The outer segments of photoreceptive pinealocytes in the pineal organ of the funa,Carassius gibelio langsdorfi

Summary The form and size of the outer segments of photoreceptive pinealocytes in the pineal organ of the funa, Carassius gibelio langsdorfi, were observed with the scanning electron microscope. The height of the outer segments measures between 1 and 3 m and the diameter varies widely from 1.5 to 8 m. Various forms of outer segments, i.e. a slender type, a dome-like type, a cap-like type and a helical type, were demonstrated. The parallel-oriented filamentous processes of the inner segments have the same length as the outer segments and a diameter of approximately 100 nm; they are projections from the apical border of the inner segment and surround the cone-like outer segments. The processes make a right angle with the lamellar disks. The distance between two processes averages 100 nm. The lamellar disks of the outer segments are oriented at right angles to the modified cilium in the basal part, but the angle often changes in the peripheral part, where the lamellar disks are raised and become parallel to the cilium.Supported by a fellowship from the Japan Society for the Promotion of Science and a grant from the Deutsche Forschungsgemeinschaft to M. UeckSupported by a grant from the Ministry of Education of Japan to K.Wake     

The pineal organ of Raja clavata: Opsin immunoreactivity and ultrastructure

Summary The pineal organ of Raja clavata was studied by light and electron microscopy, including the immunocytochemical antiopsin reaction. The pineal organ of the ray consists of three portions: (i) a large proximal pineal, (ii) a long tube-like connecting stalk, and (iii) a short distal terminal enlargement. This latter end-vesicle lies in the deep connective tissue layers of the braincase. All portions of the pineal are composed of pinealocytes, intrinsic neurons, ependymal/glial cells, and bundles of nerve fibers embedded in thin neuropil formations. The inner segments of the pinealocytes protrude into the lumen in all parts of the organ and usually contain basal bodies and numerous mitochondria. Often, two outer segments were found to arise from the basal bodies of a single inner segment. By means of light-microscopic immunocytochemistry the outer segments showed a strong antiopsin reaction.The axons of the pinealocytes form ribbon-containing synapses on dendritelike profiles, which appear to belong to the intrinsic pineal neurons. There are other axo-dendritic synapses established by presynaptic terminals lacking ribbons and containing granular and synaptic vesicles. Pineal neurons may contain granular vesicles approximately 60–100 nm in diameter; their processes contribute to the bundles of unmyelinated axons.The fine structural organization of the pineal organ and the opsin immunoreactivity of the outer segments of the pinealocytes indicate a photoreceptive capacity of the organ. The double outer segments represent a peculiar multiplication of the photoreceptor structures.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft to A. Oksche (Ok 1/24; 1/25: Mechanismen biologischer Uhren)On leave from the 2nd Department of Anatomy, Semmelweis OTE, Budapest, Hungary     

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.     

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.)     

Electron microscopic and experimental studies of the pineal organ in the white-crowned sparrow,Zonotrichia leucophrys gambelii

Summary The structure of the pineal organ of Zonotrichia leucophrys gambelii, as revealed by light- and electron-microscopy, resembles that of Passer domesticus (Oksche and Kirschstein, 1969; Ueck, 1970). The typical cellular element is the pinealocyte with certain basic structural features of the pineal photoreceptors of lower vertebrates (see Oksche, 1971). However, instead of the characteristic, cone-like outer segments, there are, as in other species of birds, only bulbous cilia with ectopic whorls of lamellae. This structure of the outer segment is, in a sense, contrary to the demonstration of synaptoid contacts, numerous unmyelinated, and occasional myelinated nerve fibers by electron microscopy. In Nissl preparations it was possible to demonstrate typical nerve cells. The pinealocytes of Z. l. gambelii are secretory; their Golgi complex forms granulated vesicles (800–1,400 Å in diameter) that belong to the group of granular inclusions characteristic of monoamines. Autonomie nerve fibers course within the connective tissue capsule of the pineal organ. In many pinealocytes of Z. l. gambelii, the granular endoplasmic reticulum contains extensively expanded cisternae that are filled with a flocculent material and closely associated with bundles of filaments. In a number of cases such loop-like structures are selectively stainable with aldehyde fuchsin. It was not possible to demonstrate specific secretory activity in the supporting cells. Extirpation of the pineal organ in Z. l. gambelii had no definitely detectable influence on the photoperiodic control of testicular growth.Aves, Passeriformes, Fringillidae.Supported by grants from the Deutsche Forschungsgemeinschaft to Professor Oksche and by the National Science Foundation (GB 11905) to Professor Farner. A part of this investigation was effected while Professor Kobayashi held a Visiting Professorship at the University of Giessen.     

