Immunocytochemical demonstration of retinal S-antigen in the pineal organ of four mammalian species

By means of immunocytochemistry retinal S-antigen is selectively demonstrated in retinal photoreceptor cells of the rat and in pinealocytes of the hedgehog, rat, gerbil and cat. Brain areas surrounding the pineal organ are immunonegative. The immunoreactive material is evenly distributed in the perikarya of the cells. Occasionally, inner segments of retinal photoreceptors and processes of pinealocytes are also stained. The outer segments of retinal photoreceptors display a strong immunoreaction. In both pinealocytes and retinal photoreceptors the intensity of the immunoreaction varied considerably among individual cells. The immunocytochemical demonstration of retinal S-antigen in mammalian pinealocytes indicates that these cells still bear characteristics of photoreceptors. This finding is in accord with the concept that mammalian pinealocytes are derived from pineal photoreceptor cells of poikilothermic vertebrates.     

Analysis of pore-rich areas on the nuclear envelope of spermatocytes

The S-antigen is a protein of photoreceptors, mainly known for its autoantigenic properties in mammals, which is widely distributed in the retina of vertebrates and in photoreceptor organs of invertebrates. Using three monoclonal antibodies specific for different epitopes of S-antigen, this study complements our previous data on retinal rods and cones and presents new results on the photosensory cells of the pineal complex. Immunoreactivity was found in (i) retinal rods and cones, (ii) cone-like and modified photoreceptor cells, and pinealocytes of the pineal organ of vertebrates, (iii) cone-like photoreceptors of the frontal organ of the frog and of the third eye of the lizard. According to the species and the antibody used, some differences were found at the level of the cellular compartments of the pineal photoreceptor cells.     

Opsin-like immunoreaction in the retinae and pineal organs of four mammalian species

Summary Opsin-like immunoreactivity was observed in the retinae and pineal organs of the mouse, rat and guinea pig, and the pineal organ of the cat. In the retina the immunoreaction was restricted to photoreceptor cells, which displayed immunostaining in their perikarya and outer and inner segments. Distinct pinealocytes endowed with characteristic processes were labelled in the pineal organs of the mouse and cat. However, in the cat the number of immunoreactive pinealocytes was very limited. In the pineal organs of the rat and guinea pig immunoreaction was very weak and diffuse. No immunoreaction was observed when the antibody was preabsorbed with purified bovine (rhod)opsin. These findings are in accord with the results of previous studies indicating molecular similarities between retinal photoreceptors and pinealocytes in mammals.Supported by grants from the Deutsche Forschungsgemeinschaft to HWK, the European Science Foundation to RGF, the Alexander-von-Humboldt Stiftung to PE, and the Dutch Foundation for the Advancement of Basic Research. The authors are greatly indebted to Dr. Willem de Grip, Nijmegen, and Profesor Andreas Oksche, Giessen, for their critical interest in this study     

Differentiation of pinopsin-immunoreactive cells in the developing quail pineal organ: an in-vivo and in-vitro immunohistochemical study

The avian pineal organ contains several types of photoreceptors with different photopigments: rhodopsin, iodopsin, and pinopsin. We have previously examined the differentiation of both rhodopsin-like and iodopsin-like immunoreactive cells during pineal development in quail embryos to determine the onset of synthesis of specific proteins and their cellular localization. In the present study, we have performed pinopsin immunohistochemistry on in-vivo developing and in-vitro cultured pineal organs of quail embryos. The results were compared with those obtained with rhodopsin and iodopsin immunohistochemistry. In the developing pineal organs, pinopsin immunoreactivity was detected at embryonic day 8, i.e. five days earlier than rhodopsin-like and iodopsin-like immunoreactivities. It was localized exclusively in the protrusions extending into the lumen throughout development, whereas rhodopsin-like and iodopsin-like immunoreactivities were usually found both in cell bodies and processes. These differences were also observed under two different types of culture conditions (dissociated cell culture and organ culture) indicating that, in the avian pineal organ, the expression pattern of the pinopsin gene is basically different from those of the other two pineal photopigments. The present study suggests that pineal cells have a mechanism for the polarized transport of pinopsin molecules.     

