Substance P-like-immunoreactive neurons in the photosensory pineal organ of the rainbow trout,Salmo gairdneri Richardson (Teleostei)

Summary Restricted numbers of substance P-like-immuno-reactive (SPL-IR) neurons were demonstrated in the photosensory pineal organ of the rainbow trout. The small parapineal organ of this teleost species receives a distinct SPL-IR innervation via the habenular nuclei, but displays no intrinsic SPL-IR neurons. Intrapineal SPL-IR neurons were located in the rostral portion of the pineal end-vesicle. Neuronal somata were found in a lateral position with smooth axonal processes extending mediad. Immunoreactive somata and axonal processes were observed intraparenchymally as well as in the pineal lumen. The pattern of immunoreactivity was not changed in excised pineal organs that had been incubated in tissue culture medium in the dark for 18 h. The possibility that the intrapineal SPL-IR neurons are not part of the neural circuitry involved in the transduction of photic information, but may have other functions, is discussed.Fellow of the Alexander von Humboldt-Stiftung, Bonn, Federal Republic of GermanySupported by research funds from the Deutsche Forschungsge-meinschaft (Ko 758/2-4)     

Microvasculature of the pineal organ of the rainbow trout (Salmo gairdneri)

Summary The angioarchitecture of the pineal organ of the rainbow trout (Salmo gairdneri) was investigated by means of the corrosion-cast preparation method and scanning electron microscopy. Two main arteries (aa. epiphyseales) supply the pineal parenchyma. They emerge from the aa. cerebri anteriores and run in the fissure between the prosencephalon and the mesencephalon. After entering the pineal stalk, the aa. epiphyseales branch off into several arterioles, most of which extend to the pineal end-vesicle where they give rise to a lobular, bilaterally symmetric capillary network. Capillaries establishing the main portion of the pineal vessels appear widened in comparison to those supplying other portions of the brain and resemble capillaries in other endocrine organs. In Salmo gairdneri, no specialized system of portal vessels appears to exist between the pineal organ and other portions of the brain.     

Cytochemical demonstration of acid phosphatase activity in the pineal organ of the rainbow trout,Salmo gairdneri

Summary Activity of acid phosphatase (ACP) was investigated cytochemically in the pineal organ of the rainbow trout, Salmo gairdneri. Intense reaction product for ACP activity was observed (1) in lysosomes varying in size and shape and (2) in endoplasmic reticulum associated with the Golgi complex of (i) the pineal photoreceptor and supporting cells, (ii) vascular endothelial cells, and (iii) macrophages inhabiting pineal lumen, parenchymal epithelium and perivascular spaces. This localization of ACP is discussed with particular reference to the capacity for lysosomal digestion in a pineal organ combining photoreceptive and secretory functions, and lacking a blood-brain barrier, as holds true for the pineal of the rainbow trout. Taking advantage of its capacity for endocytotic uptake and lysosomal digestion, the pineal organ of the rainbow trout may serve as a barrier between the blood circulation and the cerebrospinal-fluid compartment. Furthermore, the macrophages may be considered as an essential component in pineal function of fish.Fellow of the Alexander von Humboldt Foundation.Fellow of the Alexander von Humboldt Foundation.     

Vascular permeability (problem of the blood-brain barrier) in the pineal organ of the rainbow trout,Salmo gairdneri

Summary The problem of the blood-brain barrier in the pineal organ of the rainbow trout, Salmo gairdneri, was investigated following intraperitoneal or intracardial injections of several tracers and dyes with different molecular weights. As demonstrated at the light-microscopic level, repeated injections of trypan blue or horseradish peroxidase (HRP) resulted in an accumulation of these substances in the pineal epithelium (parenchyma). By use of the electron microscope, HRP was found in electron-dense bodies, probably lysosomes, in (i) the endothelial cells and perivascular macrophages 4 h after intraperitoneal injection, (ii) the supporting cells and intrapineal or luminal macrophages 8 h after injection, and (iii) the receptor cells 24 h after injection of the tracer. Ferritin particles penetrated the fenestrated endothelium of pineal capillaries. They were confined to vesicles, vacuoles and the smooth endoplasmic reticulum of the supporting cells as well as to the synaptic vesicles and the smooth endoplasmic reticulum of the pineal photoreceptors. The intercellular passage of tannic acid mixed with the fixative was blocked at the luminal junctional complex separating the pineal lumen from the basal portion of the pineal epithelium. The passive intercellular transport of substances with high molecular weight from the bloodstream to the cerebrospinal-fluid compartment is thus prevented. However, no blood-brain barrier exists for exogenously administered proteins, which are rapidly taken up by pineal cells and actively transported in a transcellular manner.The findings on the blood-brain barrier of the pineal organ of the rainbow trout are discussed with particular reference to the endocrine capacity of pineal sensory organs.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany.     

