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.     

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

Summary The MSH producing cells in the pars intermedia of Sarotherodon mossambicus have been shown to be involved in background adaptation processes. Reflected light received by the eyes affects the activity of these cells. In the present study the hypothesis has been tested that also the pineal organ, as a second photoreceptor, is involved in regulation of the metabolic activity of the MSH cells. The pineal organ appears to contain photoreceptor cells and is considered to be capable of transferring information about light conditions to the animal. Removal of the pineal organ of fish kept on a black background has no effect on activity of MSH cells, whereas the activity of these cells in fish kept in darkness is increased. Thus it seems that the pineal organ exercises its influence on MSH cells only in darkness and that this influence results in a reduced activity of these cells. It is therefore concluded that the metabolic activity of MSH cells is inhibited not only by reflected light received by the eyes, but also by the action of the pineal organ as a result of the absence of illumination.No structural signs of secretory activity can be observed in the pineal, which might indicate synthesis or release of substances like melatonin. However, administration of melatonin reduces the activity of MSH cells. Neither pinealectomy nor treatment with melatonin has any influence on the second cell type of the pars intermedia, the PAS positive cells.     

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.     

Interactions between ultraviolet light and interleukin-1 on MSH binding in both mouse melanoma and human squamous carcinoma cells

Interactions between beta-melanotropin (MSH), interleukin 1-a (IL-1), and ultraviolet light (UV) were examined in Cloudman S91 mouse melanoma and RHEK human squamous carcinoma cell lines. The following points were established: 1) both cell lines produced IL-1 and their production was stimulated by exposure of the cells to UV; 2) both cell lines possessed high affinity binding sites for MSH, and their ability to bind MSH was modulated by IL-1; 3) IL-1 exhibited both stimulatory and inhibitory effects on MSH binding to Cloudman cells; and 4) the stimulatory effect of IL-1 on MSH binding to melanoma cells was reflected in enhanced cellular responsiveness to MSH regarding tyrosinase activity (E.C. 1.14.18.1) and melanin content. The findings raise the possibility that interactions between keratinocytes and melanocytes may be regulated by IL-1 and MSH, and suggest a possible mechanism for stimulation of cutaneous melanogenesis by solar radiation: enhancement of MSH receptor activity by induction of IL-1.     

An immunocytochemical study of effects of light deprivation on prolactin cells in the adenohypophysis of the golden hamster.

Population ratio and morphology of prolactin cells were studied by employing immunohistochemical methods in the adenohypophysis of normal and experimental golden hamsters of both sexes at 16 weeks of age. Prolactin cells occupied 29% of the total adenohypophyseal cells in the intact males exposed to 14/10 h light/dark schedule. After stimulation of the pineal activity by binding or exposure of males to continuous darkness for eight weeks, prolactin cells became atrophic and were reduced in population to 17% and 13%, respectively. Pinealectomy prevented to some extent the effects of the above treatments; thus, prolactin cells constituted 27% in the pinealectomized and blinded hamsters, and 19% in the pinealectomized and darkness-treated group; and their morphology was comparable with that of the intact controls. Prolactin cells in the normal females were apparently larger in size and more numerous as compared with those of the normal males, comprising 47% of cell population in the anterior pituitary. In response to light deprivation, prolactin cells were atrophic with a diminished cytoplasm and decreased in cell number as reflected in the population ratio of 27% in the blinded and 21% in the darkness-treated groups. In pinealectomized females combined with binding or darkness-treatment, prolactin cells contained an abundance of secretory granules in the cytoplasm and maintained the population ratio comparable to that in the intact females. The present study revealed that light deprivation exerts an inhibitory effect on the secretory activity of prolactin cells and also causes hypoplasia of prolactin cells in the hamster adenohypophysis, the effects being mediated by the pineal gland.     

