Tectectomy in the cyclopean salamander

Physiology&Behavior,Vol. 50, pp. 305-309. ©PergamonPress plc, 1991. Printedin the U.S.A.

0031-9384/91 $3.00 + .00

Tectectomy in the Cyclopean Salamander I P A U L P I E T S C H A N D C A R L W. S C H N E I D E R

Department of Visual Sciences, School of Optometry and the Medical Sciences Program School of Medicine, Indiana University, Bloomington, IN 47405 and Department of Psychology, Indiana University of Pennsylvania, Indiana, PA 15705 Received 9 N o v e m b e r 1990 PIETSCH, P. AND C. W. SCHNEIDER. Tectectomyin the cyclopeansalamander. PHYSIOL BEHAV 50(2) 305-309, 1991.--In an Ambystoma larva with both natural eyes removed and one eye grafted atop the head (Cyclops preparation), vision-dependent behavior usually recovers from the enucleation inherent in the operation, but the optically activated skin blanching reaction reappears in a very small number of instances. In the present studies, while the latter trend continued for the conventional Cyclops preparation, tectectomy concurrent with the ectopic eye transplantation resulted in a several-fold increase in the recovery of blanching competency. Some 60 percent of the tectectomized Cyclops animals exhibited the same Hogben-Slome pigmentation indices as larvae with one natural eye intact (controls). As measured planimetrically with an image analyzer, the pigment spots (melanosome containing portions of dermal melanocytes) contracted to the same extent in the blanch-competent Cyclops animals as in controls with a single natural eye. Eye transplants Tectum Tectectomy Midbrain Axolotl Blanching reaction Camouflage reactions

Mesencephalon Skin pigmentation

Ambystoma Amblystoma Optic nerve regeneration

METHOD

WHEN an eye is transplanted atop the head of a bilaterally enucleated Ambystoma larva the resulting "Cyclops" has a better than 99 percent chance of eventually regaining the vision and recovering the sight-dependent learning capabilities it loses in the operation (34). Salamander larvae also depend on the optic system for the adaptive modulations of their dermal melanocytes that afford camouflage in the wilds; i.e., the neuroendocrine reactions that alter the distribution of melanosomes within dermal melanocytes and thereby result in the skin's transformation from light to dark coloration, or vice versa [see general treatment in (2)]. Bilaterally enucleated recipients of an orbitally transplanted eye have a better than even chance of fully recovering the blanching reaction, even with a xenogenic donor (29). By contrast, the Cyclops animals regain blanching ability in a very small percentage of cases, the recovery of vision qua vision notwithstanding (28). The poor recovery hampers the full exploitation of blanching as an independent, quantitative measure of the functionality of ectopic eyes. Based upon our own eye transplant data as well as the observations of others (1, 3-5, 21, 31, 35), we speculated that the tectum of the Cyclops acted to attenuate the efficiency of the donor's regenerating optic nerve fibers in searching out and reconstructing the blanch-mediating pathways. Now the tectum need not be present for a completely normal range of skin pigmentation changes (28) or for long-term survival of the subject; nor is tectal regeneration in the larval salamander vigorous or rapid (6, 22, 25, 28). Accordingly, we reasoned that tectectomy should improve the recovery rate of the blanching reaction in the Cyclops preparation.

Animals and Environment All experimental animals were Ambystoma punctatum larvae brought into the laboratory in early gastrulation and periodically monitored to insure that test subjects simultaneously developed through given Harrison (H) stages (33). Animals were 31 mm in overall length at surgery. Larvae were kept in 5 percent Holtfreter's solution, changed each day, and were fed daily rations of newly hatched brine shrimp (Artemia). Postoperatively, animals were individually maintained in bright white Styrofoam cups and in a light chamber described elsewhere (28). The intent was to expose the initially blinded (and therefore blanchincompetent) experimental subjects perpetually to conditions that would normally induce light skin coloration. The threshold for the light phase of the reaction is below 3 tux; illuminance in the light chamber was approximately 1400 lux. More precise measurements of light using a Tektronix J6523 luminance probe, in the chamber, showed the luminance of the interior walls of the cups, below the fluid line, to be 340 (---20 s.d.) nits (cd/m2). Lighting, including the chamber, was on an automatically timed circuit; the entire colony was exposed to alternating 12 h lightdark cycles (prolonged, continuous exposure to light reduces colony longevity); General Electric FC12T fluorescent lamps were adopted as the standard light source for the entire quarters. Subjects judged to be blanch competent were also checked to insure the viability of the skin darkening reaction, done by placing the test animal in a brown cup for 24--48 hours [see (28) for rationale and details].