Glomerular binding of angiotensin II in the rainbow trout,Salmo gairdneri

Summary Isolated glomeruli of the rainbow trout have been exposed in vitro to¹²⁵I-angiotensin II (0.88 × 10^–9 M) and binding sites located by light-microscopic autoradiography. These studies provide evidence of specific binding of angiotensin II by glomeruli. Binding was significantly inhibited by excess (10^–5 M) unlabelled angiotensin II, but a high degree of non-specific binding also occurred. The mammalian competitive antagonist, saralasin (3 × 10^–7 M) did not influence¹²⁵I-angiotensin II binding to fish glomeruli. Intense binding of¹²⁵I-angiotensin II was noted at the vascular pole of some glomeruli.     

Immunoreactivity to gonadotropin-releasing hormone and gonadotropic hormone in the brain and pituitary of the rainbow trout Salmo gairdneri

Summary Immunoreactivity to gonadotropin-releasing hormone (GnRH) and gonadotropic hormone (GTH) was studied at the light-microscopical level in the brain and pituitary of rainbow trout at different stages of the first reproductive cycle using antisera against synthetic mammalian GnRH and salmon GTH. GnRH perikarya were localized exclusively in the preoptic nucleus, both in the pars parvicellularis and the pars magnocellularis. A few somata contacted the cerebrospinal fluid. Not all neurosecretory cells were GnRH-positive, indicating at least a bifunctionality of the preoptic nucleus. We recorded no differences between sexes or stages of gonadal development in the location of GnRH perikarya, whereas gradual changes were found in staining intensity during the reproductive cycle. GnRH fibres ran from the partes parvicellularis and magnocellularis through the hypothalamus and merged into a common tract at the transverse commissure before entering the pituitary. In the pituitary, GnRH was localized in the neural tissue of the neurointermediate lobe and, to a lesser extent, in the neural protrusions penetrating the proximal pars distalis. The bulk of GTH-positive cells was situated in the proximal pars distalis. Some cells were found more rostrally amidst prolactin cells or in the neurointermediate lobe. Only a limited number of GTH cells appeared to be in close contact with GnRH-positive material.     

Direct effects of angiotensin II on glomerular ultrastructure in the rainbow trout,Salmo gairdneri

Summary Slices from the kidneys of the rainbow trout which were exposed to 10^-6 or 10^-5 M angiotensin II (AII) and isolated glomeruli exposed to 10^-7 or 10^-5 M AII showed ultrastructural changes compared to control tissues incubated without AII. The studies indicate that angiotensin II has a direct action on glomerular ultrastructure, flattening the epithelial podocytes and broadening the primary processes with fusion of pedicels in extreme cases. These changes suggest a probable effect of AII on water permeability of the trout glomerulus, an intrarenal action which is believed to form an essential part of the antidiuretic adaptation to increased environmental salinities.     

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.     

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.     

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     

Autoradiographic localization of 3H-5-HTP and 3H-5-HT in the pineal organ and circumventricular areas in the rainbow trout,Salmo gairdneri Richardson

Summary The time course of incorporation and cellular localization of ³H-5-hydroxytryptophan (³H-5-HTP) and ³H-5-hydroxytryptamine (³H-5-HT) in the pineal and some brain regions in rainbow trout, Salmo gairdneri, were studied by quantitative and qualitative autoradiography.Among the tissues examined, the pineal shows the highest and most rapid uptake of the two isotopes. Maximum incorporation of ³H-5-HTP is achieved by 2 h and that of ³H-5-HT by 20 minutes post injection. At the end of the six-hour experimental period, a significantly high amount of radioactivity is still detectable in the pineal. The results indicate a much slower turnover of the two indoles, especially 5-HTP, in the trout than is known for mammalian tissues.Both the ependymal supporting cells and the receptor cells of the pineal localize these isotopes. In contrast, the intrapineal neurons remain unlabeled. This is taken to suggest lack of capacity of these cells to metabolize 5-HTP and 5-HT.In the circumventricular regions, the two indoles occur in the ependyma of the recessus lateralis and the recessus praeopticus. The label is also localized in the neuropil and the neurons of the nuclei recessus lateralis and praeopticus. Semiquantitative estimates reveal a significant labeling of these areas only 20 minutes after injection, although a weak but inconsistent labeling of the ependyma is evident at 5 minutes. The significance of these results is discussed in regard to (a) normal capacity of circumventricular areas to metabolize indoles and (b) a possible chemical interaction between the pineal and the brain involving a direct pineal-cerebrospinal fluid pathway.Supported in part by a grant to the senior author from the University of Kentucky Research FoundationThe authors wish to thank the Department of Fish and Game, Kentucky, for supplying rainbow trout. The technical assistance of Mrs. Munira Nasser is gratefully acknowledged     

Myogenesis and contraction in the early embryonic heart of the rainbow trout

Summary Myogenesis in the embryonic heart of the rainbow trout, Salmo galrdneri (Rich.), was investigated electron microscopically from the 29th to the 41st somite stage. Thick and thin myofilaments are formed simultaneously as well as precursors of Z-lines, to which the thin filaments are attached. The genesis of filaments takes place in the region around the intracellular yolk droplets. The first myofibrils appear by the 33rd somite stage, probably formed by a mechanism of self-assembly in which the binding sites of actin and myosin participate. A- and I-bands do not develop before the 38th somite stage. The contraction already begins during the 33rd somite stage in the middle of the tubular heart. Gradually, the peristaltic waves spread increasingly to other parts of the heart. In the 41st somite stage the entire heart is contractile and all myocytes contain myofibrils.     