The effects of pinealectomy and blinding on the circadian locomotor activity rhythm in the Japanese newt,Cynops pyrrhogaster

We examined the effects of pinealectomy and blinding (bilateral ocular enucleation) on the circadian locomotor activity rhythm in the Japanese newt, Cynops pyrrhogaster. The pinealectomized newts were entrained to a light-dark cycle of 12 h light and 12 h darkness. After transfer to constant darkness they showed residual rhythmicity for at least several days which was gradually disrupted in prolonged constant darkness. Blinded newts were also entrained to a 12 h light/12 h dark cycle. In subsequent constant darkness they showed free-running rhythms of locomotor activity. However, the freerunning periods noticeably increased compared with those observed in the previous period of constant darkness before blinding. In blinded newts entrained to the light/dark cycle the activity rhythms were gradually disrupted after pinealectomy even in the presence of the light/dark cycle. These results suggest that both the pineal and the eyes are involved in the newt's circadian system, and also suggest that the pineal of the newt acts as an extraretinal photoreceptor which mediates the entrainment of the locomotor activity rhythm.Abbreviations circadian period - DD constant darkness - LD cycle, light-dark cycle - LD 12:12 light-dark cycle of 12 h light and 12 h darkness     

Responses of pineal photoreceptors in the brook and rainbow trout

Summary Electron microscopy of the pineal receptor cells in light- and dark-adapted brook trout, Salvelinus fontinalis and the rainbow trout, Salmo gairdneri, revealed no significant differences in the tubular and filamentous elements of the inner segment, neck and supranuclear regions. However, changes in synaptic relations between the photoreceptor and nerve cell were induced by light and darkness. In the light-adapted state, the synaptic relationship between axon terminals and photoreceptor basal processes predominates, while in darkness the synapses between photoreceptor basal processes and ganglion cell dendrites are more prominent. Further, in darkness, the photoreceptor basal processes show a number of synaptic vesicles and synaptic ribbons. These findings suggest that the sensory function of the fish pineal is enhanced during darkness but inhibited by light, and that the synaptic relationships are involved in the control of sensory activity in the pineal photoreceptor and ganglion cells. These results corroborate those of electrophysiological studies in that the maximal spontaneous discharge frequency of the ganglion cells occurs in the dark, and it also shows a burst when light is removed. The typical chemical synapse between the axon terminal and the photoreceptor basal process in light seems to function as an inhibitor.The authors thank Dr. Mary Ann Klyne for her assistance in several aspects of this work. Financial assistance was provided by the NSERC of Canada and the Ministry of Education of Québec     

Responses of the PAS-positive pars intermedia cells in the cichlid fish Sarotherodon mossambicus to ambient calcium and background adaptation

Summary The function of the PAS-positive pars intermedia cells in fish has been connected with control of background adaptation and of plasma calcium levels. Since background reflectivity and illumination influence calcium metabolism in S. mossambicus, we have tested the hypothesis that the effects of background reflectivity on the PAS-positive cells are mediated by changes in plasma calcium. However, total plasma calcium does not correlate with the activity of the PAS-positive cells as estimated by morphometrical criteria. Transfer of S. mossambicus to a white background leads to a drop in plasma calcium, and a marked reduction of the activity of the PAS-positive cells. Adaptation to low-calcium freshwater, on a neutral background, has the same effect on plasma calcium but has no effect on the PAS-positive cells. The characteristic structural features of highly active PAS-positive cells in fish from a black background are not due to the slight hypocalcemia that occurs in these fish, since addition of calcium to the water in concentrations that fully prevent the drop in plasma calcium does not suppress the PAS-positive cells. These findings make it very unlikely that these cells produce a hypercalcemie factor in S. mossambicus.     

