Dopamine and background adaptation in bullfrog tadpoles (Rana catesbeiana): a pharmacological and histofluorescence study.

Bromocryptine, a dopamine agonist, effectively inhibited the black-adaptation of prometamorphic tadpoles of the bullfrog Rana catesbeiana. The melanophore index (MI) of bromocryptine-injected tadpoles was significantly lower than that of controls. Bromocryptine was also effective in lightening the skin color of the hypophysectomized tadpoles bearing a pituitary graft. When pimozide, a dopamine receptor blocker, was given to the tadpoles adapted to a white background, the MI increased significantly and their skin color became dark. However, pimozide was not effective in darkening the skin color of the hypophysectomized tadpoles. The injection of α-methyl-p-tyrosine(α-MPT), an inhibitor of tyrosine hydroxylase, markedly attenuated the background response of the tadpoles. After being transferred from a black to a white background, the skin color of α-MPT-treated tadpoles was still significantly darker than that of non-injected controls. On histofluorescence examination, numerous green catecholamine fluorescent nerve terminals were found among the glandular cells of ƒ¿-MPT-injected and control tadpoles adapted to a black background. This green fluorescence in the pars intermedia(PI) of ƒ¿-MPT-treated tadpoles disappeared 6h after exposure to a white background. However, catecholamine fluorescence in the PI of the controls did not decrease in a white background. The present results suggest the possible involvement of dopamine in the background response of the bullfrog tadpoles. Since the background response of the tadpole skin color is dependent on the pituitary MSH, dopamine presumably inhibits MSH secretion by acting on the glandular cells of the PI when the animals are exposed to a white background. Amphibians change their skin color in response to a light or dark colored background. In this response, the dispersion and concentration of melanin granules in the dermal melanophores play the most important role, and these are dependent on levels of circulating melanophore stimulating hormone (MSH) (Hadley and Bagnara, 1975). Since the transection of the hypothalamo-hypophyseal tract or the injury to the hypothalamus in both larval and adult amphibians leads to hyperactivity of the pars intermedia (PI) of the pituitary gland, the secretion of MSH is generally believed to be under inhibitory control by the hypothalamus (Etkin, 1962; Ito, 1968). Electron microscopic and electrophysiological studies showed that nerve terminals form synaptic contacts with glandular cells of the anuran PI (Iturriza, 1964; Saland, 1968; Imai, 1969; Nakai and Gorbman, 1969; Oshima and Gorbman, 1969). By the histofluorescence technique, numerous catecholaminergic varicose fibers have also been demonstrated in the anuran PI (Enemar and Falck, 1965; Received December 4, 1981. 106 MIYAKAWA et al. Endocrinol. Japon. February 1982 Bartels, 1971; Terlou and van Kooten, 1974; Aronsson, 1976). On the basis of a microspectrofluorometric analysis of the fluorescent nerve fibers, double innervation by dopaminergic and noradrenergic fibers has been suggested in the PI of Rana temporaria (Prasada Rao and Hartwig, 1974). Dopamine, noradrenaline and adrenaline have been reported to be potent inhibitors of MSH release from the frog PI in vitro (Terlou et al., 1974; Iturriza and Kasal-Iturriza, 1972; Bower et al., 1974; Jenks, 1977). Results obtained with classical pharmacological methods, however, give the impression that the possible catecholaminergic inhibition of MSH release in vitro is mediated through ƒ¿-adrenergic and/ or dopamine receptors (Bower et al., 1974; Morgan and Hadley, 1976). Lightening of the skin color following the administration of dopamine agonists to amphibians (Platt and Norris, 1974; Smith, 1975) also points to the possible involvement of dopamine in regulating the release of MSH from the intermediate lobe. In the present experiments, as one step in elucidating the mechanisms involved in catecholaminergic regulation of MSH release in bullfrog tadpoles, changes in catecholamine fluorescence in the fibers distributed in the PI were examined during the adaptation to environmental changes. In this connection, the effect of dopamine agonist and antagonist and catecholamine synthesis inhibitor on background response was also studied. Materials and Methods Prometamorphic tadpoles of the bullfrog (Rana catesbeiana) weighing 6-20g were used. Tadpoles purchased from an animal dealer were kept for a week or more prior to the experiments in our laboratory at 22•Ž, and fed with boiled spinach. During the course of the experiments, all the animals were subjected to constant illumination with a fluorescent light. The background to which experimental animals were exposed was changed before or after the injection(s) of drugs, according to the experiment plan. The melanophores in the dorsal skin were observed under a microscope (•~100) immediately after the animals were killed by decapitation, and staged according to the melanophore index (MI) of Hogben and Slome (1931). Hypophysectomy was performed 7days prior to the experiments. About a half of the hypophysectomized tadpoles received a pituitary transplant subcutaneously from the same stage of the tadpoles 2days after hypophysectomy. (The presence of a pituitary graft was verified macroscopically or histologically at autopsy.) In order to examine the effect of dopamine agonist and antagonist on background response, 25ƒÊg of bromocryptine (2-bromo-ƒ¿-ergocryptine, Sandoz) was injected i.p. to 8 intact tadpoles and 8 hypophysectomized tadpoles with a pituitary graft. The drug was dissolved in a small volume of ethanol which was then diluted with 2000 volumes of saline. The injection volume was 0.05ml. Seven intact tadpoles and 5 hypophysectomized tadpoles bearing a pituitary graft received the vehicle only. All the animals were kept in black containers for 24h before and 5h after injection. MI was recorded 5h after injection. Nine intact and 8 hypophysectomized tadpoles which had been kept in white containers for 24h were given an injection of 25ƒÊg of pimozide (Janssen Labs.), a dopamine antagonist, dissolved in 0.05ml saline containing 0.3% tartaric acid. Eight intact tadpoles received vehicle only. All the animals were kept in white containers for another 5h following the injection. The MI was recorded for each animal at autopsy. To examine the effect of an inhibition of catecholamine synthesis on background response, tadpoles which had been kept in black containers received an i.p. injection of 40ƒÊg/g b.w. ƒ¿-methyl-p-tyrosine (ƒ¿MPT; DL-ƒ¿-methyl-p-tyrosine methyl ester HCl, Sigma) dissolved in 0.05ml D.W. (28 animals), or the same volume of D.W. (24 animals), and were transferred into white containers. Another group of tadpoles kept in black containers were treated with ƒ¿-MPT twice at 4h interval, then placed in white containers for 4-6h (20 animals) or 20h (19 animals). Four hypophysectomized tadpoles kept on a black background were also injected with ƒ¿-MPT. They were exposed to a white background after injection. MI was recorded at autopsy. To investigate the effect of dopamine agonist on background response of the animals treated with ƒ¿MPT, 25ƒÊg bromocryptine was given to 8 tadpoles kept in a black container simultaneously with 40ƒÊg/g b.w. ƒ¿-MPT. They were transferred into a white container after injections. Six h after injections, the animals were decapitated and MI was examined. For histofluorescent observations, 44 tadpoles kept in black containers were injected with ƒ¿-MPT or D.W. Half of them were placed in white containers. The remaining tadpoles were left in black containers. Sacrifice was performed 1, 3 and 6h after injections. The pituitary region was removed and processed for fluorescent microscopy according to Falck-Hillarp (Dahlstrom and Fuxe, 1964). Ten ƒÊm sections were cut Vol.29, No.1 DOPAMINE AND MSH RELEASE IN TADPOLES 107 from paraffin embedded specimens and observed under a fluorescent microscope (Olympus FLM).