Sensory cells of the “rod-” and “cone-type” in the pineal organ of Rana esculenta,as revealed by immunoreaction against opsin and by the presence of an oil (lipid) droplet

Summary The pineal organ of the frog, Rana esculenta, was studied by use of light- and electron-microscopic methods including immunoreaction against opsin. Most of the morphologically classified cone-type outer segments of the pineal photoreceptors reacted with antisera against opsin of the bovine retina that is dominated by rods. Some of the outer segments of pineal photoreceptor cells remained unstained in accord with the reference tissue, the frog retina, where generally the rods were opsin-positive and most of the cones opsin-negative.The opsin-negative outer segments of pineal photoreceptors were found in continuity with inner segments each containing a large oil (lipid) droplet. These oil droplets stained intensely with osmic acid, Sudan III, Sudan Black B or Scharlach R in cryostat sections, and were soluble in lipid solvents. In ultrathin sections of osmicated material, the oil droplets were homogeneous and of varying electron density. Approximately one tenth of the pineal photoreceptors contained oil droplets and at the same time possessed opsin-immunonegative outer segments.Since in the retina oil droplets and a negative immunoreaction against bovine opsin are characteristic of cones, we suggest that in the pineal organ they also mark conetype photoreceptors scattered among rod-type photo-receptors, the latter displaying a positive immunoreaction with the antisera used.Support from the Deutsche Forschungsgemeinschaft (Ok 1/25-3) is gratefully acknowledged     

The pineal organ is the first differentiated light receptor in the embryonic salmon,Salmo salar L.

Summary The initial appearance of S-antigen, -transducin, opsin and 5-HT during embryogenesis of the pineal organ and retina was studied by means of immunocytochemistry in the Atlantic salmon, Salmo salar L. The presence of these substances may be taken as a good indication of photoreceptor differentiation; -transducin and S-antigen are involved in the phototransduction process, opsin is the proteinaceous component of the photopigment rhodopsin, and 5-HT is a neurotransmitter or neurohormone produced by pineal photoreceptors. Two days after the retinal pigment layer became visible in the eggs, the outer segments of a few pineal photosensory cells showed immunoreactivity to opsin and -transducin. At the same time S-antigen and serotonin were present in pineal cells of the photoreceptor type. The number of immunoreactive cells in the pineal organ increased up to hatching. In the differentiating retina of the salmon, no immunoreactivity to antibodies raised against the mentioned substances was detectable until after hatching. These results indicate that in ontogeny the developing pineal organ of the salmon embryo has the ability to perceive light information much earlier than the retina.A preliminary account of this work was presented at the Tenth European Neuroscience Congress, Marseille, France, September 14–18, 1986     

Complex relationships between the pineal organ and the medial habenular nucleus-pretectal region of the mouse as revealed by S-antigen immunocytochemistry

Summary S-antigen-immunoreactive pinealocytes located in the deep portion of the pineal organ of inbred and wild pigmented mice give rise to long, beaded processes penetrating into the habenular and pretectal regions. In addition, the medial habenular nuclei and the pretectal area contain S-antigen-immunoreactive perikarya, which resemble pinealocytes in size, shape and immunoreactivity and are considered as pinealocyte-like epithalamic cells. Immunoblotting techniques reveal that a single protein band of approximately 48 kDa molecular weight accounts for this immunoreactivity. As shown with the use of the electron microscope, the majority of the S-antigen-immunoreactive processes is closely apposed to immunonegative neuronal profiles and perikarya of the habenular and pretectal regions. S-antigen-immunoreactive processes and perikarya of both pinealocytes of the deep pineal organ and pinealocyte-like epithalamic cells may form the postsynaptic element in conventional synapses involving axons provided with clear synaptic vesicles. Thus, certain mammalian pinealocytes may receive and transmit signals via point-to-point connections resembling neuro-neuronal contacts. These results challenge the concept that the mammalian pineal organ exerts its influence exclusively via the release of melatonin into the general circulation. Furthermore, they provide evidence (i) that neuronal circuits not involving the sympathetic system participate in the regulation of pineal functions in mammals, and (ii) that intimate histogenetic and functional relationships exist between the pineal organ and the habenular-pretectal nuclei in mammals.     

