Fluoreszenzmikroskopischer Nachweis biogener Monoamine in der Epiphysis cerebri von Rana esculenta und Rana pipiens

Zusammenfassung Die Epiphysis cerebri von Rana pipiens und Rana esculenta wurde fluoreszenzmikroskopisch auf das Vorhandensein bestimmter biogener Amine untersucht. Unter normalen Bedingungen lassen sich im Parenchym der Froschepiphyse keine Amine fluoreszenzmikroskopisch darstellen. Nach Vorbehandlung der Tiere mit dem Monoaminoxydase-Hemmer Nialamid ist aber eine intensive, durch Formaldehyd-Behandlung induzierte Gelbfluoreszenz in den Sinnes- und Stützzellen zu beobachten. Mikrospektrofluorometrische Messungen zeigen, daß das Fluorophor mit 5-Hydroxytryptamin identisch ist, obwohl das Vorkommen von anderen verwandten Indolen — z.B. 5-Hydroxytryptophan und Melatonin — nicht ausgeschlossen werden kann. Grünfluoreszierende adrenerge Nervenfasern sind im meningealen Hüllbindegewebe der Epiphyse zu erkennen; einige dieser Fasern scheinen auch in die Epiphyse einzudringen.Der Nachweis von 5-Hydroxytryptamin wird im Zusammenhang mit der Frage einer Melatoninsynthese diskutiert. Das 5-Hydroxytryptamin könnte außerdem in einer funktionellen Beziehung zu einem bisher noch unbekannten Protein- der Polypeptid-Hormon des Epiphysenparenchyms stehen.

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Fluorescence microscopic studies of biogenic amines in the pineal organ of Rana esculenta and Rana pipiens

Summary The pineal organ of Rana pipiens and Rana esculenta was studied by fluorescence microscopy for the histochemical demonstration of certain biogenic monoamines. Under normal conditions, no fluorogenic amines were visible in the organ. After pretreatment of the animal with a monoamine-oxidase inhibitor, nialamide, an intense yellow formaldehyde-induced fluorescence appeared both in the sensory cells and in the supporting cells. Microspectrofluorometric analysis indicated that the fluorophore is identical with 5-hydroxytryptamine; the presence of other closely related indoles, such as 5-hydroxytryptophan and melatonin, however, cannot be excluded. Fluorescent adrenergic nerves were found in the connective tissues surrounding the pineal organ; fluorescent fibers were observed also in the pineal parenchyma.The presence of 5-hydroxytryptamine in the anuran pineal organ is discussed with regard to the role that the amine plays in melatonin synthesis and with regard to a possible functional relation to some as yet unidentified protein- or polypeptid-hormone within the pineal parenchyma.

Finanzierung eines Studienaufenthaltes an der Universität Lund durch die Deutsche Forschungsgemeinschaft (Forschungsvorhaben — Ok 1/15 — von Prof. Dr. A. Oksche u.a.).     

Degenerationsstudien am Nervus pinealis von Rana esculenta L. nach stirnorgannaher und -ferner Durchtrennung

