Cell Tiss. Res. 182, 383-399 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
The Organum vasculosum laminae terminalis A Cytophysiological Study in the Duck, Anas platyrhynchos * O. Bosler Laboratoire de Neuroendocrinologie, E.R.A. 85 du C.N.R.S., Universit6 des Sciences de Montpellier, France (Professor I. Assenmacher, Director), and D6partement de Neurobiologie Cellulaire, I.N.P., C.N.R.S., 31 chemin 3.-Aiguier, Marseille, France (Dr. A. Calas, Director)
Summary. The Organum vasculosum laminae terminalis (OVLT) of the duck is lined innerly by specialized ependymal cells (tanycytes) and outwardly by a well-developed superficial vascular network, the capillaries of which often show a fenestrated endothelium. The OVLT also includes glial cells, internal non-fenestrated capillaries, bundles of fine nerve fibers and three groups of axonal swellings. One type contains granulations of 1000-1400 A. in diameter as well as 300-500/~ clear vesicles. The second type exhibits granulations and dense core vesicles of 500-800/~ in diameter along with small electron-lucent vesicles having diameters of 300-400/~. In the third type, exclusively clear vesicles 300-600 A in diameter are found. Asymmetrical synapses on dendrites and neuronal perikarya are found at every level of the organ. In the most external zone, the interposition of tanycyte endings sometimes allows neurosecretory axons to reach the parenchymal basement membrane (basal lamina). When tritiated molecules (amino acids or monoamines) are administered either in vitro by incubation or in vivo by intraventricular injections, radioautographic grains are observed over the tanycyte perikarya. Although this labeling is observed at every time point following the administration of the tracers, within three minutes only 3H-GABA appears to be concentrated in the cytoplasmic processes of the tanycytes. 3H-noradrenaline and 3H-serotonin are taken up and retained by some axons of the second type described above. Noradrenergic fibers are primarily localized in the inner zone of the OVLT where they display axodendritic synaptic contacts. Serotonergic fibers appear sparsely distributed in the OVLT but are more numerous in the lateral edges of the organ where synaptic differentiations on dendrites or on dendritic spines are also observed. Send offprint requests to: Dr. Olivier Bosler, Laboratoire de Neuroendocrinologie, Universit6 des
Sciences, Place Eug+ne-Bataillon, F-34060 Montpellier, Cedex, France * Supported by the D6partement de Biologie du C.E.A., and the I.N.S.E.R.M. (C.R.A.T., 74.1.438.45)
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It is concluded that the duck OVLT probably displays a neuroendocrine activity. Uptake and selective transport of exogenous molecules by tanycytes are also suggested by the present radioautographic observations. Finally, monoaminergic innervation is discussed at the OVLT level with special reference to the occurrence of serotonergic synapses. Key words: Organum vasculosum laminae terminalis - Radioautography Tanycytes - Monoaminergic innervation - Domestic mallard.
Introduction
The ventricular walls of the vertebrate brain include specialized regions which appear to constitute privileged sites of exchange between nervous tissue, blood and cerebrospinal fluid (CSF) (Oksche, 1973; Knigge et al., 1975). One of these highly differentiated structures is the Organum vasculosum laminae terminalis (OVLT) originally named the supraoptic crest (Wislocki and King, 1936). Morphological observations have suggested a neurosecretory activity in the OVLT as in other "circumventricular organs" (Hofer, 1958; Weindl, 1973), in mammals (Weindl et al., 1967, 1968; R6hlich and Wenger, 1969; Le Beux, 1972; Weindl and Schinko, 1975) and in birds (Mikami et al., 1976; Mikami, 1976). Moreover, recent studies with complementary techniques have suggested the possible intervention of this organ in pituitary gonadotropic function in mammals (Barry et al., 1973; Zimmerman et al., 1974; S6t/tld et al., 1976; Kawakami et al., 1973; Kawakami and Sakuma, 1976; Brownstein et al., 1976; Wenger, 1976). A suitable model system for the study of such a regulation has been the bird, especially the photosensitive species such as the duck (Benoit and Assenmacher, 1955). Following the works of Benoit and Assenmacher (1955) and of Calas (1974) on the median eminence (ME) of the duck and those of Vors (1970) and Alonso (1973) on the posthypophysis of the duck, a cytophysiological approach was employed for the study of the OVLT in the same avian family. Initially, this organ is considered from a histological and ultrastructural point of view. Secondly, the intra-organ distribution of exogenous radioactive molecules is studied since previous studies have demonstrated that specialized ependymal cells, the tanycytes (Horstmann, 1954), are capable of transferring substances across circumventricular organs (Horstmann, 1954; Knigge et al., 1975; Reese and Brightman, 1968; Weindl, 1969). In the present study, tritiated monoamines or amino acids were administered either in vivo by stereotaxic intraventricular injections or in vitro by incubation of the isolated organ. Since the cerebral monoamines are probably involved not only in hypothalamic neuroendocrine mechanisms (Kordon, 1970) but also in tanycyte function in the ME (Calas, 1975; Nozaki, 1975), the localization and possible function of monoaminergic innervation at the OVLT level is discussed.