Coho Salmon (Oncorhynchus kisutch) Prefer and Are Less Aggressive in Darker Environments

Fish are capable of excellent vision and can be profoundly influenced by the visual properties of their environment. Ambient colours have been found to affect growth, survival, aggression and reproduction, but the effect of background darkness (i.e., the darkness vs. lightness of the background) on preference and aggression has not been evaluated systematically. One-hundred Coho salmon (Oncorhynchus kisutch), a species that is increasing in popularity in aquaculture, were randomly assigned to 10 tanks. Using a Latin-square design, every tank was bisected to allow fish in each tank to choose between all the following colour choices (8 choices in total): black vs. white, light grey, dark grey, and a mixed dark grey/black pattern, as well as industry-standard blue vs. white, light grey, dark grey, and black. Fish showed a strong preference for black backgrounds over all other options (p < 0.01). Across tests, preference strength increased with background darkness (p < 0.0001). Moreover, having darker backgrounds in the environment resulted in less aggressive behaviour throughout the tank (p < 0.0001). These results provide the first evidence that darker tanks are preferred by and decrease aggression in salmonids, which points to the welfare benefits of housing farmed salmon in enclosures containing dark backgrounds.     

MSH receptors in immortalized human epidermal keratinocytes: A potential mechanism for coordinate regulation of the epidermal-melanin unit

Receptors for melanotropin (MSH) were found to be expressed by immortalized primary human epidermal keratinocytes (RHEK-1). Using ¹²⁵I-βMSH as a probe, the MSH receptors from mouse melanoma cells and human keratinocytes were found to be remarkably similar. In each cell line, there were high and low affinity receptors, with the high affinity classes showing positive cooperativity. Competition of ¹²⁵l-βMSH for binding with non-radioactive MSH revealed similar profiles. Cross-linking studies, followed by gel electrophoresis and autoradiography, showed almost identical gel migration patterns. Both cell types expressed internal as well as plasma membrane binding sites. MSH receptors on both cell types were up-regulated by ultraviolet light and by MSH itself. Although the function of MSH receptors expressed by the immortalized keratinocytes is unknown, the results are consistent with recent reports that proliferation of epidermal keratinocytes is stimulated by MSH and that proopiomelanocortin genes are expresed in the epidermis. These results support a model in which keratinocytes and melanocytes, interacting in an “epidermal-melanin unit,” each respond to UV light signals with increased MSH receptor activity. © 1993 Wiley-Liss, Inc.     

Structural studies of nerve terminals containing melanin-concentrating hormone in the eel,Anguilla anguilla

Summary Eels were adapted to black- or white-coloured backgrounds and the pituitary glands were prepared for light and electron microscopy. Immunocytochemical staining was used to study the distribution of the neurohypophysial melanin-concentrating hormone in the neurointermediate lobe. The hormone was located in small, elliptical, electron-opaque neurosecretory granules, measuring approximately 120×90 nm. The neurones terminated on blood vessels in the centre of the neurohypophysis and on the basement membrane separating neural and intermediate lobe tissues. The results of both light and electron immunocytochemistry and of radioimmunoassay are consistent with a higher rate of hormone release from eels adapted to white backgrounds than from those adapted to black backgrounds. In addition to this, when fish that had been adapted to white tanks were transferred to black tanks, there was an accumulation of irMCH in the gland and an increased numerical density of secretory granules at nerve terminals. These results reinforce the proposal that MCH is released during adaptation to a white background, to cause melanin concentration and to inhibit MSH release, and that its release is halted in black-adapted fish.     

Effects of Ultraviolet Irradiation on the Cell Cycle

Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20–30 mJ/cm² for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M “injury responses” of other cell types might be expected, consistent with their differentiated phenotypes.     

Two light‐activated neuroendocrine circuits arising in the eye trigger physiological and morphological pigmentation

Two biological processes regulate light‐induced skin colour change. A fast ‘physiological pigmentation change’ (i.e. circadian variations or camouflage) involves alterations in the distribution of pigment containing granules in the cytoplasm of chromatophores, while a slower ‘morphological pigmentation change’ (i.e. seasonal variations) entails changes in the number of pigment cells or pigment type. Although linked processes, the neuroendocrine coordination triggering each response remains largely obscure. By evaluating both events in Xenopus laevis embryos, we show that morphological pigmentation initiates by inhibiting the activity of the classical retinal ganglion cells. Morphological pigmentation is always accompanied by physiological pigmentation, and a melatonin receptor antagonist prevents both responses. Physiological pigmentation also initiates in the eye, but with repression of melanopsin‐expressing retinal ganglion cell activity that leads to secretion of alpha‐melanocyte‐stimulating hormone (α‐MSH). Our findings suggest a model in which eye photoperception links physiological and morphological pigmentation by altering α‐MSH and melatonin production, respectively.     