Zusammenfassung Am Nervus pinealis von Rana esculenta wurde der Verlauf der Wallerschen Degeneration nach Durchtrennung des Nervenstrangs unterhalb des Stirnorgans studiert.30 Std nach der Läsion tritt im distalen Stumpf der markhaltigen Nervenfasern eine Anhäufung von Glykogen auf; granulär-vakuolige Veränderungen zeigen diese Faserabschnitte am 4. Tag. Veränderungen der Myelinscheide beginnen am 7. postoperativen Tag an den Innenlamellen und greifen dann auf die ganze Markscheide über. Die Desintegration der einzelnen markhaltigen Fasern eines Nervus pinealis verläuft verschieden schnell; nach 25 Tagen ist sie an den meisten Fasern abgeschlossen.Am 7. Tag sind die ersten Veränderungen an den marklosen Fasern zu beobachten. Dabei werden die peripher in der Hüllzelle liegenden Fasern durch Retraktion des Hüllzellzytoplasmas ins Interstitium ausgestoßen und gehen dort zugrunde. Die mehr zentral in den Hüllzellen lokalisierten Fasern bleiben hingegen vom Hüllplasma umgeben. Die Degeneration dieser marklosen Elemente ist nach 14 Tagen abgeschlossen; es resultiert ein mit homogenem Inhalt geringer Elektronendichte gefüllter Kanal, der auf Querschnittsbildern eine Vakuole vortäuscht.Weiterhin ist für den degenerierenden Nervus pinealis eine starke Fibrosierung sowie der Schwund der intra- und perineuralen Lymphspalten charakteristisch. Nach etwa 60 Tagen treten im distalen Nervenstumpf von Büngnerschen Bändern umgebene marklose Nervenfasern auf. Dieses Auswachsen von Nervenfasern bestätigt elektrophysiologische Ergebnisse von Morita (s. Dodt und Morita, 1967), daß im Nervus pinealis erregungsleitende Elemente in beide Richtungen ziehen (frontofugale und frontopetale Fasern).Bindegewebsstrukturen, Degenerationsablauf und Strukturbild der Hüllzellen lassen den Nervus pinealis als peripheren Nerven erscheinen.

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Degeneration pattern of the pineal nerve of rana esculenta after transection at different anatomical levels

Summary Wallerian degeneration was studied in the pineal nerve of Rana esculenta with the electron microscope after transection of the nerve beneath the frontal organ.30 hours after the operation an accumulation of glycogen was found in the distal stump of the myelinated axons. Four days after interruption these axons displayed signs of granular and vaculoar disintegration. Myelin sheath changes started at the seventh day after the operation at the level of the inner lamellae and were subsequently followed by changes in the outer lamellar systems. The velocity of disintegration varied for individual myelinated fibers. 25 days after transection of the pineal nerve most of its myelinated fibers were completely degenerated.Initial changes of the unmyelinated fibers were recognized at the seventh postoperative day. Fibers located peripherally in the satellite cell were exposed by retraction of the satellite cytoplasm and degenerated rapidly. On the other hand, unmyelinated fibers which were located more centrally remained within the satellite cell and completed their degeneration only after 14 days. The residual structure observed was a channel occupied by a homogeneous material of low electron density.The number of collagen fibers increased and the intra- and perineural lymph spaces were obliterated in the distal nerve stump. Approximately 60 days after the operation chains of satellite cells and unmyelinated nerve fibers reappeared in the distal nerve stump. This result confirms the electrophysiological data of Morita (cf. Dodt and Morita, 1967) that nerve fibers run in both directions within the pineal nerve (i.e. they are frontofugal and frontopetal with respect to the frontal organ).The properties of the connective tissue between the pineal nerve fibers, the pattern of degeneration and the fine structure of the satellite cells suggest that the pineal nerve of Rana esculenta has the structure of a peripheral nerve rather than that of a central nervous tract.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. Ein Druckkostenzuschuß wurde aus Institutsmitteln zur Verfügung gestellt.     

Elektronenmikroskopische Untersuchungen des ventralen Diaphragmas von Locusta migratoria und der langsamen Kontraktionswelle nach Fixation durch Gefriersubstitution

Zusammenfassung Die Muskulatur des ventralen Diaphragmas von Locusta besteht aus parallel verlaufenden Muskelfasern, die über glanzscheibenartige Strukturen in den Querverbindungen miteinander verbunden sind. Jede Muskelfaser ist von einer dicken Bindegewebshülle umgeben. Die Fasern sind über Hypodermiszellen mittels Tonofibrillen in der Cuticula verankert.Unkontrahierte Sarkomere haben eine Länge von 5 m und mehr. Eine H-Zone ist angedeutet, eine M-Linie nicht vorhanden. Aktin- und Myosinfilamente (Durchmesser 72 bzw. 160 AE) liegen nicht im Register. Daneben existiert ein dritter, sehr dünner Filamenttyp. Die Z-Zone hat einen gewellten Verlauf und faßt die Aktinfilamente in Bündeln zusammen. Mitochondrien liegen beiderseits der Z-Zone. Das T-System faltet sich in Form von Sarkolemmkerben in das Faserinnere ein und setzt sich in Tubuli nach innen fort. Z-Zonen und Sarkolemmkerben sind miteinander verbunden. T-System und sarkoplasmatisches Retikulum treten durch unregelmäßig verteilte Diaden miteinander in Kontakt.Begrenzter Ca⁺⁺-Entzug läßt Kontraktionswellen von der Länge mehrerer Sarkomere entstehen.Die Fixation durch Gefriersubstitution erzeugt gegenüber Standardverfahren Veränderungen wie Schrumpfung des Sarkoplasmas, Verdickung der Myosinfilamente, Vakuolisierung von Mitochondrien und vesikulärem System. In der Kontraktionswelle verkürzt sich die A-Zone mit zunehmender Sarkomerenkontraktion. Der Durchmesser der Myosinfilamente beträgt 172 AE, die Periodik der Cross-bridges von 305 AE bleibt im mittleren Bereich der Filamente konstant.

