Evidence for a direct role of α-MSH in morphological background adaptation of the skin in Sarotherodon mossambicus

Summary The skin colour of the cichlid teleost Sarotherodon mossambicus adapted rapidly to changes in background colour. The physiological adaptation was associated with morphological changes in the dermis. Differences in the dermis were found between fish adapted to a black or white background for 14 days. Number and size of the melanophores as well as the amount of pigment in the cytoplasm of the melanophores were significantly increased in fish adapted to a black background. Changes in the dermis parallelled changes in the state of activity of the two endocrine cell types in the pars intermedia of the pituitary. Both the PAS positive cells and the MSH producing cells were more active when the fish were exposed to a black rather than a white background. Fish continuously infused with -MSH, using an osmotic minipump, had more melanophore cytoplasm and pigment per dermis surface unit area than untreated fish. The activity of the MSH cells in MSH-infused fish exposed to a black background was reduced to a level comparable to the MSH cell activity of untreated fish on a white background. -MSH treated fish that were exposed to a white background had many disintegrating MSH cells. These findings point to inactivation of these cells by exogenous -MSH. The activity of the PAS positive cells was not influenced by treatment with -MSH.     

Interaction and developmental activation of two neuroendocrine systems that regulate light‐mediated skin pigmentation

Lower vertebrates use rapid light‐regulated changes in skin colour for camouflage (background adaptation) or during circadian variation in irradiance levels. Two neuroendocrine systems, the eye/alpha‐melanocyte‐stimulating hormone (α‐MSH) and the pineal complex/melatonin circuits, regulate the process through their respective dispersion and aggregation of pigment granules (melanosomes) in skin melanophores. During development, Xenopus laevis tadpoles raised on a black background or in the dark perceive less light sensed by the eye and darken in response to increased α‐MSH secretion. As embryogenesis proceeds, the pineal complex/melatonin circuit becomes the dominant regulator in the dark and induces lightening of the skin of larvae. The eye/α‐MSH circuit continues to mediate darkening of embryos on a black background, but we propose the circuit is shut down in complete darkness in part by melatonin acting on receptors expressed by pituitary cells to inhibit the expression of pomc, the precursor of α‐MSH.     

The distribution of monoamine oxidase and acetylcholinesterase in the brain of Xenopus laevis tadpoles

Summary The distribution of monoamine oxidase (MAO) in the brain of Xenopus laevis tadpoles (stage 52–56) was studied histochemically with a modified Glenner's tryptamine-tetrazolium method. A moderate activity was observed in fibre regions of the striatum and septum (including the medial and lateral forebrain bundles), in the neuropil of the nucleus amygdalae, in the commissura anterior and commissura hippocampi, in the fibre regions of the diencephalon (including the optic chiasma), in the fibre regions of the tectum opticum and the tegmentum of the mesencephalon and in the white substance of the ventral half of the medulla oblongata. A greater MAO activity was found in the neuropil of the entire nucleus praeopticus. In the partes anterior and magnocellularis of this nucleus, MAO positive fibres are present in close contact with the perikarya, indicating a monoaminergic innervation of these neurons. The perikarya themselves did not show MAO activity. In the neurons of the nucleus praeopticus epichiasmaticus, the paraventricular organ (PVO) and nucleus infundibularis dorsalis (NID), only a slight MAO activity has been demonstrated in the perikarya, whereas a strong MAO positivity was found in the intraventricular protrusions and the neuropil. These data indicate the aminergic character of the neurons of these nuclei. From the postoptic fibre region a MAO positive tract was observed towards the developing median eminence and pars intermedia of the hypophysis. The pars nervosa and some cells of the pars distalis also contained MAO. Along the border of the aquaeduct of Silvius and the fourth ventricle, MAO positive liquor-containing neurons are also present.The distribution of acetylcholinesterase (AChE) was investigated in the hypothalamohypophysial region. AChE activity was found in the neuropil of the nucleus praeopticus magnocellularis, in the fibres of the optic chiasma and in the postoptic fibre region. The neurons of the PVO and NID were AChE negative. An AChE positive tract could be traced from the postoptic fibre region to the developing median eminence and pars nervosa. The pars distalis did not show AChE activity. However, in tadpoles reaching the metamorphic climax, ChE activity appeared in certain cells of the pars distalis; this might be related to degenerative phenomena in the acidophilic cells. The absence of AChE activity in the pars intermedia indicates a regulation of MSH release by peptidergic nerves to be unlikely.The stimulating interest and helpful advice of Prof. Dr. P. G. W. J. van Oordt is gratefully acknowledged. Thanks are also due to Mr. H. van Kooten and his co-workers for making the photographs.     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo1