Ultrastructure of serotonin-containing cells in the pineal organ of Lampetra planeri (Petromyzontidae)

Summary The ultrastructure of the cells containing residual bodies (Collin, 1969) was investigated in the pineal organ of Lampetra planeri. These cells are characterized by their indoleamine metabolism (Meiniel, 1978; Meiniel and Hartwig, 1980). Morphologically, they belong mainly to two types: (1) a photoreceptor cell type, and (2) a pinealocyte cell type. The first type is present in the pineal sensory epithelium and in the atrium, while the second is observed in the deep part of the atrium. Intermediate cell types are rare. All these cells are characterized by the presence of voluminous dense bodies, the 5-HT-storing structures, in their cytoplasm.The elongated cone-type photoreceptor cells show a segmental organization and well-developed outer segments consisting of short disks (2–3 m), while their basal pedicles form synapses with the dendritic processes of neurons. The pinealocytes are spherical or oval in shape, their receptor poles being regressed to cilia of the 9+0 type. In these cells, no synaptic ribbons have to date been observed. In both cell types a Golgi apparatus is present producing dense granules 130 nm in diameter and a polymorphous dense material.The photoreceptor cells most probably respond to light and transmit a sensory (i.e., nervous) message. In addition, they produce and metabolize indoleamines, probably including, melatonin (Meiniel, 1978; Meiniel and Hartwig, 1980). The pinealocytes, in spite of their loss of direct photosensitivity, retain their capacity to metabolize indoleamines (Meiniel, 1978; Meiniel and Hartwig, 1980).The presence, in the same pineal organ, of another photoreceptor cell type (cf. Collin, 1969–1971) differing morphologically as well as biochemically (no detectable indoleamine metabolism) from the photoreceptor cell type described in the present investigation, points to the existence of two different sensory cell lines: (1) a pure photoreceptor line, and (2) a photoneuroendocrine line. The phylogenetic evolution of these two cell lines is discussed in terms of functional analogy.     

Weitere elektronenmikroskopische Untersuchungen am Pinealorgan vonPhoxinus laevis (Teleostei,Cyprinidae)

Zusammenfassung Auch im Pinealorgan adulter Elritzen (Phoxinus laevis) sind die Außenglieder der Sinneszellen nicht degeneriert. Der Vergleich wurde zwischen adulten Exemplaren (8,6 cm) und 19,5 Monate jüngeren Tieren (3 cm) durchgeführt. Im Verhältnis zu den Jungtieren sind die Neuropilformationen und die synaptischen Strukturen adulter Elritzen stärker ausgeprägt. Bei den letzteren enthalten die Endfüße der pinealen Rezeptoren zahlreiche synaptische Bänder und 300–400 Å große Bläschen. Die Neuropilzonen sind außerdem reich an verdichteten Membranstellen (tight junctions). Im Pinealorgan des adulten Phoxinus finden sich eigenartige Einschlußkörper, die aus konzentrischen Lamellen bestehen; ähnliche Membran wirbel hat Takahashi (1969) bei zweijährigen Goldfischen beschrieben. Häufig lassen sich in den Epiphysen adulter Elritzen Axone beobachten, die mit 500–1000 Å großen granulierten Vesikeln gefüllt sind und im Verband bahnartig zusammengefaßter markloser Nervenfasern verlaufen. Die Herkunft und Bedeutung dieser granulierten Fasern werden diskutiert, ebenso wie die funktionelle Rolle des pinealen Sinnesorgans.

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Further electron microscopic studies of the pineal organ inPhoxinus laevis (Teleostei, Cyprinidae)

Summary The outer segments of pineal receptor cells of adult (8.6 cm) European minnows (Phoxinus laevis), like the lamellated outer segments of 19.5 months younger (3 cm) specimens, did not show signs of degeneration (see also Oksche and Kirschstein, 1967). Neuropile formations and synaptic structures were more prominent in adult than in youngPhoxinus. In the adult group, the end-feet of pineal receptors contained numerous synaptic ribbons and vesicles (300–400 Å in diameter). The neuropile zones were rich in tight junctions. A peculiar structure of the pineal organ of agingPhoxinus was the inclusion body formed by concentric arrangement of lamellae. Membrane whorls similar to these inclusion bodies were described in the pineal organ of two years old goldfish (Takahashi, 1969). In the pineal organ of adultPhoxinus considerable numbers of axons containing dense-core vesicles (500–1,000 Å in diameter) were observed within tract-like bundles of unmyelinated nerve fibers. The origin and significance of these granulated fibers and the functional role of the pineal sense organ have been discussed.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

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.     

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.     

