HOW DOES A SALAMANDER CHANGE ITS SPOTS?

Paul Pietsch and Carl W. Schneider

Indiana University, Bloomington Indiana 47405 and Department of Psychology, Indiana University of Pennsylvania, Indiana Pennsylvania 15705

Adapted and revised from an invited article, Readout mechanisms for the optically activated skin camouflage reactions of salamander (Ambystoma) larvae, in the Axolotl Newsletter, 20:20-23, 1991.

Web contact pietsch@indiana.edu

Key words: skin pigmentation, camouflage reaction, metachrosis, melanophores, melanocytes, salamanders, axolotl, hypophysectomy, Ambystoma larvae

Abstract

Salamander larvae can adapt their skin color to the background. Pigment cells of the skin, dermal melanophores, mediate the changes. It is generally believed that the darkening phase of the reaction depends on the release of melanophore stimulating hormone (MSH) by the pituitary gland. Experiments described here show that, indeed, darkening cannot occur without the pituitary. Animals begin to blanch immediately after either hypophysectomy or the part of the brain containing the pituitary is removed. But replacing the detatched pituitary or the piece of brain containing it, restores the animal's ability to darken. In addition, experiments were conducted to test the hypothesis that blanching after removal of the pituitary is tantamount to a spastic paralysis.

Introduction

Few wild species of Ambystoma would survive larvahood without the ability to adapt their skin color to changes in the immediate photic background. The adaptations, formally known as metachrosis, afford camouflage from swift and vicious predators who share the salamander larva's native waters. Historically, the reactions have been regarded as neuro-endocrine in character (see Bargnara and Hadley, 1973 for general treatment of pigment biology).

In the laboratory, the animals typically blanch when placed in white cups. They progressively darken when transferred to a black receptacle, eventually becoming all but visibly undetectable if the incubation period is protracted over several days. Early in the 20th century, Henry Laurens at Yale (1914, 1916, 1917), demonstrated that the camouflage reactions in question become activated via the visual system. We have been exploiting these reactions as a non-invasive, behavior-independent test of the functional incorporation into the host site of grafted eyes (Pietsch and Schneider, 1985, 1988, 1990). While our focus has been mainly on the sensory side of the reaction, and on its bright phase, some of our experiments have dealt parenthetically with output, and we routinely test darkening as well.

In the present communication, we turn our attention to the readout for the system, specifically to experiments illustrating the indispensability of the hypophysis to the darkening phase.

Definitions and usages

This photograph shows what we mean by blanching and darkening: image
Fig. 1: Two sibling, 22-mm A. tigrinum larvae whose forebrains (including the pituitary) had been swapped and then re-swapped. {for a light micrograph, go here}

The spots, which contract into puncta during blanching, (allowing stationary brightly colored pigments to show through) and expand in darkening (progressively obscuring the otherwise straw-yellow field) are actually melanosomes, pigment bodies, within dermal melanophores. (See Pietsch and Tokarski, 1993.)

image
Fig. 2: Dermal melanophore of an Ambystoma tigrinum larva. Epidermal melanocytes, which slowly react to prolonged radiation changes and can fine-tune an animal's apparent coloration, are not part of the reaction under consideration. During brightening, the melanosomes concentrate in the cell body. During darkening, the pigment bodies disperse into dendrites of the cells. Whether the dendrites contract and expand or are permanently fixed in position has not been completely answered (see discussion in op. cit.). To do justice to both sides of the controversy, we currently apply the term "spot" rather "cell" to the effectors.

Inhibiting the dark phase of the reaction

In one series, our principal subjects were 28 mm sibling A. tigrinum larvae collected in Tennessee by Charles Sullivan and received in a fortuitously large (for tigrinum) jelly mass at Harrison stage 24; i. e. siblings whose development we could monitor from early embryonic stages up to the time experimentation. All potentially painful procedures were carried out with animals under MS 222 narcosis. As stock, the animals were maintained on 12-hour cycles of light and dark (which enhances long-term viability) but during the actual experiments were keep in continual illumination of approximately 1400 Lux in a light chamber described elsewhere (Pietsch and Schneider, 1985). Controls for lighting conditions included groups of larvae maintained throughout in either black or white pans. Lighting controls showed no unanticipated signs.

The test subjects were initially divided into three groups. Using the map shown in Fig. 3, we subjected them to one of the following procedures:

image
Fig. 3: A is roughly the forebrain; B the midbrain and ventral diencephalon and C the hindbrain; AB is the combined fore- and midbrain. See Pietsch and Schneider, 1985 for additional details.

Immediately after surgery, animals were placed in black pans; i. e., stimulus conditions for darkening (op. cit.).

What happened to the B-less and hypophysectomized animals?