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long‐term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore‐fibroblast co‐culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast‐derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane‐enclosed melanosomes in a process that was upregulated by α‐melanocyte‐stimulating hormone (α‐MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane‐proteins seemed to be of importance, as the cluster‐like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long‐term colour changes and for studies of factors that affect pigmentation.     

Hypothalamic control of the pars intermedia in Xenopus laevis tadpoles

Summary In Xenopus laevis tadpoles the relation between a paired nucleus of bio-amine producing neurons in the caudal hypothalamus and the pars intermedia of the hypophysis was studied.Treatment of the animals (stage 49 to 50 of Nieuwkoop and Faber's normal table) with reserpine caused aggregation of the skin melanophores within one hour, followed by redispersion five to six hours after the beginning of the experiment. This was at exactly the same time as the bio-amines in the caudal hypothalamus disappeared. However, the drug was ineffective if the nuclei had been removed. This indicates that reserpine acts via these nuclei and does not influence the skin melanophores directly.It was concluded that the initial aggregation of the melanophores may be the result of a reduced extrusion of MSH from the pars intermedia, caused by an increased output of a MIF by the bio-amine producing nuclei. The redispersion was explained by assuming that the bio-amines were depleted and the nuclei stopped with the extrusion of the MIF. This does not mean that the production of a MIF is the only function of the paired bio-amine producing nucleus in the caudal hypothalamus.The author thanks Prof. Dr. P. G. W. J. van Oordt for his helpful comments and criticism. Mr. J. H. I. J. M. ten Berge and Mr. E. W. A. Kamperdijk provided great assistance during the course of the experiments. Mr. H. van Kooten made the diagram and the photograph.     

Effects of 6-hydroxydopamine and 5-hydroxydopamine onlthe teleost melanophores innervation

Intraperitoneal injections of 6-OH-dopamine (80 mg kg^−l) promote, in toad fish, killifish, lsummer and winter flounders, a darkening of their colour and loss of capacity to adapt tolthe colour of the background. This condition persisted for three weeks after which thelanimals gradually returned to normal. The study of the skin of 6-hydroxydopamineltreated killifish showed degenerative lesions in the fine nerves and synapses of its melanophores, 124 h after the administration of the drug. These lesions progressed and 4 days later, lno synaptic structures could be detected in these cells. This condition persisted up to thel20th day. These results suggest that melanophores have a single monoaminergic innervaion.     

Unusual Leucophore‐Like Cells Specifically Appear in the Lineage of Melanophores in the Periodic Albino Mutant of Xenopus laevis

In the periodic albino mutant (a^p/a^p) of Xenopus laevis, peculiar leucophore‐like cells appear in the skins of tadpoles and froglets, whereas no such cells are observed in the wild‐type (+/+). These leucophore‐like cells are unusual in (1) appearing white, but not iridescent, under incident light, (2) emitting green fluorescence under blue light, (3) exhibiting pigment dispersion in the presence of α‐melanocyte stimulating hormone (αMSH), and (4) containing an abundance of bizarre‐shaped, reflecting platelet‐like organelles. In this study, the developmental and ultrastructural characteristics of these leucophore‐like cells were compared with melanophores, iridophores and xanthophores, utilizing fluorescence stereomicroscopy, and light and electron microscopy. Staining with methylene blue, exposure to αMSH, and culture of neural crest cells were also performed to clarify the pigment cell type. The results obtained clearly indicate that: (1) the leucophore‐like cells in the mutant are different from melanophores, iridophores and xanthophores, (2) the leucophore‐like cells are essentially similar to melanophores of the wild‐type with respect to their localization in the skin and manner of response to αMSH, (3) the leucophore‐like cells contain many premelanosomes that are observed in developing melanophores, and (4) mosaic pigment cells containing both melanosomes specific to mutant melanophores and peculiar reflecting platelet‐like organelles are observed in the mutant tadpoles. These findings strongly suggest that the leucophore‐like cells in the periodic albino mutant are derived from the melanophore lineage, which provides some insight into the origin of brightly colored pigment cells in lower vertebrates.     