Material and Methods Thirty adult male Pekin ducks, Anas platyrhynchos, were used in this study. They were reared in natural temperature and photoperiodicity conditions and were sacrificed throughout the year.
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Morphological Techniques The animals were perfused intravascularly under Numbutal anesthesia first with a physiological saline solution and then with an appropriate fixative. For light microscopic examination the brains were fixed with Bouin-Hollande fluid, embedded in paraffin, sagitally sectioned at 8 Ilm, then stained with Cresyl violet. For electron microscopic investigation large pieces containing the OVLT were excised from the brains after perfusion and immersed in 3.64~ glutaraldehyde in 0.01 M phosphate buffer, pH 7.4. Carefully dissected blocks were post-fixed with 2 ~ osmium tetroxide in the same buffer, dehydrated in ethanol and embedded in Epon. The polymerized pieces were sectioned with a Reichert OmU2 ultramicrotome. Control semithin sections were stained with toluidine blue for light microscopic examination. The ultrathin sections of the OVLT were contrasted with a 5 ~ alcoholic solution of uranyl acetate and then with lead citrate, and were observed with a JEM-100 B electron microscope.
Radioautography The two radioactive monoamines used in this study were 3H-noradrenaline (3H-1,1' NA, specific activity (SA): 20Ci/mmole, C.E.A. Saclay, France) and 3H-serotonin (5-hydroxytryptamine, 3H-5 HT, G, S.A.: 5-14 Ci/mmole, Radiochemical Centre Amersham, England). Six hours before administering the tracers, the animals were intraperitoneally injected with nialamide, 200 mg/kg (generously supplied by Pfizer). The other tritiated molecules used were: 3H-L-histidine (3H-2,5 His, SA: 20 Ci/mmole), 3H-L-proline (3H-5 Pro, SA: 20 Ci/mmole) and 3H-~-aminobutyric acid (3H-2,3 GABA, SA: 43 Ci/mmole) from C.E.A. Saclay, France. For in vivo experiments, the tracers were dried under nitrogen flow, then reconstituted in 50 ~tl of artificial CSF (Merlis, 1940) to obtain a final concentration of 10-5 M monoamine or amino acid. They were then stereotaxically introduced into the third cerebral ventricle. The brains were fixed after 15 or 30min for monoamines and after 10 or 90min for amino acids by intravascular perfusion with glutaraldehyde as described above. For the in vitro experiments, the animals were sacrificed by decapitation and the OVLT, rapidly dissected out, was incubated for 30 rain at 37~ in oxygenated Tyrode solution containing the tritiated monoamine at a concentration of 10-5 M to 10 -7 M. For incubation with tritiated amino acids, a shorter time was employed (3min) and the concentration of the amino acid was 10 -6 M. After rinsing in Tyrode solution the pieces were fixed with glutaraldehyde. Following the in vivo and in vitro administration of the tracers, the specimens were prepared for electron microscopy as described above. For radioautography semithin sections were affixed to glass slides and coated by dipping in Ilford Emulsion K5 diluted 1:1. These light microscopic radioautographs were developed in Kodak D I 9 after 8 to 20 days of exposure and were then stained with toluidine blue. For high resolution radioautography, ultrathin sections on a celloidin film deposited on glass slides were stained with uranyl acetate and lead citrate and lightly vaporized with carbon. After dipping in Ilford emulsion L4 diluted 1:4 the radioautographs were exposed for 20 to 60 days at which time they were processed in Kodak Microdol X or in Phenidon and collected on grids. After the celloidin membranes had been thinned by isoamyl acetate, the grids were observed with a Jem 100-B electron microscope.