1Supported by PHS Grant 507RR7031H of the BMRG, Indiana University, Bloomington and a Faculty Grant from the University Senate of Indiana University of Pennsylvania. 305

306

Operations The investigation involved five types of subjects: 1) Normal: animals with both natural eyes intact but anesthetized concurrent with and for the same duration as the experimental groups; 2) One-Eyed: subjects with one natural eye removed but the other intact, a control necessitated by the fact that the extent of blanching seems to be affected by the animal's having one versus two eyes (30); 3) Eyeless: larvae with both eyes removed; 4) Cyclops-I: bilaterally enucleated animals with an eye transplanted atop the head but without tectectomy; 5) Cyclops-X: like Cyclops-I but with tectectomy, as described in the next paragraph. Surgery was carried out under a stereoscopic microscope, a Petri dish lid lined with Vermont marble clay serving as the operating platform. Animals were anesthetized in 1:5000 MS 222 (see under Tricalne in the Merck Index). A prospective host was braced on the clay with insect pins, a decussating pair gently cinching the back of the head; another set were inserted at a low angle into either side of the mouth, eased through the branchiohyoids and flattened, lightly but f'mnly anchoring the lower jaw onto the soft surface. The host's eyes were then bilaterally extirpated. Skin on the dorsum of its head was incised in the midline and reflected laterally; the dorsal neurocranium (still unossified in young larvae) was subtotally removed, exposing the entire roof of the midbrain and the dorsal diencephalon. In operations involving tectectomy (Cyclops-X), a mid-sagittal incision was made along the entire length of the tectum, bisecting the structure and totally exposing the relatively large, gaping cerebral aqueduct. The resulting tectal halves were excised down to the floor of the cerebral aqueduct. Of the subject's midbrain, only the tegmentum remained. When the operation did not involve tectectomy (Cyclops-I), the pia-arachnoid was removed above the anterio-dorsal aspect of the tectum. (Although the latter membrane is totally transparent, its outline can be visualized by gently teasing its blood vessels so as to create minute hemorrhages within it.) The head wound was temporarily closed with the skin flap while the donor was being prepared. The donor animal was secured alongside the host and its left eye excised with an asymmetrical flap of periorbital skin remaining attached, the asymmetry for orientation purposes. The donor eye was positioned with its ventral pole facing rostrally on the host and the stump of its optic nerve aimed, by employing an eyepiece reticle, at a point just posterior to the host's conspicuous pineal body. The donor eye was secured with a transparent (Tygon), semi-flexible Briicke (33), these two properties permitting fine adjustments of the grafted eye's optical axis. After 15-20 minutes the MS-222 was diluted to 1:7000, and the host was kept immobile for 4-5 hours.

Measurements The evaluations were made 8-12 weeks postoperatively and the results were analyzed in three independent ways: 1) by matching and sorting the subjects, under a stereoscopic microscope, into one of three groups: A, B and C, corresponding to the overall pigmentation patterns of Normal, One-Eyed and Eyeless animals, respectively (i.e., the three control types); 2) by ascertaining Hogben-Slome (HS) pigmentation indices (17) of the dermal melanocytes in each subject's dorsal occipital-anterior cervical region, as far laterally as the roots of the last pair of external gills; 3) by electronic planimetry of the pigmented portion of dermal melanocytes in microphotographic negatives of each subject. (Unpigmented portions of the latter cells were not visible in the micrographs.) The micrographs were taken with