Pineal organ-like organization of the retina in megachiroptean bats

Phylogenetically originated from photoreceptive structures, the pineal organ adapts the organism to circadian and circannual light periodicity of the environment, while the retina develops to a light-based locator. Bats have a nocturnal life and an echolocator orientation presumably modifying the task of photoreception. Looking for morphological basis of the special functions, in the present work we compared the fine structure and immunocytochemistry of the retina and pineal organ in micro- and megacrochiroptean bats. We found that there is a high similarity between the retina and pineal organ in megachiropterans when compared to other species investigated so far. Besides of photoreceptor derived pinealocytes, the pineal organ of both micro- and megachiropterans contain intrapineal neurons and/or ganglionic cells as well as glial cells. Like spherules and pedicles of retinal photoreceptors, axon-type processes of pinealocytes form synaptic ribbon containig terminals. Similar to retinal photoreceptors and neurons, pinealocytes and pineal neurons contain immunoreactive glutamate and aspartate. In addition, excitatory amino acids accumulate in the pineal neurohormonal endings and might have a role in the hormonal (serotonin?) release of the organ. Concerning the structure of the retina the highest similarity to the organization of the pineal organ was found in the megachiroptean fruit eating bats Cynopterus sphinx and Rusettus niloticus. The retina of these species forms folds and crypts in its photoreceptor layer. This organization is similar to the folds of the pineal wall successively developed during evolution. Since a folded photoreceptor layer is not viable for a photolocator screen in decoding two-dimensional images, we suppose that this peculiar organization of the megachiropteran retina is connected to a "pineal-like" photometer task of the eye needed by these species active at night.     

Rod-type transducin alpha-subunit mediates a phototransduction pathway in the chicken pineal gland

The chicken pineal gland is a photosensitive neuroendocrine organ producing melatonin in circadian clock-regulated and light-sensitive manners. To understand the relationship between the photoreceptive molecule pinopsin and the light-dependent melatonin suppression that is sensitive to pertussis toxin treatment, we have searched for pertussis toxin-sensitive G protein alpha-subunits expressed in the chicken pineal gland. Here we report the cDNA cloning of the pineal transducin alpha-subunit (Gtalpha), which is highly homologous to human retinal rod cell-specific Gt(1)alpha. Concurrent cDNA cloning of chicken retinal Gt(1)alpha and Gt(2)alpha (rod and cone cell-specific alpha-subunits of transducin, respectively) revealed that the chicken pineal Gtalpha is identical to the retinal Gt(1)alpha. Double-immunostaining analysis of the chicken pineal sections localized Gt(1)alpha-immunoreactivity in the rudimentary outer segments of both follicular and parafollicular pinealocytes that were immunopositive to anti-pinopsin antibody. To examine whether pineal Gt(1)alpha is involved in the pineal phototransduction pathway, trypsin protection assay was applied for detecting the conversion of GDP-bound Gt(1)alpha into the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound form in the pineal membrane homogenate. It was clearly demonstrated that the pineal Gt(1)alpha is activated in a light-dependent manner in the presence of GTPgammaS. These data together suggest strongly that pineal Gt(1)alpha mediates the phototransduction pathway triggered by pinopsin in the chicken pinealocytes.     