ResuRs A. Light Microscopic Examination ( F i g . 1 A a n d B) W h e n o b s e r v e d i n a s a g i t t a l p l a n e , t h e lamina terminal& o f t h e d u c k s t r e t c h e s obliquely from the optic chiasma to the anterior commissure and borders on the p r e o p t i c r e c e s s o f t h e t h i r d v e n t r i c l e . O n t h e o u t e r side, it is l i m i t e d b y t h e r i c h v a s c u l a r s u p p l y o f t h e c&terna praechiasmatica. T h e O V L T is t h a t v e r y t h i n p a r t o f t h e lamina terminal& w h i c h e x h i b i t s s p e c i a l i z e d e p e n d y m a l cells, t h e t a n y c y t e s ( H o r s t m a n n , 1954).
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Fig. 1. A Sagittal section of the duck OVLT. Cresyl violet stain. POR preoptic recess; OC optic chiasma; SP superficial plexus; N nerve ceil bodies, x 100. B Semithin section of the duck OVLT. Internal zone. Toluidine blue stain. Ep ependyma; N neuron; GL glial cell; IC internal capillary. x 1200
The p e r i k a r y a of these cells display a typical elongated a n d regularly shaped n u c l e u s a n d constitute a n i n t e r n a l m o n o c e l l u l a r layer. A m o r e diversified sube p e n d y m a l layer c o n t a i n s n u m e r o u s r o u n d e d glial cells, as well as several n e u r o n s , nerve fibers a n d small capillaries. I n the thicker external zone, these O V L T comp o n e n t s are separated by cytoplasmic processes of tanycytes which extend toward the vascular network.
Fig. 2 A - D . Internal (A, B, C) and external zone of the duck OVLT. A OVLT tanycytes joined by apical junctional complexes (arrows) show microvilli (MV) and protrusions (P) into the lumen of the 3rd ventricle (3rd V) and cytoplasmic processes (TP). G Golgi apparatus; M mitochondria; L lysosome. • 6500. B Cytoplasmic branched process of a tanycyte rich in microfilaments. M mitochondria. • 19,000. C Non-fenestrated internal capillary surrounded by a narrow perivascular space (PS). • 4200. D A glial cell body (GL) abuts on the peripheral basement m e m b r a n e (B) lining the perivascular space (PS) of an external fenestrated capillary (arrows). P tanycytic or glial processes; FB fibroblastic processes. • 10,000
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Fig. 3. Subependymalperikaryonof the duck OVLT. Note the preferentiallocation of neurosecretory granules (NG) within the Golgi area (G). RER rough endoplasmic reticulurn; M mitochondria; Nnucleus. x 17,000
B. Electron Microseopic Examination 1. Ependyma. In the vicinity of the OVLT (lateral, rostral and caudal edges) typical ependymal cells are observed. The ependymal tanycytes, however, contain nuclei which are more elliptical and have a more irregular size distribution (Fig. 2 A). Their apical membranes, presenting some bulbous extensions and a few microvilli into the third ventricle, are generally devoid of cilia. In their most apical part, the interdigitated lateral membranes display zonulae adhaerentes and desmosomes but the identification of tight junctions above these junctional complexes is impossible without high molecular weight tracer experiments (Brightmann and Reese, 1969). The cytoplasmic organelles are confined to the supranuclear region of the cells and include numerous ribosomes which are either free or bound to fragments o f endoplasmic reticulum, a well developed Golgi apparatus, mitochondria, some inclusions (lysosomes, lipofuscin) and many micro filaments. Below the somatic region, the ependymal tanycyte consists of a long and branched process (Fig. 2 B) projecting from the soma through the neuropil and abutting with foot-like endings on the peripheral basal lamina (Fig. 4B). These processes, composed of many
Fig. 4 A - F . Innervation of the duck OVLT. A Internal zone. Bundles of small unmyelinated axons (*) are isolated from each other by the cytoplasmic processes (P) of tanycytes and glial cells (GL). x 5000. B External zone. A type a neurosecretory axon reaches the parenchymal basement membrane (arrow). A x a other axons of the first type; P S perivascular space; GL glial cell. x 8000. C Two nerve fibers of type b display asymmetrical synaptic contacts with the same dendrite (D 1). One of them also constitutes a synaptic afference on another dendritic profile (D2). Arrows indicate dense core vesicles. • 23,000. D Asymmetrical synaptic contact between a type c axon and a dendrite (arrow). M mitochondria, x 23,000. E A neuronal perikaryon receives a synaptic afference of type b (arrow). • 16,000. F A synaptic differentiation (arrow) is also observed on a dendrite belonging to a neuron of the organ (N). • 12,000