Hide, rest or die: a light-mediated diapause response in Daphnia magna to the threat of fish predation

1. In a laboratory batch culture experiment, a diapause response of Daphnia magna to a simulated threat of fish predation was tested at various light intensities, which under natural conditions determine potential vulnerability of Daphnia to visual planktivorous fish. 2. Under moderate light intensity (1.4 μmol m^?2 s^?1) that allows effective predation by fish, the proportion of females producing dormant eggs was significantly higher than under dim light conditions (0.001 μmol m^?2 s^?1) that are not favourable for visual detection of prey. Production of dormant eggs was not observed in complete darkness or in treatments missing fish kairomones, irrespective of tested light conditions. 3. The observed phenomenon is interpreted as a flexible response of prey to the conditional risk of predation assessed by Daphnia according to the presence of fish‐derived cues on the one hand and the presence of dark refugia on the other. Irrespective of the presence of fish kairomones, Daphnia may not produce resting eggs as long as a safe, dark, bottom zone is accessible.     

Influence of light and darkness on the ultrastructure of the pineal organ in the blind cave fish,Astyanax mexicanus

Ultrastructural changes of the pineal organ were investigated in the blind cave fish, Astyanax mexicanus, kept under continous artificial light (5000 lux), in continuous darkness, and under natural light conditions. The pineal end-vesicle of the fish kept under natural photoperiod consisted of photoreceptor cells and supporting cells mixed with a few ganglion cells. The photoreceptor cells possessed well-developed outer segments with regularly arranged lamellar membranes. The supporting cells contained a number of lipid droplets and large globular cisternae filled with fine granules. In the fish kept under continuous light or in darkness, the pineal end-vesicle displayed a dilated lumen, and the outer segments of the receptors showed signs of degeneration. Furthermore, alterations of cell organelles were observed in the photoreceptor and supporting cells.     

The development of a hypothalamic monoaminergic system for the regulation of the pars intermedia activity in Xenopus laevis

Summary In Xenopus laevis the development of hypothalamic monoaminergic cells was studied in relation to adaptation to background colour. The first melanophores appear at stage 33/34 (normal table of Nieuwkoop and Faber, 1956), gradually increasing in number. The melanine granules are dispersed throughout the cell, irrespective of the background colour. The dispersion apparently is caused by MSH released by the developing pars intermedia cells. Between stage 39 and stage 41, larvae placed on a white background changed colour from black to white due to aggregation of the melanine granules within the melanophores. With Falck's method for demonstrating monoamines, a small number of fluorescent cells was observed in the hypothalamus simultaneously with the first background-dependent colour change. These cells were arranged in a paired nucleus, bordering the third ventricle. Initially, the nucleus extends from 50 microns behind the optic chiasma to the lateral dilatations of the third ventricle; 8–10 hours later, similar cells were also found at the lateral dilatations and in the dorso-lateral part of the infundibular lobe. The cells have apical processes protruding in the ventricular lumen. Fluorescent axons, originating from the cells, were occasionally observed. Considering the above-mentioned results in combination with the electron microscopical data of Nyholm (1972), it is concluded that the MSH producing cells are under monoaminergic nervous control from the beginning of background colour adaptation.This paper was presented at the Fourth Joint Meeting of the Dutch and British Societies for Endocrinology (Terlou et al., 1973).The authors wish to thank Prof. Dr. P.G.W.J. van Oordt for his stimulating interest and helpful suggestions. The photographs were made by Mr. H. van Kooten and his co-workers.     

Studies on the structure,ultrastructure and function of the subcommissural organ-reissner's fibre complex of the European eel Anguilla anguilla L.

