500 EYE FIELD OPERATIONS 



ENUCLEATION AND PIGMENTARY RESPONSES 



Scharrer (1932) in reference to salamander larvae, stated that "• . . .In addition 

 to sight and smell, the lateral line sense organs may play a role in obtaining food" and 

 Nicholas (1922) claimed that in the absence of the eyes, the sense of smell became para- 

 mount in the feeding reaction as evidenced by the animal's positive response to substances 

 diffusing in the water. Detwller and Copenhaver (19'+0) state: "We wish to emphasize the 

 fact that in the absence of both the eyes and the nasal placodes the larvae feed as well 

 as do the normal animals." Utilizing these facts, it ia possible to enucleate (i.e., re- 

 move the eyes) Amblystoma larvae (stages #25 to #27) and rear them on Enchytrea (white 

 worms) in light, darkness, and even under various concentrations of monochromatic light- 

 ing, to determine the relation of the eyes to both growth and pigmentary responses of the 

 skin. 



1. Enucleate Amblystoma larvae (stages #25 to #27-) and keep them in adequate 

 aquaria for 5 to U days in order to select those individuals which survived 

 the operation most satisfactorily. If necessary, anesthetize them, before 

 enucleation, in l/3,000 MS 222. 



2. Prepare a completely darkened environment (e.g., photographic dark room) but 

 one in which the temperature does not vary from that of the light environment. 

 This may require the circulation of air with a fan, for any dark Cover will 

 absorb radiant energy more rapidly that a light colored cover. The temperature 

 must be checked at least once dally. Another and most satisfactory method is 

 to coat the outside (sides and bottom) of finger bowls with flat black paint, 

 and provide an overlapping black-painted cover. These darkened finger bowls, 

 along with the controls (unpainted finger bowls), can then be kept together in 

 a conatant-temperature water bath. Daylight (but not sunlight) should be pro- 

 vided. If this is not possible, controlled artificial light (without heat) 

 should be provided. 



5. Place in each of 10 blackened finger bowls a single enucleated Amblystoma with 



50 cc. of growing medium. Place in each of 10 more blackened finger bowls a 



single normal (unoperated) Amblystoma. 

 1|. In a similar manner prepare 10 unpainted finger bowls, placing in each a single 



enucleated Amblystoma and in each of 10 more unpainted finger bowls, place a 



single normal (unoperated) Amblystoma. 



5. All kO finger bowls should be kept at the same temperature. The water should 

 be changed on alternate days. The young larvae may be fed first on small 

 Daphnla and, as they grow, on small and finally on large Enchytreid worms. The 

 feeding should be identical for all larvae, experlmentals and controls, in dark 

 and in light. 



6. The darkened animals may be changed and fed in a photographic darkroom, with a 

 very dim red (photographic) light, in minimum time. The quickest procedure ia 

 to pour the larva and medium through a coarse sieve, to eliminate faecal 

 material. 



7. Keep the larvae to the time of metamorphosis (50 to 60 days), using this change 

 as one criterion of growth rate. Meaaurementa of all larvae should be taken at 

 bi-weekly intervals, and notations made on general coloration. Measurements 

 can be made rapidly by pasting millimeter graph paper on the underaide of a 

 flat-bottomed finger bowl (or Petri dish) into which the larva is placed for a 

 quick total length measurement. 



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 If facilities and time allow, there are other variables to this experiment: 



1. Instead of using a black-painted cover for the darkened finger bowls, cover 

 them with glass (of Wratten celluloid) color filters which allows various mono- 

 chromatic radiations through to the larvae to determine the relative value of 

 parts of the spectrum in pigmentary response. 



2. The eyes may be removed and transplanted to heterotopic positions, where they 

 do not acquire nerve connections with the central nervous system, to determine 

 whether they then function in pigmentary response. 



