sidney, 1970).
Jakway, J. S. and Riss, W. Retinal projections in the tiger salamander, Ambystoma tigrinum. Brain, Behav. Evol. 5:401-442 (1972).
Laurens, H. The reactions of normal and eyeless amphibian larvae to light. J. Exp. Zool. 16:194-210 (1914).
Pietsch, P. and Schneider, C. W. Vision and the skin camouflage reactions of Ambystoma larvae: the effects of eye transplants and brain lesions. Brain Res. 340: 37-60 (1985)
Pietsch, P. and Schneider, C. W. 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).
Pietsch, P. and Tokarski, T.R. The dermal melanophore of the larval salamander, Ambystoma tigrinum. Cytobios 69: 107-131 (1992).
Stone, L.S. Heteroplastic transplantation of eyes between larvae of two species of Amblystoma. J. Exp. Zool. 55:193-261 (1930).
Stone, L.S. and Ellison, F. S. Exchange of eyes between adult hosts of Amblystoma punctatum and Triturus viridescens. Proc. Soc. Exp. Biol. Med. 45:181-182 (1940).
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1. Unoperated A. opacum.[primary magnification for figs. 1-4 = 7.6 x]
2. Eyeless (bilaterally enucleated) A. opacum.
3. Xenograft (blanch competent): successful blanching reaction in an A. opacum 85 days after bilateral enucleation and transplantation of left eye of an A. tigrinum to its left orbit.
4. Xenograft (blanch incompetent): identical to that in fig. 3 but with permanent failure of blanching reaction; host here was sibling of host in fig. 3; transplant operation performed during same session as case in fig. 3.
Figures 9,10 show the same subjects, both siblings from the same volley: A. opacum larvae, one an unoperated control, the other the enucleated recipient in its left orbit of A. tigrinum eye. In 9, the latter is on the left side; both subjects were receiving a "brown" test at the time the record photograph was taken; i.e., had spent the previous 4 days in a brown cup (which partially darkens the animal), in order to evaluate the "darkening" phase of their camouflage reactions; fig. 9 was taken 141 days postoperatively. In fig. 10, at 184 days postoperatively, the two subjects were undergoing a bright test (had been in a white polystyrene cup) just prior to the photograph. The apparent darkness of the subjects in 10 is a result of pigmentation and shadows in the brain and deep tissues, visible (under the stereomicroscope) because the pigment spots have contracted. Note, though, that the changes in the size of the dermal pigment spots are of the same magnitude in both subjects; this was true throughout their life. Notice, however, the differences in the comparative size of the eyes, more noticeable in 10 than in 9, doubtless because of the 41 days of additional growth. The Xenograft subject is the same animal shown in figure 3, at 85 days postoperatively; i. e., three months earlier. A. tigrinum grows more rapidly and attains greater size than A. opacum.Ideally, a transplant should exhibit the growth potential of donor.
Here we see fully darkened A. opacums, the one on the left the recipient of the left of an A. tigrinum, the animal on the right an Eyeless control. The photo is useful first or all for comparison with fig. 10; it also faithfully represents the few eye-transplant recipients in which the camouflage reaction never recovered. The animal on the left, the recipient of the foreign eye, is a sibling of the subject of fig. 10; belong to the same operational volley; was also maintained in a white for the 184 day observation period; and was like 10 in all respects except that it never recovered the camouflage reaction. It was being photographed here within a few minutes of fig. 10. The animal on the right is an Eyeless A. opacumcontrol.
Fig. 12 shows two A. punctatum, each a host of an A. tigrinum eye, and both having fully recovered the camouflage reaction prior to the test. On the afternoon of the photograph, the eye of the animal on the left was removed while that of the animal on the right was left in place. The animals were place in a white cup, wherein the animal on the left darkened while the one on the right blanched (cf. with the eyeless animal on the fight in fig. 13). The animal on the left in 13 is an unoperated control A. punctatumin the same volley as the subjects in 12 [primary mag. 7.6 x].