PIETSCH AND SCHNEIDER

TABLE 1 PIGMENTATION:MATCHING,INDICES,GROUPDISTRIBUTIONS Group

N

A

B

C

% Competent*

Normal One-Eyed Eyeless Cyclops-I Cyclops-X§

5 6 8 6 10

5 0 0 0 0

0 6 0 1 6

0 0 8 5 4

100 100 0 16 60

HS Indext 1.08 2.00 4.89 4.30 3.46

+ 0.20 _ 0.00 +_ 0.22 +__ 1.30 +__ 1.51

ill: 1 1 1 1 2

A - - H S 1.5; B -< HS 2.0; C >- HS 4. *Percent blanchingcompetent. fHogben-Slome (17) PigmentationIndex: means (---standard deviation). ~:fl= distribution,where 1 is unimodaland 2 is bimodal. §Tectectomized subjects.

an automatic Zeiss Photomax system, mounted on the stereoscopic microscope; the photographic negative was magnified 5 times with an enlarger and the target was circumscribed with a stylus on the digitizing pad of a Zeiss Videoplan image analyzing computer; the computer was programmed to store and analyze the data as the area per cell, in arbitrary units, "cell" meaning that portion of the melanocyte directly observable owing to pigment. Statistical analyses were performed with Videoplan software supplemented by calculations with RS/1 and Speakeasy programs, on a VAX 8650 digital computer. RESULTS Normal Ambystoma larvae fully blanch within about 20 minutes after their transfer from a clear receptacle to a white cup. Eyeless animals darken when illuminated in a container of any reflectance [see (28) for details and for other literature]. All experiments in this study were conducted well within the thresholds and reaction times of the test system. Table 1 provides an intuitive overview of the experimental findings. All Normal subjects exhibited the extreme contraction of pigment observed at HS indices approaching 1.0. The animals in the One-Eyed group, represented by Fig. 1, showed HS indices of 2.0. The HS value of 2 (that of the One-Eyed group) became the principal criterion for the recovery of the blanching reaction among the Cyclops groups. Eyeless animals (Fig. 2) presented HS indices ->4; pigmentation responses equivalent to those of Eyeless were adopted as the criterion for judging blanch incompetency. As was true of the One-Eyed controls, no Cyclops of either type showed HS indices characteristic of the Normals. Among the Cyclops-X (tectectomized) animals, 6 were very similar in pigmentation patterns to the One-Eyed larvae (Fig, 3) and 4, clearly lacking in a blanching reaction, resembled Eyeless subjects. Of the Cyclops-I animals (intact tectum) one resembled the One-Eyed animals while the remaining 5 presented the dark skin coloration and expanded melanocytes of the Eyeless subjects (Fig. 4). Distribution analysis of the HS indices (column tq in Table 1) confLrmed what had been suspected intuitively, namely the Cyclops-X group was bimodal; i.e., the group in question consisted of two distinctly different populations, provisionally assumed to be one with and the other without a blanching reaction. The assumption was tested during image analysis, and these data are presented below. The other 4 groups were unimodal. Table 2 shows the results of quantitative image analysis; a

TECTECTOMY IN CYCLOPEAN SALAMANDERS

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FIGS. 1-4. Melanocytes (pigmented portions) in Ambystoma punctatum larvae. (1) One-Eyed. (2) Eyeless. (3) Cyclops-X-plus. (4) Cyclops-I-minus. See text for descriptions. Primary magnification x 16.2.

TABLE 2 IMAGE ANALYSIS OF DERMAL MELANOCYTES* Group Normal One-Eyed Eyeless Cyclops-X-plust Cyclops-X-minus~ Cyclops-X (all) Cyclops-I

Cells (N) 200 175 296 100 99 274 145

*Area/spot in planimetric units. tBlanch-competent subjects. ~Blanch-incompetent subjects.

Mean -+ S.D. 1.41 2.12 16.23 1.92 12.66 6.52 8.21

_+ 0.74 -+ 1.18 -+ 6.26 -+ 0.90 _+ 6.68 -+ 6.45 +- 5.01

TABLE 3 GROUP COMPARISONS: MELANOCYTE SIZES

Median 1.36 1.97 15.42 1.90 13.91 3.22 7.12

Comparison One-Eyed vs. Normal Cyclops-X [all] vs.Cyclops-I Cyclops-X-plus vs. Cyclops-X-minus One-Eyed vs. Cyclops-X-plus One- Eyed vs. Cyclops-I Cyclops-X-minus vs. Eyeless

df

t

§

373 361 101 251 318 393

7.0 3.0 15.9 1.6 15.6 4.8

yes yes yes no yes yes

dr. degrees of freedom. t: two-sided Student's t-values. §: significantly different at the 95% confidence level. X: tectectomized; plus: blanch-competent; minus: blanch-incompetent.