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Electron microscope studies of the ventral diaphragm of Locusta migratoria and of the slow wave of contraction after fixation by freeze substitution

Summary The muscular system of the ventral diaphragm of Locusta consists of parallel muscle fibers, which are connected by structures like intercalated discs within transverse bridges. Each muscle fiber is enveloped by a thick sheath of connective tissue. The fibers are attached to the cuticule by means of hypodermic cells with tonofibrils.Uncontracted sarcomeres have a length of 5 m and more. The H-band is slightly indicated, a M-line is not visible. Actin and myosin filaments (diameter 72 respectively 160 AE) are out of register. Moreover there is a third and very thin type of filaments. The Z-band has an undulating shape and collects the actin filaments into bundles. Mitochondria lie on either side of the Z-band. The T-system invaginates as sarcolemmal clefts and continues its course inwards as tubuli. The sarcolemmal clefts are connected with the Z-band. The T-system and the sarcoplasmic reticulum are joined by diads of irregular distribution.Limitated deprivation of Ca⁺⁺ causes waves of contraction with the length of several sarcomeres.Contrary to standard methods the freeze-substitution causes some modifications such as shrinking of the sarcoplasm, thickening of the myosin filaments, vacuolization of mitochondria and vesicular system. Within the waves of contraction the A-band shortenes with increasing sarcomere contraction. The diameter of the myosin filaments measures 172 AE, the 305 AE-period of the cross-bridges remains constant within the middle of the filaments.

Auszug aus der Dissertationsarbeit Elektronenmikroskopische Untersuchungen der Muskulatur des ventralen Diaphragmas von Locusta migratoria und der langsamen Kontraktionswelle unter Anwendung der Gefriersubstitution (Mathematisch-Naturwissenschaftliche Fakultät der Universität Göttingen). Auf Anregung von Herrn Prof. Dr. Schlote und mit Unterstützung durch die Deutsche Forschungsgemeinschaft und die Göttinger Akademie der Wissenschaften.     

Opsin-immunoreactive outer segments and acetylcholinesterase-positive neurons in the pineal complex of Phoxinus phoxinus (Teleostei,Cyprinidae)

Summary The pineal complex of the teleost, Phoxinus phoxinus L., was studied light-microscopically by the use of the indirect immunocytochemical antiopsin reaction and the histochemical acetylcholinersterase (AChE) method.Opsin-immunoreactive outer segments of photoreceptor cells were demonstrated in large numbers in all divisions of the pineal end-vesicle and in the pineal stalk. Moreover, they were found in the roof of the third ventricle, adjacent to the orifice of the pineal recess as well as scattered in the parapineal organ. These immunocytochemical observations provide direct evidence of the presence of an opsin associated with a photopigment in the photosensory cells of the pineal and parapineal organs of Phoxinus. By means of the AChE reaction (Karnovsky and Roots 1964) inner segments of pineal photoreceptors, intrinsic nerve cells, several intrapineal bundles of nerve fibers, and a prominent pineal tract were specifically marked. The pineal neurons can be divided into two types: one is located near the pineal lumen, the other near the basal lamina. The latter perikarya bear stained processes directed toward the photoreceptor layer. A rostral aggregation of two types of AChE-positive nerve cells occurs in the ventral wall of the pineal end-vesicle. The main portion of the AChE-positive pineal tract, which lies within the dorsal wall of the pineal stalk, can be followed to the posterior commissure where some of the nerve fibers course laterally. A few AChE-positive pineal nerve fibers run toward the lateral habenular nucleus via the habenular commissure. In the region of the subcommissural organ single AChE-positive neurons accompany the pineal tract. The nerve cells of the parapineal organ exhibit a moderate AChE activity.These findings extend the structural basis for the remarkable light-dependent activity of the pineal organ of Phoxinus phoxinus. To the memory of Professor Karl von Frisch, pioneer and master in the field of photoneuroendocrine systemsThis investigation was supported by grants from the Deutsche Forschungsgemeinschaft to A.O. (Ok 1/24; 1/25: Mechanismen biologischer Uhren) and to H.-W. K. (Ko 758/1; 758–2)On leave from the 2nd Department of Anatomy, SOTE, Budapest, Hungary     