The distribution of aminergic neurones and their projections in the brain of the teleost,Myoxocephalus scorpius

Summary Fluorescent histochemistry was carried out on the brain of the teleost Myoxocephalus scorpius to show the distribution of monoaminergic neurones and their projections.Posterior to the obex of the fourth ventricle, at the junction of the spinal chord and medulla, there is an unpaired dorsal nucleus of catecholaminergic cells. A second group of catecholaminergic perikarya are scattered lateral to the vagal and glossopharyngeal motor nuclei. Both groups of aminergic cells contribute to a tract which crosses the fourth ventricle at the obex and runs along the lateral wall of the medulla towards the diencephalon.At the level of the isthmus there is a lateral nucleus composed of large catecholaminergic cells with prominent fluorescent axons and its possible homology with the locus coeruleus is considered. Medially, in the same region a nucleus of serotonergic neurones lies between the paired tracts of the fasciculus longitudinalis medialis.In the diencephalon there are three paraventricular nuclei, the nuclei recessus posterioris and lateralis and the paraventricular organ pars anterior. Ventral to the lateral recess there is a further nucleus less closely associated with the ependyma.The distribution of fluorescent fibres is described and the dispositions of the aminergic nuclei compared to those of other teleosts.     

Effect of background color on the pigmentation of Xenopus laevis larvae

Larvae of Xenopus laevis (stage 39, after hatching) were reared in the 0.95-L (10-cm height; 9.5-cm diameter) containers with all combinations of white, grey, and black color of bottom and walls (side background). The containers were kept in a closed box at 12-hours illumination per day. After two months (stage 55), the number of pigment cells was counted on lateral side of larval body. Both bottom and side background had a significant effect on number of dermal melanophores.     

Monoaminergic Systems in the Hypothalamus of the Acanthopterygian Chelon labrosus (Risso, 1826), with Special Reference to the Organon Vasculosum Hypothalami

Distribution of biogenic amines in the diencephalon of the advanced teleost Chelon labrosus was investigated by formaldehyde-induced fluorescence. We have found three closely interrelated bright yellow-green fluorescent monoaminergic cell groups having numerous cerebrospinal fluid-contacting cells with dendritic processes that protrude into the lumen of the third ventricle. The most rostral of them, the organon vasculosum hypothalami, located dorsally at the mid and caudal hypothalamus level, showed under electron microscopy some monoaminergic cells and others with an abundant smooth endoplasmic reticulum. The cerebrospinal fluid-contacting processes of both cell types, in association with numerous fibres, terminal buttons and some capillaries, constitute a thick and complex intraventricular mat. The other two fluorescent regions, nucleus recessi lateralis and nucleus recessi posterioris, border the lateral and posterior recesses of the hypothalamus. The ultrastructural characteristics of the organon vasculosum hypothalami and its intraventricular mat suggest a function in the regulation of chemical changes in the cerebrospinal fluid. These monoaminergic regions probably represent three cell masses originated from a single region in primitive fish.     