Immunocytochemical demonstration of rod-opsin,S-antigen,and neuron-specific proteins in the human pineal gland

Summary The aim of this study was to examine whether rod-opsin and S-antigen immunoreactions were present in the pineal organ of adult man and how these immunoreactions were correlated with neuronal markers, e.g., synaptophysin, and neurofilaments L, H and M. Three perfusion-fixed epithalamic regions including the pineal organ and five pineal glands obtained at routine autopsy were used. The specimens were taken from female or male patients, 25 to 85 years of age. All immunoreactions were performed using highly specific, well-characterized antibodies. Rod-opsin and S-antigen-immunoreactive pinealocytes occurred in all pineal organs investigated; however, the immunoreaction was restricted to small subpopulations of pinealocytes (rod-opsin immunoreaction: approximately 3%–5%; S-antigen immunoreaction: approximately 5%–10% of the total population). In contrast, immunoreactions for synaptophysin and neurofilaments M and H were present in numerous pinealocytes. Immunoreactivity for neurofilament L was not found. These data suggest that the cellular composition of the human pineal organ is heterogeneous. Moreover, the presence of rod-opsin and S-antigen immunoreactions in the human pineal organ indicates that it may be affected by autoimmune retinal diseases that are provoked by antibodies against these proteins, as is the case in rodents and non-human primates.     

Cerebrospinal fluid-contacting neurons,sensory pinealocytes and Landolt's clubs of the retina as revealed by means of an electron-microscopic immunoreaction against opsin

Opsin-immunoreactive sites of hypothalamic cerebrospinal fluid (CSF)-contacting neurons, pinealocytes and retinal cells were studied in various vertebrates (Carassius auratus, Phoxinus phoxinus, Triturus cristatus, Bombina bombina, Rana esculenta) by means of postembedding immuno-electron microscopy with the use of the protein A-gold labeling method. The retina of the rat served as a general reference tissue for the quality of the immunocytochemical reaction. A strong opsin immunoreaction (rat-antibovine opsin serum) was obtained in the rod-type outer segments of photoreceptors in the retina of all species studied. Cone-type outer segments exhibited only very few antigenic binding sites. In the pineal organ of the goldfish and the frog, outer segments of the photoreceptor cells displayed strong immunoreactivity. No immunoreaction was found in hypothalamic CSF-contacting neurons and Landolt's clubs of nerve cells of the bipolar layer of the retina. The morphological similarity between the ciliated dendritic terminal of the Landolt's club and the intraventricular dendritic ending of the CSF-contacting neurons is emphasized.     