Neurone und zentralnervöse Verbindungen des Pinealorgans der Anuren

Zusammenfassung Das Problem der Zwischenneurone nimmt bei der Deutung der chromatischen und achromatischen Antworten der pinealen Sinnesorgane eine Schlüsselstellung ein. Frühere Ergebnisse schienen darauf hinzuweisen, daß der nervöse Apparat des Pinealorgans (Epiphysis cerebri) vonRana temporaria undRana esculenta bineuronal organisiert ist. Mit modifizierten Methylenblau- und Golgi-Methoden gelang es jetzt, die nervösen Strukturen der pinealen Rezeptoren und Nervenzellen präziser darzustellen. Das neurohistologische Bild der Epiphysis cerebri enthält auch Nervenzellen, die an Zwischenneurone denken lassen. Diese diffus verstreuten kleinen Elemente unterscheiden sich von den klassischen Typen der retinalen Bipolar-, Horizontal- und Amakrinzellen. Der Tractus pinealis vonR. temporaria undR. esculenta wird von Axonen großer multipolarer und kleiner, wenig verzweigter Ganglienzellen gebildet. Die zentrale Projektion dieser pinealofugalen (afferenten) Bahn ist von großem funktionellem Interesse. Nach vollständiger Unterbrechung des Tr. pinealis finden sich degenerierende Faserelemente innerhalb und am unteren Rand der Comm. posterior, in der Area praetectalis und in den Kernarealen des sog. Zentralen Graus. Keine degenerierenden Tractusfasern sind in der subependymalen Schicht und in den aminergen Kerngebieten des Mesencephalon zu beobachten. Die beschriebenen Verbindungen des Tr. pinealis könnten die anatomische Basis einiger lichtabhängiger (phototaktischer) Reflexe darstellen. Weitere Untersuchungen über die zentralnervöse Projektion des Tr. pinealis sind im Gang.

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Neurons and central nervous connexions of the pineal organ in anura

Summary The problem of interneurons appears to be very important for the functional interpretation of the chromatic and achromatic responses of pineal sense organs. Previous results seemed to indicate a bineuronal chain in the pineal organ (epiphysis cerebri) ofRana temporaria andRana esculenta. Precise images of pineal receptors, neurons and their connexions were obtained in the present studies using methylene-blue and Golgi methods. A limited number of interneurons probably exist in theepiphysis ofR. temporaria andR. esculenta: the images of these scattered neurons were observed to differ from the classical bipolar, horizontal and amacrine cells of the amphibian retina. The pineal tract of R. temporaria andR. esculenta is formed by the axons of large multipolar and smaller scarcely ramified nerve cells. The central projection of this pinealo-fugal (afferent) pathway is of great functional interest. After complete surgical interruption of the pineal tract degenerating nerve fibers were traced in Nauta (Fink-Heimer) preparations. Degenerating fibers were observed within and beneath the posterior commissure, in the pretectal region and in the nuclear areas of the periventricular gray. The subependymal layer and the basal aminergic nuclei of the frog mesencephalon were always free of degenerating fibers. The anatomical connexion of the pineal tract described in this paper could serve as a basis for some light-dependent (phototactic) reflexes. Further investigations concerned with the central projections of the pineal tract are in progress.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. — Eine Kurzfassung der Befunde wurde auf dem Symposium The Pineal Gland der Ciba Foundation in London (30. 6.–2. 7. 1970) vorgetragen (s. Oksche, 1971).     