Chromatophores and color change in the lizard,Anolis carolinensis

Summary The skin of the lizard, Anolis carolinensis, changes rapidly from bright green to a dark brown color in response to melanophore stimulating hormone (MSH). Chromatophores responsible for color changes of the skin are xanthophores which lie just beneath the basal lamina containing pterinosomes and carotenoid vesicles. Iridophores lying immediately below the xanthophores contain regularly arranged rows of reflecting platelets. Melanophores containing melanosomes are present immediately below the iridophores. The ultrastructural features of these chromatophores and their pigmentary organelles are described. The color of Anolis skin is determined by the position of the melanosomes within the melanophores which is regulated by MSH and other hormones such as norepinephrine. Skins are green when melanosomes are located in a perinuclear position within melanophores. In response to MSH, they migrate into the terminal processes of the melanophores which overlie the xanthophores above, thus effectively preventing light penetration to the iridophores below, resulting in skins becoming brown. The structural and functional characteristics of Anolis chromatophores are compared to the dermal chromatophore unit of the frog.This study was supported in part by GB-8347 from the National Science Foundation.Contribution No. 244, Department of Biology, Wayne State University.The authors are indebted to Dr. Joseph T. Bagnara for his encouragement during the study and to Dr. Wayne Ferris for his advice and the use of his electron microscope laboratory.     

Structural changes in the pars intermedia of the cichlid teleost Sarotherodon mossambicus as a result of background adaptation and illumination

Summary The pars intermedia of Sarotherodon mossambicus contains two structurally different endocrine cell types. The predominant cell type is assumed to synthesize MSH and related peptides. The second cell type is PAS positive; its function and products are unknown. In this second cell type changes occur in relation to background colour and illumination. Thus, PAS positive cells of fish adapted to a white background are less numerous and metabolically less active than those of fish adapted to a black background, and are most active in fish kept in total darkness. In blinded fish, whether in light or in darkness, the activity of the PAS positive cells is similar to that of the black background-adapted animals. The significance of these responses in relation to the control of background adaptation is discussed.     

Physiological Color Changes in Reptiles

SYNOPSIS. The physiological regulation of color changes in reptilesas studied in the lizard, Anolis carolinensis, is discussed.In Anolis, the ability to adapt to a background is dependentupon the level of circulating MSH, therelease of which is dependenton information received through the eyes. Blinded (or intact)lizards are brown under conditions of strong illumination andgreen under conditions of lower light intensities, and, again,these color changes are regulated by MSH. According to Kleinholz,color changes in the blinded lizard are regulated by dermalphotoreceptors. High or low temperatures directly affect thecolor of Anolis skins and alter the rate at which skins respondto hormones. Aggregationof melanin granules within Anolis melanophoresin response to sympathomimetic stimulation is regulated throughalpha adrenergic receptors whereas dispersion of melanin granulesin response to such stimulation is controlled through beta adrenergicreceptorspossessed by the melanophores. Most Anolis melanophores possessboth alpha and beta adrenergic receptors, but some melanophorespossess only beta adrenergic receptors. In the normal physiologyof the lizard, under conditions of stress, stimulation of alphaadrenergic receptors by catecholamines leads to an "excitement—pallor"followedby an "excitement—darkening" resulting from stimulationof beta adrenergic receptors which causes dispersion of melaningranules within localized populations of melanophores. Thus,in Anolis, dispersion of melanin granules within melanophoresis regulated by both MSH and by catecholamines. Evidence ispresented that the intracellular level of cyclic 3', 5'-AMPwithin melanophores may be responsible for the regulation ofmovement of melanin granules.     

Melanopsin photoreception in the eye regulates light‐induced skin colour changes through the production of α‐MSH in the pituitary gland

How skin colour adjusts to circadian light/dark cycles is poorly understood. Melanopsin (Opn4) is expressed in melanophores, where in vitro studies suggest it regulates skin pigmentation through a ‘primary colour response’ in which light photosensitivity is translated directly into pigment movement. However, the entrainment of the circadian rhythm is regulated by a population of melanopsin‐expressing retinal ganglion cells (mRGCs) in the eye. Therefore, in vivo, melanopsin may trigger a ‘secondary colour response’ initiated in the eye and controlled by the neuro‐endocrine system. We analysed the expression of opn4m and opn4x and melanin aggregation induced by light (background adaptation) in Xenopus laevis embryos. While opn4m and opn4x are expressed at early developmental times, light‐induced pigment aggregation requires the eye to become functional. Pharmacological inhibition of melanopsin suggests a model whereby mRGC activation lightens skin pigmentation via a secondary response involving negative regulation of alpha‐melanocyte‐stimulating hormone (α‐MSH) secretion by the pituitary.     