Evolution of photosensory pineal organs in new light: the fate of neuroendocrine photoreceptors

Pineal evolution is envisaged as a gradual transformation of pinealocytes (a gradual regression of pinealocyte sensory capacity within a particular cell line), the so-called sensory cell line of the pineal organ. In most non-mammals the pineal organ is a directly photosensory organ, while the pineal organ of mammals (epiphysis cerebri) is a non-sensory neuroendocrine organ under photoperiod control. The phylogenetic transformation of the pineal organ is reflected in the morphology and physiology of the main parenchymal cell type, the pinealocyte. In anamniotes, pinealocytes with retinal cone photoreceptor-like characteristics predominate, whereas in sauropsids so-called rudimentary photoreceptors predominate. These have well-developed secretory characteristics, and have been interpreted as intermediaries between the anamniote pineal photoreceptors and the mammalian non-sensory pinealocytes. We have re-examined the original studies on which the gradual transformation hypothesis of pineal evolution is based, and found that the evidence for this model of pineal evolution is ambiguous. In the light of recent advances in the understanding of neural development mechanisms, we propose a new hypothesis of pineal evolution, in which the old notion 'gradual regression within the sensory cell line' should be replaced with 'changes in fate restriction within the neural lineage of the pineal field'.     

Characteristic pattern of monoaminergic nerve fibers in the pineal organ of the monkey,Macaca fuscata

Summary Monoaminergic nerve fibers were studied in the pineal organ of the monkey, Macaca fuscata, by use of fluorescence and immunohistochemical procedures. Abundant formations of noradrenergic nerve fibers were observed in the pineal organ. They entered the parenchyma in the form of several coarse bundles via the capsule in the distal portion of the organ and spread throughout the organ after branching into smaller units. The density of the autonomic innervation decreased gradually toward the proximal portion of the organ. In the distal portion, numerous nerve fibers formed perivascular plexuses around the blood vessels and some fibers ran as bundles unrelated to the blood vessels in the stroma. Fine varicose fibers and bundles derived from these plexuses penetrated among the pinealocytes. However, only a few intraparenchymal fluorescent fibers were detected in the proximal third of the gland. With the use of serotonin antiserum serotonin-immunoreactive nerve fibers were clearly restricted to the ventroproximal part of the pineal organ. Although the somata of the pinealocytes showed intense immunoreactivity, their processes were not stained. In one exceptional case, clusters of pinealocytes displaying very intense immunoreactivity were found in an area extending from the distal margin of the ventral portion of the pineal stalk to the proximal portion of the pineal organ proper; these cells were bipolar or multipolar and endowed with well-stained processes.     

Pineal neurons projecting to the brain of the rainbow trout,Salmo gairdneri Richardson (Teleostei)

Summary The morphology of intrapineal neurons that give rise to the pineal tract and project to the brain in the rainbow trout was visualized by the use of neuronal backfilling with horseradish peroxidase (HRP). The tracing was performed on excised pineal organs under in-vitro conditions at 4° C, with filling times ranging from 6 to 24 h. Large multipolar, bipolar and unipolar neurons were visualized in the rostral tip of the pineal organ (pineal ganglion). These neurons possessed extended dendritic trees participating in the formation of a circumscribed neuropil-like area. Throughout the pineal organ small bipolar elements were the most ubiquitous type of neuron, however, with markedly smaller numbers in the proximal portion of the pineal end-vesicle. In the pineal stalk, some bipolar neurons were observed to contact the pineal lumen, which is continuous with the third ventricle, via dendritic processes of various types. It could not be established whether any of these CSF-contacting processes were identical with photoreceptor outer segments. The basal processes of the bipolar neurons sometimes possessed distally projecting collaterals. In conclusion, it has been shown that (i) different types of neurons displaying varied patterns of regional distribution contribute to the pineal tract, and (ii) certain CSF-contacting neurons in the pineal organ send axonal processes directly toward the brain.Supported by Research grant Ko 758/2-4 from the Deutsche ForschungsgemeinschaftFellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany     

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.     

The pineal complex of the three-spined stickleback,Gasterosteus aculeatus L.

Summary The pineal complex of the three-spined stickleback (Gasterosteus aculeatus L.) was investigated by light and electron microscopy, as well as fluorescence histochemistry for demonstration of catecholamines and indolamines. The pineal complex of the stickleback consists of a pineal organ and a small parapineal organ situated on the left side of the pineal stalk. The pineal organ, including the entire stalk, is comprised mainly of ependymal-type interstitial cells and photoreceptor cells with well-developed outer segments. Both unmyelinated and myelinated nerve fibres are present in the pineal organ. Nerve tracts from the stalk enter the habenular and posterior commissures. A small bundle of nerve fibres connects the parapineal organ and the left habenular body. The presence of indolamines (5-HTP, 5-HT) was demonstrated in cell bodies of both the pineal body and the pineal stalk, and catecholaminergic nerve fibres surround the pineal complex.     

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.     