Melatonin modulates the neural activity in the photosensory pineal organ of the trout: Evidence for endocrine-neuronal interactions

Summary Hormonal and neural signals transmitted from the pineal organ to the brain in cold-blooded vertebrates presumably convert information about the ambient illumination into signals which may be used to mediate photoperiodic and circadian responses. The possible intrapineal function of melatonin was investigated by recording intra- and extracellularly from photoreceptors and second-order neurons in the isolated superfused pineal organ of the trout (Salmo gairdneri). Melatonin added through the perfusion bath to the explanted pineal organ caused a dose-related and reversible inhibition of ganglion cells of the luminance type whereas the hormone did not significantly affect the membrane potential of photoreceptors and their light-evoked response. The observed effects seem to be independent from photoperiod and adaptation conditions. These results suggest that melatonin provides a feedforward signal to intrapineal neurons regulating the neural output of the organ.Laboratory of Fish Biology, School of Agriculture, Nagoya University, Chikusa, Nagoya 464 Japan     

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.     

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     

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.     

Further investigations on the structure and function of the saccus vasculosus of the rainbow trout,Salmo gairdneri Richardson

Alkaline phosphatase activity in coronet cells of the saccus vasculosus of the rainbow trout was localized ultracytochemically. Deposits of reaction product were found in varying amounts on the membranes of primary vesicles in the globules. This observation is discussed in relation to other morphological data and the possible resorptive function of the coronet cells in the homeostasis of the CSF.     

Effects of cortisol on gill chloride cell morphology and ionic uptake in the freshwater trout,Salmo gairdneri

Summary Daily intramuscular injection of cortisol (4 mg kg^–1 body weight) in rainbow trout,Salmo gairdneri, for 10 days caused significant increases in the number and individual apical surface area of gill chloride cells per mm² of filament epithelium. Concomitantly, whole body influxes of sodium (Na⁺) and chloride (Cl^–) increased. Acute (3 h) intra-arterial infusion of cortisol did not affect whole body Na⁺ or Cl^– influx. A significant correlation was observed between both Na⁺ and Cl^– influxes and the fractional apical surface area of filament chloride cells in control, sham (saline-injected) and experimental (cortisol-injected) fish. The chloride cells displayed similar ultrastructural modifications in trout undergoing cortisol treatment as in trout transferred to ion-deficient water. These findings suggest the existence of structure/function relationships in which branchial chloride cell morphology is an important determinant of Na⁺ and Cl^– transport capacity. We conclude that chronic cortisol treatment enhances whole body Na⁺ and Cl^– influxes by promoting proliferation of branchial chloride cells. The results of correlation analysis indicate that the chloride cell is an important site of NaCl uptake in freshwater rainbow trout.     

Arterio-venous anastomoses in rainbow trout gill filaments

Summary The origin of arterio-venous anastomoses, connecting the efferent filament artery (EFA) with the central venous sinus (CVS) of gill filaments can be well discerned by scanning electron microscopy in the rainbow trout. Corresponding vessels between the afferent filament artery and the CVS could not be detected with the techniques applied. AVA-specific endothelial cells are characterized by their bulky shape and their microvillous surface. The general morphology of AVA's in Salmo gairdneri is very similar to that of AVA's in Tilapia mossambica (Vogel et al., 1974) but they are much longer in the trout. No filament whorls have been encountered in AVA endothelia of Salmo gairdneri.This study is dedicated to Prof. Dr. W. Graumann, Director of the Institute of Anatomy, University of Tübingen, on the occasion of his 60th birthday. It was supported by the Deutsche Forschungsgemeinschaft     

Photoreceptor development in the pineal organ and the eye of Plecoglossus altivelis and Paralichthys olivaceus (Teleostei)

Summary The ontogenetic apperance of pineal photo-receptors was compared with that of retinal photoreceptors in the ayu Plecoglossus altivelis and the lefteye flounder Paralichthys olivaceus, which hatched 10 days and 3 days after fertilization, respectively. Despite the disparity in incubation time, the outer segments (containing membranous lamellae) of the pineal photoreceptors first appeared from 3 to 4 days after fertilization in both species. In contrast, the outer segments of the retinal photoreceptors first became visible 5 to 6 days after fertilization, although a characteristic retinal stratification and the optic tract leaving the ganglion cell layer were already found 4 days after fertilization in both species. The functional significance of these temporal disparities and/or similarities in photoreceptor development are discussed with special reference to the timing of daily rhythmic activities during the early developmental period of the teleosts.The results were presented at the Annual Meeting of the Japanese Society of Scientific Fisheries on April 2, 1990 (Tokyo)