308

PIETSCH AND SCHNEIDER

summary of the important group-to-group comparisons can be found in Table 3. The pigmented portions of the dermal melanocytes of Normal subjects were more extensively contracted than those of One-Eyed animals, as indicated by their smaller planimetric area (see Table 2); the difference was statistically significant (see Table 3). Thus, as in the pigmentation matchings, the One-Eyed rather than the Normal values were adopted as the positive control criteria. Pigmented portions of melanocytes were appreciably larger in Cyclops-I than in Cyclops-X, even without subdividing the latter group for the presence or absence of a blanching reaction: the median melanocyte area in Cyclops-I was over twice that of Cyclops-X subjects. To refine the analysis, the Cyclops-X group was sorted into Cyclops-X-plus and Cyclops-X-minus subgroups, respectively representing those capable and incapable of blanching in white cups. The medians and means of these two subgroups leave little doubt that they represent separate populations (see Table 2), thus vindicating the provisional assumption as to the underlying cause of the distributional bimodality: where the plus groups showed medians and means of approximately 1.9 units, those of the minus group generated values of about 13 units. Both the U-test and t-test indicated that the differences were significant well beyond the 95% criterion level adopted a priori ( U = 509; see Table 3 for degrees of freedom and t-values). The pigmented portions of melanocytes in the Cyclops-X-plus subgroup were very close in size to those of the One-Eyed controls (Table 2); the differences between the two groups were statistically insignificant (see Table 3). By contrast, the melanocytes of the Cyclops-I subjects were several times the control (OneEyed) sizes (Table 2), and the differences were statistically significant (Table 3). The pigmentation patterns just reported were evident in each subject from about 4-5 weeks postoperatively and persisted for an additional month after the data had been harvested for image analysis. Blanch-competent subjects darkened within an hour after their transfer to brown cups; i.e., the dark phase of the camouflage reaction was also intact. The animals' pigmentation reverted to pretest HS indices within an hour after the subjects were returned to white cups. DISCUSSION Tectectomy concurrent with eye transplantation dramatically

improved the recovery of the Cyclops subjects' blanching reaction in all measures we employed. Tecta in place, the Cyclops-I animals exhibited the high failure rates previously observed (28). With the Cyclops-X preparation, 60 percent of the subjects showed the same pigmentation indices as the One-Eyed controls; among blanch-competent Cyclops-X subjects (the plus subgroup), the quantitative response was identical (statistically) to the control value. The results vindicated our a priori expectations, namely that tectectomy would substantially enhance the recovery of the blanching reaction. Our investigation thus validates a means by which pigmentation changes can be expeditiously employed in the pursuit of important, still extant questions concerning the eye of the Cyclops preparation (28,34). Our data also seem consistent with the broad trends suggested in the literature concerning the vertebrate optic system (9, 10, 19, 27, 36, 41). Although our findings do not justify a protracted discussion of the latter, it is worth observing parenthetically that a welter of evidence suggests some parts of the tectum can attract and others repel embryonic or regenerating optic nerve fibers; that the attracting and repelling agents transcend phylogenetic lines among a broad spectrum of vertebrates, in vivo as well as in vitro, and in response to specific molecular entities (4, 5, 7, 8, 11-16, 18, 20, 21, 23, 31, 36-39). Our results suggest that efforts to answer the following questions concerning the Cyclops preparation in particular would be fruitful: does the tectum play an active or passive role in minimizing the recovery of blanching? Are fibers sequestered, inhibited from further growth or diverted from the appropriate course? Can the tectum, or parts of it, cause selective inhibition of collaterals that would, if the nerve were growing in from the orbit, reconstruct the blanching circuits? Given today's technology, the tectectomized Cyclops could be a valuable model for pursuing such questions. As anticipated from previous observations (30), and confirmed during the course of controlling the present experiments, quantitative differences in blanching were evident between OneEyed and Normal subjects.

ACKNOWLEDGEMENT We are grateful to Jacque E. Kubley, Director, Graphics Laboratory, School of Optometry, Indiana University, Bloomington, IN for advice and assistance with photography.

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