Summary The Subcommissural organ (SCO) of the European eel consists of tall columnar cells which secrete Reissner's fibre (RF) into the third ventricle and store colloid-like materials of differing electron densities in their hypendymal regions. The cells of the SCO carry large cilia and numerous microvilli.Function of the SCO-RF complex has been investigated by an in situ staining technique using aldehyde fuchsin and by the intracranial injection of ³⁵S-cysteine combined with autoradiography. Osmotic stimuli had no effect on stainability of the complex, though indications were obtained from the autoradiographic studies that it was rendered more active by transferring eels from sea water to fresh water. The effects of changes in background colouration and total illumination were investigated using the in situ technique and it was found that a change from a black background to a white one and from a black background to darkness appeared to stimulate the complex. It is shown that activity of the SCO-RF complex, unlike that of the hypothalamo-neurohypophysial system does not appear to be affected by stimuli that may be regarded as merely stressful.The findings are evaluated and discussed in the light of previous work and it is suggested that the use of radioactive tracers offers the best chance of success in establishing the function of the complex.     

A comparative study of the relationship between light intensity and feeding ability in brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus)

1. Field observations indicate that the ability to feed at different light intensities may differ between brown trout and Arctic charr, and this is the first study to test this experimentally. To establish a background level of feeding in daylight at midday, trout and charr in two size groups were kept in tanks (one fish per tank) at three constant temperatures (5.0, 10.8 and 13.0 °C) and each fish was offered, one at a time, 50 freshly killed shrimps (Gammarus pulex), the number eaten being recorded. Shrimps could only be taken in the water column because a metal mesh prevented access to dead shrimps on the tank bottom. In a first series of experiments, individual fish were kept at one of 10 natural light intensities (range 0.001–50 lx). In a second series, conditions were similar except that the fish tank was covered in black polyethylene and had a light‐tight lid with a shutter so that light levels could be kept constant, using artificial illumination. In a third series, the fish were fed in total darkness, but the false bottom was removed, allowing access to dead shrimps on the tank bottom as well as in the water column. 2. The results of the first and second series differed interspecifically but were very similar intraspecifically, with no significant differences between the food intake for the two size groups or in the experiments at 10.8 and 13.0 °C. Food intake remained fairly constant at light intensities between 50 lx (dusk or dawn) and 0.03 lx and was similar to that of fish feeding at midday. At 10.8 and 13.0 °C, food intake between 0.03 and 50 lx was higher for trout than for charr, mean values for shrimps eaten per fish being 39.9 for trout (range 36–44, n = 100 fish) and 32.0 for charr (range 28–38, n = 100), but at 5.0 °C, the situation was reversed with mean values of 15.1 for trout (range 11–18, n = 50 fish) and 19.8 for charr (range 17–22, n = 50). 3. As light intensity decreased from 0.04 to 0.001 lx, feeding rate decreased exponentially but was always higher for charr than for trout, with a mean number of shrimps eaten at 0.001 lx of 9.3 for trout (range 5–13, n = 20 fish) and 13.6 for charr (range 9–20, n = 20) at 10.8 and 13.0 °C, and 2.0 for trout (range 1–4, n = 10 fish) and 5.5 for charr (range 2–8, n = 10) at 5.0 °C. In total darkness (false bottom fitted), none of the 50 shrimps was taken by either species. When the false bottom was removed in the third series, the mean number of shrimps consumed over 24 h was eight for trout (range 3–11, n = 20 fish) and 14.9 for charr (range 9–20, n = 20) at 10.8 and 13.0 °C, and two for trout (range 0–4, n = 10 fish) and five for charr (range 3–8, n = 10) at 5.0 °C. 4. Therefore, the feeding ability of trout was superior to that of charr when using photopic vision in daylight and mesotopic vision at dusk and dawn, but inferior to that of charr when using scotopic vision at low light intensity. Charr were also superior at low temperatures and when foraging for food in total darkness. Therefore, as light intensity decreases after dusk in their natural habitat, the advantage in feeding will shift from trout to charr, with the reverse occurring as light intensity increases after dawn.