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microfilaments, contain longitudinally oriented mitochondria and some dense inclusions, more frequently observed in their endings. 2. Neurons. The OVLT of the duck contains some neuronal perikarya 8-10 ~tm
in diameter. They are scattered in the subependymal and external regions and appear as large cells with a clear cytoplasm and nucleoplasm and an irregularly shaped nucleus. The endoplasmic reticulum is predominantly granular and a welldeveloped Golgi apparatus is also present. Free ribosomes, large and circular mitochondria, multivesicular bodies, lysosomal bodies and lipid droplets are seen throughout the cytoplasm. Neurosecretory granules, 1000-2000 A in diameter, can sometimes be observed, primarily in the Golgi area of the cell (Fig. 3). 3. Glial Cells. Glial cells are found scattered throughout the entire organ, being
more numerous in the subependymal layer. Glial and ependymal cells have a similar appearance, but the former present a more rounded cell body and nucleus. In addition, glial cells seem to give rise to cytoplasmic processes toward the superficial region of the OVLT, where a glial cell body can occasionally be seen to contact the parenchymal basal lamina (Fig. 2 D). 4. Nerve Fibers. The OVLT of the duck exhibits a peculiar arrangement of different
types of nerve fibers. Numerous bundles of small axons (0.1 to 0.5 ~tm in diameter), generally unmyelinated and containing microtubules, run in a rostro-caudal direction in the thickness of the organ (Fig. 4A). Some larger axonal sections containing vesicles with dense material and/or clear vesicles are also found throughout the OVLT. They can be classified into three principal groups (Fig. 4): a) This category includes axons containing granulations of about 1000-2000/~ in diameter (the electron dense material occupies the whole vesicle) and some clear vesicles (300-500 A in diameter). b) Granulations 500-800 A, in diameter as well as other vesicles with a smaller dense core are observed in the numerous axons of this category. Some electron lucent vesicles (300400 A) are either intermingled with them or confined to a restricted area, namely the vicinity of the axonal membrane. c) This group includes the smallest number of axons which contain only clear vesicles 300 to 600 A in diameter.
Fig. 5 A - E . Light microscopic (A-B) and high resolution (C-E) radioautographs of duck OVLT 30min after intraventricular injections of tritiated monoamines. A 3H-NA. Sagittal section. The reaction is more intense over the inner zone (In Z) of the OVLT. Note the prominent labeling of ependyma and the occurrence of clusters of silver grains over the subependymal zone (arrows). Ex Z external zone. • 300. B 3H-5HT. Frontal section. Scattered silver grains have no preferential localization. Dense accumulations are more numerous over the lateral edges of the lamina terminalis. O V organum vasculosum. • 200. C 3H-NA. A large varicosity of the subependymal zone (type b) exhibits an intense labeling. Non-labeled fibers belonging to the same axonal group are also present. T tanycyte nucleus. TP tanycyte process. • 12,000. D 3H-NA. Only one type b fiber is labeled and displays an axodendritic synaptic contact. TP tanycyte process. • 18,000. E 3H-5HT. A synaptic contact is observed lateral to the OVLT between a labeled type b axon and a dendritic spine (DS). x 28,000
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All these fibers are present at each level of the organ although the type (a) is preferentially observed in the most external zone. Certain types (b and c) occasionally establish synaptic contacts, generally of the asymmetrical type (type 1 according to Gray, 1959), with neuronal perikarya or, more frequently, with dendrites (Fig. 4C, D, E, F). In the external region, the foot-like endings oftanycyte processes sometimes allow type (a) axonal endings to abut on the outer basement membrane of the OVLT.