Within a few hours, during which time the unoperated animals darkened, all B-less and hypophysectomized subjects had blanched.

On the following day, using previously unoperated subjects as the donors, one third of the B-less group received a transplant of a freshly excised region B; another third received an hypophysis, implanted into the still-gaping mouth of the IVth ventricle; the status quo was maintained for the remaining group of animals, now serving in the role of the B-less controls.

Four hours post-operatively, all recipients of either region B or of the hypophysis alone had darkened. The B-less (now controls) remained blanched.

Extending the experiment

The experiment just described was extend as follows. On the subsequent morning, a region B was transferred back to the void in the cranium of its original donor, the latter animals having blanched in the interim. By that evening, these aniamals, the original donors, were now dark while the transitory hosts, lacking B now, had blanched. The exchange was repeated among half the subjects (the balance serving as controls). By morning the pigment patterns had again reversed while the unexchanged half (controls) showed no changes. The animals in Fig. 1 were among the subjects of the re-reversal experiment.

Flipping the brain

Another series of experiments were performed in which young A. mexicanum (axolotl) larvae were the principal subjects. Hypophysectomy from inside the oral cavity cause the subjects to blanch. However when the animal's hypophysis was inserted into the IVth ventricle, instead of being discarded, the animals darkened.

When we removed either B or region AB as a unit (including the hypophysis), the subjects blanched. Returning either B or AB reinstated darkening. Animals with AB removed remained blanched after the return of only A.

Induced blanching in eyeless mutants

Most Ambystoma larvae, including bilaterally enucleated animals, blanch if kept in darkness (less than 0.5 Lux) for several days. But an exception is the genetically eyeless (e/e) mutant A. mexicanium (see Epp 1972; Pietsch and Schneider, 1985); i. e., members of this strain ordinarily are permanently dark irrespective of the photic conditions. Could we induce blanching in eyeless mutants?

With B removed from the eyeless mutants, they blanched. Darkening returned when B was put back, whether orthotopically or with the ventral surface facing up.

Rotating AB and reversing the light source

In a variant of the latter experiments with eyeless mutants a lighting apparatus was constructed with the illumination coming from below rather than above. Exposures were carried, using the latter. Region AB was the object of attention; AB was reimplanted right-side-up in some animals and upside down in others. The subjects darkened equally well whether AB faced up or down. When the subjects were then transfer to the standard light chamber, thus reversing the light source rather than the AB, no changes occurred in darkening.

Blanching as spastic autonomic paralysis

The decapitated body can live for extended periods and, as might be suspected from the importance of the hypophysis, generally do so in a permanently blanched state, as in Fig. 4:

image

Fig. 4: The living body of an animal with extirpated forebrain (and ipso facto, the pituitary removed) -- as seen under the stereoscopic microscope.

However, when we cut the nerves that supply specific dermatomes (skin segments), in some cases unilaterally, within 15 minutes the zone in question darkened while innervated neighboring, or contralateral, dermatomes remained blanched:

image

Fig. 5 (see caption of Fig. 6 for description)


image
Fig.6: Higher power view of adjacent dermatomes of the debrained A. tigrinum larva shown in Fig. 5. The denervated dermatome (serial skin segment) is to the right and the dermatome with nerves intact is on the reader's left. Notice how the melanophores of the denervated right side produce confluent tiger-like patches whereas the cells on the left still appear as the individual spots typical of the blanched headless body (compare with Fig. 4). The white area on the upper right, above the tigroid patches, is a pigment-free zone of the dermatome, not generally visible to the naked eye.

In other experiments, localized cordectomy failed to alleviated blanching whereas unilateral sympathectomy, with the spinal cord left in place, was followed in moments by darkening.

Conclusion

We believe that removing the humoral output of the hypophysis, whether direct or ipso facto, and inducing permanent blanching creates spastic (pre-ganglionic) sympathetic paralysis. Both sympathectomy or denervation of a dermatome set up the equivalence of flaccid (postganglionic) sympathetic paralysis. In this connection, the camouflage reaction seems quite similar to autonomic reflexes in general, preganglionic lesions producing spasticity of the effector and postganglionic lesions causing flaccidity. Autonomic reflexes, in general, are subject to humoral influences. In the dark phase of the camouflage reaction, hormones play an indispensible role.

Our data also emphasize the dual neural and endocrine character of metachrosis in the Ambystoma larva. Post-ganglionic sympathetics doubless represent the final pathway to the melanophore. The humeral nature of the pituitary contribution would explain why the organ made its contribution even when detatched or rotated or placed in an unnatural location.

Literature cited

Carl W. Schneider is a Professor Emeritus at Indiana University of Pennsylvania, Indiana, Pennsyvania.

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