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     

Histological,histochemical and electron microscopical studies on the nervous apparatus of the pineal organ in the tiger salamander,Ambystoma tigrinum

Summary 150–190 photoreceptor cells form a basic structural component of the pineal organ of Ambystoma tigrinum. Most of the outer and inner segments of these cells project into the lumen horizontally. Only 10 percent of the total number of photoreceptor cells are located within the pineal roof which is composed of a single cell layer. The photoreceptor cells are connected with nerve cells by synapses displaying characteristic ribbons. Different types of synaptic contacts, i.e. simple, tangential, dyad, triad and invaginated, are found. They are embedded in extended neuropil zones. A particular type of synapse indicates the presence of interneurons. The basal processes of some photoreceptor cells leave the pineal organ and make synaptic contacts with nervous elements located within the area of the subcommissural organ. Employing the method of Karnovsky and Roots (1964) for histochemical demonstration of acetylcholinesterase (AChE) approximately 70 neurons (intrapineal neurons) can be discerned in the pineal organ of Ambystoma tigrinum. In analogy to the distribution of photoreceptor cells only few nerve cells are observed in the roof portion of the pineal organ. Evidently, two different types of AChE-positive intrapineal neurons are present. About 40–50 AChE-positive neurons (extrapineal neurons) are scattered in the area of the subcommissural organ. In this area two types of nerve cells can be distinguished: 1) neurons which send pinealofugal (afferent) axons toward the posterior commissure and 2) neurons which emit pinealopetal (efferent) axons into or toward the pineal organ.The nervous pathways connecting the pineal organ with the diencephalomesencephalic border area are represented by a distinct pineal pedicle and several accessory pineal tracts.Granular nerve fibers run within the posterior commissure and establish synaptic contacts in the commissural region adjacent to the pineal organ. Some of these granular elements enter the pineal organ.The morphology of the nervous apparatus of the pineal organ of Ambystoma tigrinum is discussed in context with evidence from physiological experiments.In partial fulfillment of the requirements for the degree of Dr. med., Faculty of Medicine, Justus Liebig University, GiessenThe author is indebted to Professors A. Oksche and M. Ueck for their interest in this study. Thanks are due to Professor Ch. Baumann, Giessen, and Professor H. Langer, Bochum, for stimulating discussions. The technical assistance of Miss R. Liesner is gratefully acknowledgedDedicated to Professor Berta Scharrer on the occasion of her 70th birthday. Supported by grants from the Deutsche Forschungsgemeinschaft to A.O. and M.U.     

Rasterelektronenmikroskopische Beobachtungen an pinealen Sinneszellen der Forelle,Salmo gairdneri (Teleostei)

Zusammenfassung Im rasterelektronenmikroskopischen Bild des Pinealorgans vonSalmo gairdneri kann man drei verschiedene Außengliedtypen der Photorezeptoren unterscheiden. Diese Ergebnisse werden im Hinblick auf die Ultrastrukturkonzepte von Rüdeberg (1969) und Bergmann (1971) diskutiert. Rasterelektronenmikroskopische Studien erleichtern die anatomische Klassifizierung von pinealen Sinneszellen aufgrund ihrer Außengliedform.

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Scanning electron microscopic observations of pineal photoreceptor cells in the trout,Salmo gairdneri (teleostei)

Summary The outer segments of pineal photoreceptor cells ofSalmo gairdneri were investigated with the scanning electron microscope. The scanning electron micrographs showed three different types of outer segments. These results are discussed with respect to the ultrastructural concepts of Rüdeberg (1969) and Bergmann (1971). Scanning electron microscopy permits better anatomical classification of pineal photoreceptor cells according to the form of their outer segments.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (Arbeitsgruppe Prof. Dr. A. Oksche). Herrn Prof. Dr. G. Pfefferkorn, Direktor des Instituts für Medizinische Physik an der Westfälischen Wilhelms-Universität Münster, danke ich für einen Arbeitsplatz am Rasterelektronenmikroskop, Herrn Prof. Dr. H. G. Fromme, Münster, für die Unterstützung bei der präparativen Aufbereitung des Materials.     