5. Vascularization. The duck OVLT exhibits both internal and external capillaries. Their endothelial cells are surmounted by narrow or large perivascular spaces and contain collagen fibrils and fibroblastic processes. In contrast to internal vessels, however, only the external capillaries often show a fenestrated endothelium (Fig. 2C, D).
C. Radioautographic Study of the Incorporation of Tritiated Molecules 1. Monoamines. In light microscopic radioautographs, the localization of the reaction in the OVLT is similar after an incubation in vitro and an intraventricular administration of the tracers. In addition to scattered silver grains, some intense accumulations are observed, the distribution of which differs depending on the tracer (Fig. 5 A, B). With 3H-NA the labeling is more significant over the inner zone of the OVLT and particularly over the ependyma. Clumps of silver grains are seen essentially in the subependymal zone. With 3H-5 HT, these intense accumulations of silver grains appear more evident in the lateral edges, where the organ becomes thicker; some are also present on the subependymal and outer regions. At the ultrastructural level, the intense ependymal radioautographic reaction is located primarily in the perikarya of tanycytes; the labeling of their processes is not prominent against the diffuse reaction observed in the surrounding neuropil. The aggregates of silver grains observed after light microscopic examination are found to correspond to axonal varicosities. The specific distribution for 3N-NA and 3H-5 HT is confirmed. The 3H-NA is incorporated into small or large axons which are scattered among non-labeled axons and which are contained in the second category described above (Fig. 5 C, D). Some of them establish axodendritic synaptic contacts (Fig. 5D). With 3H-5 HT, the labeled fibers are found to be exclusively small axons belonging to the same group. In addition, true asymmetrical synaptic differentiations on dendrites or on dendritic spines are also observed (Fig. 5 E). 2. Amino Acids. In addition to a significant labeling of the ependyma with 3H-His or 3H-Pro, certain neuronal cell bodies exhibit more silver grains than their surroundings. Also the radioautographic reaction is more intense on the tanycytes of the OVLT than on the lateral ependyma of the lamina terminalis, 90 min following the intraventricular administration of 3H-His (Fig. 6 A). Within three minutes of the administration of 3H-GABA in vitro the tanycytes of the OVLT exhibit a very high degree of labeling which extends to their extremities (Fig. 6 B). Glial and endothelial vascular cells are also significantly labeled.
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Fig. 6 A - C . Radioautographs of semithin (A-B) and thin (C) sections of duck O V L T after administration o f tritiated amino acids. A Intraventricular administration of 3H-His and fixation after 90 min. Frontal section. The most intense radioautographic accumulations of silver grains are seen u p o n the tanycytes of the thinner part of the lamina terminalis which constitutes the OVLT (limited by the two arrows). Note the preferential labeling of ependyma (Ep). S V superficial vessels. • 180. B and C In vitro incubation with 3H-GABA for 3 min. Frontal sections. B Ependyma (Ep), tanycyte processes (arrows) and vascular endothelium are significantly labeled. S V superficial vessels. • 180. C Note the distinct labeling of tanycyte processes (TP) at the ultrastructural level. Phenidon developer. M mitochondria, Ax a type a neurosecretory axons, x 13,500
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The light microscopic radioautographic images obtained for each tritiated molecule are confirmed at the ultrastructural level. 3H-His and 3H-Pro are primarily retained in tanycyte cell bodies, in neuronal perikarya and in dendrites. Finally, although 3H-GABA yields an intense diffuse labeling over all tissue elements, silver grains are clearly prominent on the perikarya and processes of tanycytes (Fig. 6 C).