Cyclical training enhances the melanophore responses of zebrafish to background colours

Zebrafish respond to visual stimuli to adapt their body colour to the background. If, rather than being a simple on/off reaction to visual stimulation, the colour change involves cognitive and memory-related processes, training fish with cyclical changes of the background would be expected to increase its ability to change colour. To test this, we developed a standardized procedure for quantifying the responses of melanophores to background changes in living adult specimens of leopard, a zebrafish mutant with spotted stripes. After training with 2-day cyclical alternation of white and black backgrounds for over 20 days, the proportion of the melanosome-filled area of dorsal melanophores, which was 20% on the black background before the training, increased up to 97%. In these trained fish, a rapid melanosome aggregation occurred within 10 s of the background change from black to white. The results indicate that the zebrafish melanophore responses can be modulated by learning, in which areal and speed control of melanosome movement are important for dispersion and aggregation, respectively.     

Monoamine oxidase in the hypothalamo-hypophysial region of the teleosts,Anguilla japonica and Oryzias latipes

Summary Distribution of monoamine oxidase (MAO) was histochemically examined in the hypothalamo-hypophysial region of the eel (Anguilla japonica) and the medaka (Oryzias latipes) with a modified Glenner's tryptamine-tetrazolium method. The hypothalamic neurosecretory cells showed very weak MAO activity in their perikarya. MAO-positive fibers were present in close contact with the neurosecretory cells, suggesting that monoaminergic fibers participate in the control of neurosecretory cell activity. The nucleus lateralis tuberis (NLT) contained cells exhibiting strong MAO activity. These cells must be monoaminergic neurons.In the anterior region of the neurohypophysis of both eel and medaka, two bundles of MAO-positive fibers originating from the NLT proceed down along each side of the third ventricle into the pars distalis. This suggests that monoaminergic neurons of the NLT are involved in the release of hormones from the pars distalis. In addition to these tracts, numerous MAO-positive fibers proceed backward from the post-optic area and end around the blood capillaries located between the neurohypophysis and the pars intermedia in both species.I wish to express my gratitude to Prof. H. Kobayashi for his valuable advice during the course of this study. I am indebted to Prof. S. Uchida, Ocean Research Institute, University of Tokyo, for supplying the eels.     

Chromatophoromas in a pine snake

Iridophoroma and melanophoroma were diagnosed in an adult male pine snake. Light microscopic examination of irregularly thickened white and black portions of abnormal scales demonstrated two distinctive populations of pigment-containing cells. Pigment cells within abnormal-appearing white scales had needle-shaped granules that were dark amber in color while black portions were composed of pigment cells typical of melanophores, with dark black, round granules. Both populations of cells showed junctional activity, and clusters of both neoplastic pigment cell types were found in adjoining areas of the epidermis. By electron microscopy, the pigment cell with amber-colored granules contained reflecting platelet profiles typical of iridophores while pigment cells with dark round granules contained melanosomes. At a junctional area between abnormal white and black scales, mosaic chromatophores containing reflecting platelet profiles and melanosomes were observed. At 1 1/2 years following initial diagnosis, the snake died and neoplastic iridophores were found at multiple visceral sites; there was no evidence of metastases of melanophores to any organ. The two pigment cell tumors are believed to have developed from either stem cells destined to become iridophores and melanophores or from prexisting iridophores and melanophores in the dermis.     