Nonvisual photoreceptors of the deep brain, pineal organs and retina

The role of the nonvisual photoreception is to synchronise periodic functions of living organisms to the environmental light periods in order to help survival of various species in different biotopes. In vertebrates, the so-called deep brain (septal and hypothalamic) photoreceptors, the pineal organs (pineal- and parapineal organs, frontal- and parietal eye) and the retina (of the "lateral" eye) are involved in the light-based entrain of endogenous circadian clocks present in various organs. In humans, photoperiodicity was studied in connection with sleep disturbances in shift work, seasonal depression, and in jet-lag of transmeridional travellers. In the present review, experimental and molecular aspects are discussed, focusing on the histological and histochemical basis of the function of nonvisual photoreceptors. We also offer a view about functional changes of these photoreceptors during pre- and postnatal development as well as about its possible evolution. Our scope in some points is different from the generally accepted views on the nonvisual photoreceptive systems. The deep brain photoreceptors are hypothalamic and septal nuclei of the periventricular cerebrospinal fluid (CSF)-contacting neuronal system. Already present in the lancelet and representing the most ancient type of vertebrate nerve cells ("protoneurons"), CSF-contacting neurons are sensory-type cells sitting in the wall of the brain ventricles that send a ciliated dendritic process into the CSF. Various opsins and other members of the phototransduction cascade have been demonstrated in telencephalic and hypothalamic groups of these neurons. In all species examined so far, deep brain photoreceptors play a role in the circadian and circannual regulation of periodic functions. Mainly called pineal "glands" in the last decades, the pineal organs actually represent a differentiated form of encephalic photoreceptors. Supposed to be intra- and extracranially outgrown groups of deep brain photoreceptors, pineal organs also contain neurons and glial elements. Extracranial pineal organs of submammalians are cone-dominated photoreceptors sensitive to different wavelengths of light, while intracranial pineal organs predominantly contain rod-like photoreceptor cells and thus scotopic light receptors. Vitamin B-based light-sensitive cryptochromes localized immunocytochemically in some pineal cells may take part in both the photoreception and the pacemaker function of the pineal organ. In spite of expressing phototransduction cascade molecules and forming outer segment-like cilia in some species, the mammalian pineal is considered by most of the authors as a light-insensitive organ. Expression of phototransduction cascade molecules, predominantly in young animals, is a photoreceptor-like characteristic of pinealocytes in higher vertebrates that may contribute to a light-percepting task in the perinatal entrainment of rhythmic functions. In adult mammals, adrenergic nerves--mediating daily fluctuation of sympathetic activity rather than retinal light information as generally supposed--may sustain circadian periodicity already entrained by light perinatally. Altogether three phases were supposed to exist in pineal entrainment of internal pacemakers: an embryological synchronization by light and in viviparous vertebrates by maternal effects (1); a light-based, postnatal entrainment (2); and in adults, a maintenance of periodicity by daily sympathetic rhythm of the hypothalamus. In addition to its visual function, the lateral eye retina performs a nonvisual task. Nonvisual retinal light perception primarily entrains genetically-determined periodicity, such as rod-cone dominance, EEG rhythms or retinomotor movements. It also influences the suprachiasmatic nucleus, the primary pacemaker of the brain. As neither rods nor cones seem to represent the nonvisual retinal photoreceptors, the presence of additional photoreceptors has been supposed. Cryptochrome 1, a photosensitive molecule identified in retinal nerve cells and in a subpopulation of retinal photoreceptors, is a good candidate for the nonvisual photoreceptor molecule as well as for a member of pacemaker molecules in the retina. When comparing various visual and nonvisual photoreceptors, transitory, "semi visual" (directional) light-perceptive cells can be detected among them, such as those in the parietal eye of reptiles. Measuring diffuse light intensity of the environment, semivisual photoreceptors also possess some directional light perceptive capacity aided by complementary lens-like structures, and screening pigment cells. Semivisual photoreception in aquatic animals may serve for identifying environmental areas of suitable illumination, or in poikilotermic terrestrial species for measuring direct solar irradiation for thermoregulation. As directional photoreceptors were identified among nonvisual light perceptive cells in the lancelet, but eyes are lacking, an early appearance of semivisual function, prior to a visual one (nonvisual --> semivisual --> visual?) in the vertebrate evolution was supposed.     

Immunocytochemical and electron-microscopic investigations of the pineal organ in adult agamid lizards,Uromastix hardwicki

Summary Lacertilian species display a remarkable diversity in the organization of the neural apparatus of their pineal organ (epiphysis cerebri). The occurrence of immunoreactive S-antigen and opsin was investigated in the retina and pineal organ of adult lizards, Uromastix hardwicki. In this species, numerous retinal photoreceptors displayed S-antigen-like immunoreactivity, whereas only very few pinealocytes were labeled. Immunoreactive opsin was found neither in retinal photoreceptors nor in pinealocytes. Electron microscopy showed that all pinealocytes of Uromastix hardwicki resemble modified pineal photoreceptors. A peculiar observation is the existence of a previously undescribed membrane system in the inner segments of these cells. It is evidently derived from the rough endoplasmic reticulum but consists of smooth membranes. The modified pineal photoreceptor cells of Uromastix hardwicki were never seen to establish synaptic contacts with somata or dendrites of intrapineal neurons, which are extremely rare. Vesiclecrowned ribbons are prominent in the basal processes of the receptor cells, facing the basal lamina or establishing receptor-receptor and receptor-interstitial type synaptoid contacts. Dense-core granules (60–250 nm in diameter) speak in favor of a secretory activity of the pinealocytes. Attention is drawn to the existence of receptor-receptor and receptor-interstitial cell contacts indicating intramural cellular relationships that deserve further study.Supported by the Deutsche Forschungsgemeinschaft (Ko 758/31) and the Deutscher Akademischer Austauschdienst (Senior DAAD Research Fellowship to M.A.H.)     