Discussion The 0 V L T as a Potential Site o f Neurohemal Release
The occurrence of various nerve fibers containing granulations as well as dense and/or clear vesicles in the OVLT of the duck suggests a neuroendocrine activity at this level. The conditions of fixation used in the present study result in the description of three principal groups of axonal profiles. Type a neurosecretory fibers predominate in the external zone close to the superficial capillaries, the fenestrated endothelium of which provides a morphological support for the supposition of a neurohemal release. Moreover, some of these axons are sometimes in close association with the parenchymal basement membrane which lines the perivascular space. In addition, as postulated by Kobayashi (1975), the contents of axon endings which are isolated from the capillary wall by the interposition of tanycyte processes might also be released into the blood by passing between and/or through these processes. Morphological signs of neuroendocrine activity in the OVLT have also been recently reported in other avian species, the White-crowned Sparrow (Mikami et al., 1976) and the quail (Mikami, 1976), where both internal and external capillaries exhibit a fenestrated endothelium. In the duck, the different ultrastructural features of the two kinds of vessels are identical to those described in the ME (Calas and Assenmacher, 1970). In mammals, in addition to morphological data, some experimental evidence has already suggested the participation of the OVLT in neuroendocrine mechanisms. Ultrastructural changes in the OVLT were reported in the golden hamster following castration (Weindl and Schinko, 1975) and in the rat after ovariectomy or hypophysectomy (Wenger, 1976). Moreover, the electrical stimulation of the periventricular area which includes the OVLT induces a release of LH (Kawakami et al., 1973). Additional evidence that this organ may be involved in gonadotropic regulation is furnished by biochemical assays demonstrating that significant amounts of LH-RH are present in the rat OVLT (Brownstein et al., 1976). Immunocytochemical studies at the histological level have also contributed to the identification of this neurohormone in the OVLT of mammals (Barry et al., 1973; Zimmerman et al., 1974; Barry and Carette, 1975; Weindl and Schinko, 1975; S6tfil6 et al., 1976) and of the duck (Calas, 1975) where the external localization of LH-RHcontaining fibers is analogous to that of the ME (Calas et al., 1973). In addition, other neurohormones were also detected in the OVLT of mammals: somato-
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statin (Pelletier et al., 1976), vasopressin and oxytocin (Zimmerman and Lobo Antunes, 1976). If neurohormones (and other molecules) can be released into the blood at the level of external vessels, the fenestrations of endothelial cells might also allow passage of intravascular substances in the opposite direction, as already demonstrated in the rat (Weindl, 1969). Thus, the OVLT, like the other "windows" of the brain (Knigge, 1975), might be involved in hormonal feed-back circuits directed toward the central nervous system. Large molecules, however, might be intracellularly transported through the tanycytes (Nakai and Naito, 1975).
The Function o f Tanycytes
In the last few years, the tanycytes have been considered only as an anatomical (Weindl and Joynt, 1972) or a functional link (Knigge and Silverman, 1972; Kobayashi et al., 1972; Knigge et al., 1975) between CSF and blood in the ME. From this latter point of view, the tanycytes of the ME would have the ability to take up and transfer certain substances such as ~-amino-butyric acid and thyroxine (Knigge and Silverman, 1972), peroxidase (Kobayashi et al., 1972), LH-RH (Uemura et al., 1975) and dopamine (Scott and Krobisch-Dudley, 1975). The specialized ependymal cells of the duck OVLT exhibit the same morphological features as ME tanycytes; possible functional similarities may then be discussed in relation to our radioautographic study of the incorporation of tritiated monoamines and amino acids. In the present experiments, including the use of nialamide to inhibit the major (3H-NA) or the sole (3H-5 HT) way of tritiated monoamine catabolism (monoamine oxidase), the diffuse radioautographic distribution of silver grains is to be related essentially to free 3H-NA or 3H-5HT retained by the glutaraldehyde (Descarries and Droz, 1970). Our radioautographic technique is also reliable for the detection of the sites of uptake and/or retention of tritiated amino acids (Larra and Droz, 1970). Free radiolabeled amino acids may be visualized by using glutaraldehyde as a fixative. Thus, tritiated monoamines and amino acids are shown to penetrate the duck OVLT after their administration in vitro or in vivo. The pronounced radioautographic reaction generally observed on the ependyma suggests an uptake of these radiolabeled molecules by the perikarya of tanycytes. Our present observations show, however, that although 3H-His labels the OVLT intensely and more specifically its ependyma, only 3H-GABA appears to have been clearly accumulated in the tanycyte processes. On the other hand, since data concerning a possible release of this 3H-GABA at a site different from that of its uptake are lacking, we cannot clearly relate the ubiquitous labeling of tanycyte cytoplasm to a functional transependymal transfer. If such a transport does exist in the duck OVLT, it would be very selective, as already suggested by the analogous incorporation of 3H-His (and not 3H-Pro) in the ME (Calas, 1975). Moreover, our present results along with those of Calas suggest that a functional difference probably exists between ME and OVLT tanycytes. Transependymal transport as a potential neuroendocrine route in the ME is
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supported both by the identification of several factors in the CSF, namely adenohypophyseal hormones (Linfoot et al., 1970; Kendall et al., 1975), neurohormones (Heller, 1969; Joseph et al., 1975) and neuromediators (Denker and H~iggendal, 1969) and by the effect of intraventricularly injected LH-RH on serum LH level (Weiner et al., 1972; Ben-Jonathan et al., 1974; Uemura et al., 1975) and TRH on serum TSH level (Gordon et al., 1972). The OVLT probably participates in neuroendocrine regulations according to the accepted concept of axonal transfer, storage and excretion of hypothalamic neurohormones: it might also be involved in another, transependymal, alternative route.