Synapse-like contacts between axons of the pineal tract and the subcommissural organ in Rana perezi (Anra) and their absence in Carassius auratus (Teleostei): ultrastructural tracer studies

The present study was designed to investigate the controversial subject of the existence of a neural input from the pineal organ via the pineal tract to the subcommissural organ (SCO) in teleosts and anurans. Horseradish peroxidase was injected into the pineal organ and pineal tract of Carassius auratus and Rana perezi. Within the pinealofugal fibers the tracer was visualized at the light-and electron-microscopic levels either by immunocytochemistry using an anti-peroxidase serum, or by revealing the enzymatic activity of peroxidase. In both species, labeled myelinated and unmyelinated fibers of the pineal tract were readily traced by means of electron microscopy. In R. perezi, numerous terminals contacting the SCO cells in a synapse-like (synaptoid, hemisynaptic) manner bore the label, whereas a different population of endings was devoid of the tracer, indicating that in this species the SCO receives a dual neural input, one of pineal origin, the other of unknown source and nature. In the SCO of C. auratus, neither labeled nor unlabeled synapse-like contacts were found. Thus, in this latter species, a direct neural input to the SCO is missing. It is concluded that the secretory activity of the SCO can be controlled by different mechanisms in different species, and that more than one neural input mechanism may operate in the same species.     

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.     

Postsmolt change in numbers of acetylcholinesterase-positive cells in the pineal organ of the Pacific coho salmon

Summary We have examined the occurrence of acetylcholinesterase (AChE)-positive cells in the pineal organ of different developmental stages of the Pacific coho salmon. Large numbers of AChE cells were present in fresh-water living alevins, in all stages of presmolts (n=307–544), and in adult spawners (n=696–1774) whereas seawater-living postmolts displayed a total lack of labeled cells. The AChE-reactive cells were evently distributed within the pineal end-vesicle and stalk of the presmolts and adults. However, the AChE-positive cells that occurred in the pineal stalk were of a smaller type and more uniform in shape than the cells of the pineal endvesicle. The dense populations of AChE-stained cells in the alevins, were all situated in the caudal part of the pineal end-vesicle. We conclude that changes in pineal metabolism occur in postsmolt salmon that liver in saltwater. It is not clear whether the observed change in pineal AChE expression is an unspecific change caused by life in the sea, reflecting alterations that are related to aspects of osmoregulation, and/or is involved in the visual function of the pineal organ resulting from changes in the environmental lighting conditions, e.g., photoperiod, light-intensity, or spectral composition. This study adds to our previous findings of changes that occur in the central nervous system of the salmon during the time of the parr-smolt transformation and migration between limnic and marine environments and indicates a possible central role of the pineal organ in the control of these events.     

Putative cholinergic elements in the photosensory pineal organ and retina of a teleost,Phoxinus phoxinus L. (Cyprinidae)

Summary Putative cholinergic neurons in the photosensory pineal organ of a cyprinid teleost, the European minnow, were studied by use of choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry. Pinealofugally projecting neurons were visualized using retrograde HRP-filling through their cut axons. For comparison, the distribution of choline acetyltransferase immunoreactivity (ChAT-IR) and AChE-positive elements in the retina was investigated.While the distributional patterns of ChAT-IR and strongly AChE-positive perikarya in the retina are similar and may represent the same neuronal population, ChAT-IR and AChE-positive elements in the pineal organ appear to belong to separate populations. In the retina, small- to medium-sized perikarya in the inner nuclear layer, and small perikarya in the ganglion cell layer are ChAT-IR and AChE positive. The entire inner plexiform layer is AChE positive, while only sublaminae 1, 2 and 4 are ChAT-IR. No indication of cholinergic activity was observed in the optic axon layer.In the pineal organ, ChAT-IR is restricted to small perikarya situated rostrally and dorsally in the pineal end-vesicle. AChE-positive neurons are present throughout the pineal end-vesicle and the pineal stalk. The pineal tract (the pinealofugally projecting axons of intrapineal neurons) is strongly AChE positive, but displays no ChAT-IR. The distribution of pinealofugally projecting neurons, labeled with retrogradely transported HRP, is markedly dissimilar to that of the ChAT-IR elements. It is proposed that the photosensory pineal organ transmits photic information to the brain via a non-cholinergic pathway. The possibility that the ChAT-IR neurons represent small local interneurons is discussed in the light of comparative physiological and anatomical findings.     