Rhythm and soul in the avian pineal

The avian pineal gland, like that of mammals, displays a striking circadian rhythm in the synthesis and release of the hormone melatonin. However, the pineal gland plays a more prominent role in avian circadian organization and differs from that in mammals in several ways. One important difference is that the pineal gland in birds is relatively autonomous. In addition to making melatonin, the avian pineal contains photoreceptors and a circadian clock (thus, an entire circadian system) within itself. Furthermore, avian pineals retain their circadian properties in organ or dispersed cell culture, making biochemical components of regulatory pathways accessible. Avian pinealocytes are directly photosensitive, and novel candidates for the unidentified photopigments involved in the regulation of clock function and melatonin production, including melanopsin, pinopsin, iodopsin, and the cryptochromes, are being evaluated. Transduction pathways and second messengers that may be involved in acute and entraining effects, including cyclic nucleotides, calcium fluxes, and protein kinases, have been, and continue to be, examined. Moreover, several clock genes similar to those found in Drosophila and mouse are expressed, and their dynamics and interactions are being studied. Finally, the bases for acute and clock regulation of the key enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT), are described. The ability to study entrainment, the oscillator itself, and a physiological output in the same tissue at the same time makes the avian pineal gland an excellent model to study the bases and regulation of circadian rhythms.     

Pinopsin expressed in the retinal photoreceptors of a diurnal gecko

Retinal cDNAs encoding the putative opsins, dg3 and dg4, were isolated from a diurnal gecko, Phelsuma madagascariensis longinsulae. dg3 mRNA is localized in about 20% of the thin members of type C double cones, and likely encodes an opsin of the ultraviolet-sensitive pigment. Surprisingly, dg4 is very similar to chicken pinopsin, a pineal-specific photoreceptive molecule. An anti-dg4 antiserum recognized a small population of photoreceptor outer segments in the retina and a large number of pinealocytes. Our results suggest that P. m. longinsulae expresses pinopsin in its retina, which usually plays a role as a photoreceptive molecule in the pineal organ.     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Rod-opsin immunoreaction in the pineal organ of the pigmented mouse does not indicate the presence of a functional photopigment

The aim of the present study was to characterize the rod-opsin immunoreaction in the mammalian pineal organ. Pigmented mice (strain C57BL) were selected as the animal model. Immunocytochemical investigations involving the use of highly specific polyclonal and monoclonal antibodies against bovine rod-opsin (the apoprotein of the photopigment rhodopsin) showed that approximately 25% of all pinealocytes were rod-opsin immunoreactive. Immunoblotting techniques revealed three protein bands of approximately 40, 75, and 110 kDa; these were detected by the monoclonal antibody and the polyclonal antiserum in retinal and pineal extracts. These protein bands presumably represented the monomeric, dimeric and trimeric forms of rod-opsin. The amount of rod-opsin in retina and pineal organ was quantified by means of an enzyme-linked immunosorbent assay. This yielded 570±30 pmoles rod-opsin per eye and 0.3±0.05 pmoles rod-opsin per pineal organ. High pressure liquid chromatography analysis of whole eye extracts demonstrated the chromophoric group of the photopigment rhodopsin, 11-cis retinal, and its isomer, all-trans-retinal. A shift from 11-cis retinal to all-trans-retinal was found upon light adaptation. No retinals were detected in the pineal organ. Autoradiographic investigations showed that ³H-retinol, intraperitoneally injected into the animals, was incorporated into the outer and inner segments of retinal photoreceptors, but not into the pineal organ. It is concluded that the mouse pineal organ contains the authentic apoprotein of rhodopsin but that it lacks retinal derivatives as essential components of all known vertebrate photopigments. Consequently, the photoreceptor-specific proteins of the mammalian pineal organ are not involved in photoreception and phototransduction, but may serve other functions to be explored in future studies.     