The Monoaminergic Innervation of the 0 VLT If the diffuse radioautographic distribution of silver grains is to be related essentially to free 3H-NA or 3H-5 HT in the detection of tritiated monoamines, then the intense sites of accumulation reveal the structures which have concentrated the tracers (Descarries and Droz, 1970), i.e. monoaminergic fibers (Taxi and Droz, 1969). Although these fibers belong to the same group (type b which also includes non-monoaminergic axons), some facts argue for a selective uptake of 3H-NA and 3H-5HT by distinct noradrenergic and serotonergic fibers, including the different topographic distribution of sequestering axons after the administration of either 3H-NA or 3H-5 HT. In addition, the identity of the radioautographic reaction after both in vivo experiments and in vitro incubations in Tyrode medium containing 10 7 M 3H-5HT also eliminates the possibility of a non-specific uptake by catecholaminergic axons (Shaskan and Snyder, 1970). According to Descarries et al. (1976), the specificity of the in vivo uptake of 3H-5 HT injected at a higher concentration might be accounted for by an intraventricular and intratissual dilution of the tracer. Concerning the labeling of catecholaminergic fibers by 3H-NA, the reactive axons of the duck OVLT are most probably noradrenergic since under these experimental conditions dopaminergic fibers cannot be distinctly detected by radioautography (Calas et al., 1976). Thus, the internal localization of noradrenergic fibers appears to be quite similar to that of the ME of the duck (Calas, 1973). In contrast, most of the indolaminergic fibers are more laterally distributed and do not appear to innervate specifically the OVLT. However, the occurrence of true serotonergic synapses (in addition to noradrenergic ones) on dendrites and dendritic spines at the lamina terminalis level argues for a possible direct intervention of serotonergic fibers on neurosecretory neurons. Non-monoaminergic synapses are also present throughout the organ. Based on light microscopic radioautographic observations, the monoaminergic innervation of the OVLT does not appear to be significant. This observation agrees with biochemical results of Saavedra et al. (1976) who found moderate amounts of noradrenaline and serotonin in the rat OVLT. In contrast, cholinergic structures are probably more numerous than monoaminergic structures, as assessed by the detection of high levels of choline acetyltransferase activity (Saavedra et al., 1976).
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Conclusion The OVLT of the duck appears to be a neurovascular structure which is similar to the ME not only by its constitutive elements but also by its noteworthy abilities of exchange between blood, CSF and nervous tissue. Such exchange mechanisms most probably involve a neurohemal release of molecules from neurosecretory axons. Moreover, because of their close relationship with the CSF and the external perivascular space, the OVLT tanycytes appear to be capable of taking up and selectively translocating certain substances between the ventricular and the vascular pole of the organ. Concerning its monoaminergic innervation, the occurrence of noradrenergic and serotonergic synapses supports a direct, although discrete, participation of these monoamines in neuroendocrine mechanisms at the OVLT level. In addition, this "window" of the brain appears to be a good model for the study of the synaptic control of serotonin on neurosecretory neurons.