Molecular cloning and characterization of rhodopsin in a teleost (Plecoglossus altivelis,Osmeridae)

Amplified fragments encoding exon-4 of opsin cDNAs were cloned from the retina of landlocked ayu (Plecoglossus altivelis), and sequenced. On the basis of the sequence homology to previously characterized fish visual pigments, one clone was identified as rod opsin (AYU-Rh), and two clones as green (AYU-G1, -G2), one as red (AYU-R) and two as ultraviolet (AYU-UV1, -UV2) cone opsins. The 335-amino acid sequence deduced from the full-length cDNA of AYU-Rh included residues highly conserved in vertebrate rhodopsins and showed the greatest degree (88%) of similarity with salmon rhodopsin. Southern blotting analysis indicated that ayu possess two rhodopsin genes, one encoding visual rhodopsin (AYU-Rh) and the other non-visual extra-ocular rhodopsin (AYU-ExoRh). RT-PCR experiments revealed that AYU-Rh was expressed in the retina and AYU-ExoRh in the pineal gland. In situ hybridization experiments showed that the mRNA of AYU-Rh was localized only in rod cells not in cone cells. Lake and river type landlocked ayu having different amounts of retinal and 3-hydroxyretinal in their retinas expressed a rhodopsin (AYU-Rh) of identical amino acid sequence.     

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.     

Ontogenetic development of S-antigen- and rodopsin immunoreactions in retinal and pineal photoreceptors of Xenopus laevis in relation to the onset of melatonin-dependent color-change mechanisms

Summary In Xenopus laevis Daud., the ontogenetic occurrence of two photoreceptor-specific proteins, S-antigen and rod-opsin, was investigated and correlated to the maturation of the neurohormonal effector system involved in melatonin-dependent color-change mechanisms. Tadpoles ranging from stage 12 to 57 (Nieuwkoop and Faber 1956) were fixed in Zamboni's or Bouin's solution. Frozen or paraffin sections of either total heads or dissected brains and eyes were prepared and treated with highly specific antisera against S-antigen and rod-opsin. In the retina, immunoreactive S-antigen and rod-opsin were first demonstrated in a few centrally located photoreceptors at stage 37/38. Photoreceptors of the peripheral (iridical) portions of the retina gradually became immunoreactive during further development. As in the retina, the first S-antigen-immunoreactive photoreceptors in the pineal complex appeared at stage 37/ 38. At this and all later stages investigated rod-opsin immunoreactivity was restricted to a few dot-like structures resembling developing pineal outer and inner segments. In most animals rod-opsin immunoreactivity was completely absent from the pineal complex. The analysis of retinal proteins with the immunoblotting technique (Western blot) revealed that the S-antigen antibody bound to a 48-kDa protein and the rod-opsin antibody to a 38-kDa protein. The body lightening reaction was determined with the aid of the melanophore index in larvae fixed in light or darkness, respectively. Aggregation of melanophore melanosomes in darkness (the melatonin-dependent primary chromatic response) first occurred at stage 37/38 when melanophores started to differentiate and became pigmented. These results indicate that in Xenopus laevis (i) the molecular mechanisms of photoreception develop simultaneously in retina and pineal complex; (ii) most pineal photoreceptors differ from retinal rods in that they contain immunoreactive S-antigen but essentially no immunoreactive rod-opsin; and (iii) the differentiation of phototransduction processes coincides with the onset of melatonin-dependent photoneuroendocrine regulation of color-change mechanisms.Supported by USUHS protocol C07049 (MDR) and the Deutsche Forschungsgemeinschaft (HWK)