Structural changes in the pars intermedia of the cichlid teleost Sarotherodon mossambicus as a result of background adaptation and illumination

Summary The pars intermedia of Sarotherodon mossambicus (Tilapia mossambica) contains two cell types which can be differentiated at both the light and electron microscopic level. The predominant cell type is lead haematoxyline positive, and has been shown to be the MSH producing cell type by means of immunocytochemical staining at the ultrastructural level. The changes in cellular and nuclear volume, as well as the results of stereological measurements on the cytoplasmic organelles, show that the activity of MSH cells is high on a black background and low on a white background or in total darkness. In blinded fish under a normal day-night regime the activity of the MSH cell is as high as that in black adapted fish, whereas the activity is low when the blinded fish are kept in total darkness. From the observed differences in activity of the MSH cells between the experimental groups, it is concluded that the MSH cells are not activated by the absence of reflected light, but by a high ratio between direct and reflected light. A second light-sensitive organ, supposedly the pineal gland, is also involved in the background response of the MSH producing cells.     

Catecholamine fluorescence in the pituitary of the eel,Anguilla anguilla,with special reference to its variation during background adaptation

Summary In the neuro-intermediate lobe (NIL) of the eel, Anguilla anguilla, a specific formaldehyde-induced fluorescence, indicating a catecholamine (CA) innervation, has been demonstrated in the neural lobe processes. Microspectrofluorimetric analyses and pharmacological treatments indicate noradrenaline or dopamine or both to be responsible for the fluorescence.The fluorescence in the NIL has displayed a definite tendency toward variation during the adaptation to a white and to a black background. The highest amounts of fluorescence were generally found in animals adapted to a black background, especially when adapted for a rather long period, and in animals recently transferred to a white background. The lowest amounts of fluorescence were generally found in animals adapted to a white background.This and the result of injections of CA-depleting drugs suggest that the monoaminergic nerves are active when the animal is on a white background, inhibiting the MSH release directly or indirectly or both, or in co-operation with other factors.Specific green fluorescent structures were also found in other parts of the neural lobe supplying the pars distalis.In some pharmacologically untreated specimens and in animals treated with CA-depleting drugs, the intermedia cells fluoresced. Microspectrofluorimetric analyses indicated that this fluorophore was not a CA.We wish to express our sincere thanks to Miss Ingrid Carlsen for excellent technical assistance, Mr. Lajos Erdös for the photography and the technical staff of the Department of Histology in Lund. We are also indepted to Dr. Anders Björklund for valuable discussion and advice.Supported by grants from the Swedish Natural Science Research Council, the University of Lund, and the Royal Physiographic Society of Lund.     

Localization of monoamines in the forebrain of two salmonid species,with special reference to the hypothalamo-hypophysial system

Summary In the salmon and trout aminergic cell bodies were found in the nucleus recessus lateraralis (NRL) and the nucleus recessus posterioris (NRP), both of which are situated near the third ventricle. Three cell types could be distinguished. Type 1 produces a green and type 2 a yellow fluorescence. The former type probably contains dopamine and the latter 5-hydroxytryptamine. Both types possess intraventricular protrusions in contact with the cerebrospinal fluid. The third cell type produces a less intense blue-green fluorescence; relatively few cells of this type have thick processes in contact with the ventricle. In addition, large fluorescent cells were found in the salmon, dorsal from the caudal part of the NRL. The various parts of the NRL and NRP are interconnected by thick bundles of nerve fibers; tracts leaving the nuclei could be traced for short distances only. The cells of the nucleus praeopticus (NPO), those of the medial part and to a much lesser extent also of the lateral part of the nucleus lateralis tuberis (NLT) have an aminergic innervation which probably originates from the NRL and/or NRP. All parts of the neurohypophysis contain many monoaminergic fibers, with aminergic material concentrated at the neuro-adenohypophysial interface. Fibers were not observed to penetrate the basal lamina. In the salmon and trout the fibers have a similar distribution, but differ in the intensity of fluorescence, being high in the salmon and low in the trout. Only in the trout have fluorescent cells been found in the adenohypophysis and very occasionally in the neurohypophysis. A number of these cells are basophilic and show a PAS-positive reaction.