Immunocytochemical localization of serotonin and photoreceptor-specific proteins (rod-opsin,S-antigen) in the pineal complex of the river lamprey,Lampetra japonica,with special reference to photoneuroendocrine cells

Summary The pineal complex of the river lamprey, Lampetra japonica, was examined by means of immunocytochemistry with antisera against serotonin, the precursor of melatonin, and two photoreceptor proteins, rod-opsin (the apoprotein of the photopigment rhodopsin) and S-antigen. Serotonin-immunoreactive cells were observed in both the pineal and the parapineal organ. The proximal portion of the pineal organ (atrium) comprised numerous serotonin-immunoreactive cells displaying spherical somata. In the distal end-vesicle of the pineal organ, the serotonin-immunoreactive elements resembled photoreceptors in their size and shape. These cells projecting into the pineal lumen and toward the basal lamina were especially conspicuous in the ventral portion of the end-vesicle. In addition, single serotonin-immunoreactive nerve cells were found in this location. Retinal photoreceptors were never seen to contain immunoreactive serotonin; amacrine cells were the only retinal elements exhibiting serotonin immunoreaction. Strong S-antigen immunoreactivity was found in numerous photoreceptors located in the pineal end-vesicle. In contrast, the S-antigen immunoreactivity was weak in the spherical cells of the atrium. Thus, the pattern of S-antigen immunoreactivity was roughly opposite to that of serotonin. Similar findings were obtained in the parapineal organ. The rod-opsin immunoreaction was restricted to the outer segments of photoreceptors in the pineal end-vesicle and parapineal organ. No rodopsin immunoreactive outer segments occurred in the proximal portion of the atrium. Double immunostaining was employed to investigate whether immunoreactive opsin and serotonin are colocalized in one and the same cell. This approach revealed that (i) most of the rodopsin-immunoreactive outer segments in the end-vesicle belonged to serotonin-immunonegative photoreceptors; (ii) nearly all serotonin-immunoreactive cells in the end-vesicle bore short rod-opsin-immunoreactive outer segments protruding into the pineal lumen; and (iii) the spherical serotonin-immunoreactive cells in the pineal stalk lacked rod-opsin immunoreaction and an outer segment. These results support the concept that multiple cell lines of the photoreceptor type exist in the pineal complex at an early evolutionary stage.     

Immunocytochemical localization of vitamin A in the retina and pineal organ of the frog, Rana esculenta

Vitamin A immunoreactive sites were studied in the retina and pineal organ of the frog, Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. In dark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. In light-adapted retina, cones and green (blue-sensitive) rods were immunopositive. At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photorecptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments. The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.     

Immunocytochemical identification of photoreceptor proteins in hypothalamic cerebrospinal fluid-contacting neurons of the larval lamprey (Petromyzon marinus)

Antibodies directed against different visual pigment opsins, and an antibody raised against the C terminal of the -subunit of retinal G protein (transducin) labelled cerebrospinal fluid-contacting cells located within the hypothalamus (postoptic commissural nucleus and ventral hypothalamic nucleus) of ammocoete lampreys (Petromyzon marinus). These antibodies also labelled photoreceptor cells within the retina and the pineal and parapineal organs, but no other areas of the brain. Despite considerable behavioural and physiological evidence for the existence of deep brain photoreceptors, numerous studies have failed to identify photoreceptor proteins within the basal brain. The results presented in this paper support our recent results in the lizard Anolis carolinensis, suggesting that a group of cerebrospinal fluid-contacting neurons within the vertebrate brain have a photosensory capacity. We speculate that these cells mediate extraocular and extrapineal photoreception in nonmammalian vertebrates.     