Elektronenmikroskopische Untersuchungen am Pinealorgan von Passer domesticus

Zusammenfassung Das Pinealorgan von Passer domesticus enthält Zellen mit innengliedartigem mitochondrienreichen Stift, der eine bulböse Zilie mit 9+0 Zilienfibrillen entsendet. Randzipfel solcher Zilien, die vielfach in Gruppen anzutreffen sind, können sich in etwa 200 Å starke Lamellen fortsetzen. Konzentrische und wirbeiförmige Lamellenzüge, die von mehreren solchen Zilien ausgehen, bilden im Lumen markscheidenartige oder auch ungeordnete Membranenkomplexe. Ein direkter Zusammenhang mit Zilien konnte nur bei einem Teil dieser Lamellenkörper ermittelt werden. Der Bauplan der bulbösen Zilien entspricht den frühen Entwicklungsstadien des Photorezeptoren-Außengliedes. Es fehlen aber die für die letzteren so charakteristischen Membraninvaginationen; die Lamellenkomplexe der Vogelepiphyse haben eine ektopische Lage zur bulbösen Zilie. Oft finden sich an den Lamellenkörpern Degenerationszeichen. Diese degenerativ veränderten Strukturen erinnern an die Gebilde, die in den pinealen Lichtsinnesorganen der niederen Vertebraten aus zerfallenden Außengliedplättchen hervorgehen. Im Vergleich zu voll differenzierten pinealen Sinneszellen erscheinen die rezeptorenähnlichen Pinealocyten von P. domesticus rudimentär. Definitive funktionelle Schlüsse sind aus solchen morphologischen Vergleichen aber nicht möglich. Im Epiphysenstiel von P. domesticus verlaufen kräftige Nervenbahnen, die im Material dieser Studie ausschließlich aus marklosen Nervenfasern (Durchmesser 0,12–1,5 m) bestehen. Diese Faserzüge sind von autonomen Nervenstämmchen zu unterscheiden, die perivasculär die bindegewebige Hülle des Pinealorgans durchsetzen und stellenweise an das Parenchym vordringen. Im Pinealorgan von P. domesticus findet sich auch ein Pinealocytentyp mit 800–1200 Å großen granulierten Vesikeln, die im Golgi-Apparat dieser Zellen entstehen. Die elektronenmikroskopischen und neurohistologischen Befunde werden mit Hinweis auf verhaltensphysiologische (Gaston und Menaker) und elektrophysiologische (Ralph und Dawson) Ergebnisse diskutiert. Da das Pinealorgan von P. domesticus nach Menaker eine zentrale Komponente der biologischen Uhr beherbergt, sind Fragen nach einem sensorischen Eigenapparat und nach der sekretorischen Aktivität dieses Organs besonders aktuell.

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Electron microscopic studies of the pineal organ in Passer domesticus

Summary The pineal organ of Passer domesticus contains cells with an inner segment, rich in mitochondria, from which a bulbous 9+0 type cilium originates. These cilia are often grouped together and may form 200 Å thick lamellae. Concentric or irregular whorl-like lamellar complexes arise from a number of such cilia. The structure of the bulbous cilia corresponds to that of early developmental stages of the photoreceptor outer segment. The characteristic membrane invaginations of the retinal cones are absent in the bulbous cilia of the avian pineal organ; the lamellar complexes have a position ectopic to the cilia. Signs of degeneration are present at the lamellar bodies. The degenerated forms resemble structures that arise from disintegrating outer segment plates in the pineal photoreceptor cells of lower vertebrates. The receptor-like pinealocytes of P. domesticus appear rudimentary when compared with the fully differentiated pineal sensory cells. Definitive functional interpretations are not possible from such morphological comparisons. In the pineal stalk of P. domesticus, nerve tracts are present consisting of unmyelinated fibers of 0.12–1.50 m diameter. These nerve tracts differ from autonomic nerves that traverse perivascularly the connective tissue of the pineal capsule and in places enter the pineal organ. In the pineal organ of P. domesticus, a cell type is also found containing 800–1,200 Å diameter granular vesicles which originate in the Golgi complex. The electron microscopic and neurohistological findings are discussed with reference to experiments by Gaston and Menaker (i.e. the effect of pinealectomy on the circadian locomotor rhythm of P. domesticus) and to electrophysiological results of Ralph and Dawson. Since, according to Menaker, the pineal organ of P. domesticus is a crucial component of the endogenous time-measuring system, questions concerning the presence of a sensory apparatus and secretory activity in this organ assume a special significance.

Eine kurze Zusammenfassung der Ergebnisse wurde im Seminar on Hypothalamic and Endocrine Functions in Birds, Tokio (19.–24. Mai 1969), vorgetragen.     

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.