Pinealocytes immunoreactive with antisera against secretory glycoproteins of the subcommissural organ: A comparative study

Summary By means of light-microscopic immunocyto-chemistry two polyclonal antibodies (AFRU, ASO; see p. 470) directed against secretory glycoproteins of the subcom-missural organ were shown to cross-react with cells in the pineal organ of lamprey larvae, coho salmon, a toad, two species of lizards, domestic fowl, albino rat and bovine (taxonomic details, see below). The AFRU-immunoreactive cells were identified as pinealocytes of the receptor line (pineal photoreceptors, modified photoreceptors or classical pinealocytes, respectively) either due to their characteristic structural features or by combining AFRU-immunoreaction with S-antigen and opsin immunocytochemistry in the same or adjacent sections. Depending on the species, AFRU- or ASO-immunoreactions were found in the entire perikaryon, inner segments, perinuclear area, and in basal processes facing capillaries or the basal lamina. In most cases, only certain populations of pinealocytes were immunolabeled; these cells were arranged in a peculiar topographical pattern. In lamprey larvae, immunoreactive pinealocytes were observed only in the pineal organ, but not in the parapineal organ. In coho salmon, the immunoreaction occurred in S-antigen-positive pinealocytes of the pineal end-vesicle, but was absent from S-antigen-immunoreactive pinealocytes of the stalk region. In the rat, AFRU-immunoreaction was restricted to S-antigen-immunoreactive pinealocytes found in the deep portion of the pineal organ and the habenular region. These findings support the concept that several types of pinealocytes exist, which differ in their molecular, biochemical and functional features. They also indicate the possibility that the AFRU- and ASO-immunoreactive material found in certain pinealocytes might represent a proteinaceous or peptidic compound, which is synthesized and released from a specialized type of pinealocyte in a hormone-like fashion. This cell type may share functional characteristics with peptidergic neurons or paraneurons.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, to E.M.R. and A.O.; Grant S-85-39 from the Direccion de Investigaciones, Universidad Austral de Chile, to E.M.R.; Grant 187 from FONDECYT, Chile, to C.R.Y.; and Grant Ko 758/3-1 from the Deutsche Forschungsgemeinschaft, Federal Republic of Germany, to H.W.K.     

Vertebrate ancient opsin photopigment spectra and the avian photoperiodic response

In mammals, photoreception is restricted to cones, rods and a subset of retinal ganglion cells. By contrast, non-mammalian vertebrates possess many extraocular photoreceptors but in many cases the role of these photoreceptors and their underlying photopigments is unknown. In birds, deep brain photoreceptors have been shown to sense photic changes in daylength (photoperiod) and mediate seasonal reproduction. Nonetheless, the specific identity of the opsin photopigment 'sensor' involved has remained elusive. Previously, we showed that vertebrate ancient (VA) opsin is expressed in avian hypothalamic neurons and forms a photosensitive molecule. However, a direct functional link between VA opsin and the regulation of seasonal biology was absent. Here, we report the in vivo and in vitro absorption spectra (λ(max) = ~490 nm) for chicken VA photopigments. Furthermore, the spectral sensitivity of these photopigments match the peak absorbance of the avian photoperiodic response (λ(max) = 492 nm) and permits maximum photon capture within the restricted light environment of the hypothalamus. Such a correspondence argues strongly that VA opsin plays a key role in regulating seasonal reproduction in birds.     

Immunocytochemical localization of Vitamin A in the retina and pineal organ of the frog,Rana esculenta

Summary Vitamin A immunoreactive sites were studied in the retina and pincal organ of the frog,Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. Indark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. Inlight-adapted retina, cones and green (blue-sensitive) rods were immunopositive.At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photoreceptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments.The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Hungarian OTKA grant Nr. 1619 to B.V., and a grant from the Pardee Foundation to G.H.W.