References Alonso, G.: Etude morpho-fonctionnelle du syst6me neuros6cr6toire hypothalamo-posthypophysaire chez le Canard et le Rat: R61e de l'innervation monoaminergique. Th~se Sp6c. Physiol., Montpellier (1973) Barry, J., Carette, B.: Immunofluorescence study of LRF neurons in primates. Cell Tiss. Res. 164, 163-178 (1975) Barry, J., Dubois, M.P., Poulain, P.: LRF producing cells of the mammalian hypothalamus. A fluorescent antibody study. Z. Zellforsch. 146, 351-366 (1973) Ben-Jonathan, N., Mical, R.S., Porter, J.C.: Transport of LRF from CSF to hypophysial portal and systemic blood and the release of LH. Endocrinol. 95, 18-25 (1974) Benoit, J., Assenmacher, I.: Le contr61e hypothalamique de l'activit6 pr6hypophysaire gonadotrope. J. Physiol. (Paris) 47, 427-567 (1955) Brightman, M.W., Reese, T.S.: Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol. 40, 648-677 (1969) Brownstein, M.J., Palkovits, M., Kizer, J.S. : On the origin of luteinizing hormone-releasing hormone (LH-RH) in the supraoptic crest. Life Sci. 17, 679-682 (1976) Calas, A.: L'innervation monoaminergique de l'6minence m6diane. Etude radioautographique et pharmacologique chez le Canard, Arias platyrhynchos. I. L'innervation cat6cholaminergique. Z. Zellforsch. 138, 503-522 (1973) Calas, A.: L'innervation peptidergique et monoaminergique de l'6minence m6diane. Etude cytophysiologique chez les Oiseaux. Th6se Sci., Montpellier (1974) Calas, A.: The avian median eminence as a model for diversified neuroendocrine routes. In: Brainendocrine interaction. II. The ventricular system. 2rid Int. Symp., Shizuoka 1974 (K.M. Knigge, D.E. Scott, H. Kobayashi, S. Ishii, eds.), pp. 54-69. Basel: Karger 1975 Calas, A., Assenmacher, I.: Ultrastructure de l'Eminence M6diane du Canard. Z. Zellforsch. 109, 64-82 (1970) Calas, A., Besson, M.J., Gauchy, C., Alonso, G., Glowinski, J., Cheramy, A. : Radioautographic study of in vivo incorporation of 3H-monoamine in the cat caudate nucleus. Identification of serotoninergic fibers. Brain Res. 118, 1-13 (1976) Calas, A., Kerdelhu6, B., Assenmacher, I., Jutisz, M.: Les axones/l LH-RH de l'6minence m6diane. Mise en 6vidence chez le Canard par une technique immunocytochimique. C.R. Acad. Sci. (Paris) 277, 2765-2768 (1973) Denker, S.J., H~iggendal, C.J.: Noradrenaline and cerebrospinal fluid. In: Sterba Zirkumventrikul/ire Organe und Liquor, pp. 243-247: Jena: Fischer 1969 Descarries, L., Beaudet, A., Watkins, K.C.: Serotonin nerve terminals in adult rat neocortex. Brain Res. 100, 563-588 (1975)
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Descarries, L., Droz, B.: Intraneural distribution of exogenous norepinephrine in the central nervous system of the rat. J. Cell Biol. 44, 385-399 (1970) Gordon, J.H., Bollinger, J., Reichlin, S.: Plasma thyrotropin-releasing hormone after injection into the third ventricle, systemic circulation, median eminence and anterior pituitary. Endocrinol. 91, 696 701 (1972) Gray, E.G.: Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscopic study. J. Anat. (Lond.) 93, 420-432 (1959) I-feller, H.: Neurohypophysial hormones in the cerebrospinal fluid. In: Sterba, Zirkumventrikul/ire Organe und Liquor, pp. 232-235. Jena: Fischer 1969 Hofer, H.: Zur Morphologie der circumventricularen Organe des Zwischenhirns der S/iugetiere. Verh. Dtsch. Zool. Ges., Frankfurt, pp. 202-251 (1958) Horstmann, E.: Die Faserglia des Selaehiergehirns. Z. Zellforsch. 39, 588-617 (1954) Joseph, S.A., Sorrentino, S., Sundberg, D.K.: Releasing hormones, LRF and TRF, in the cerebrospinal fluid of the third ventricle. In: Brain-endocrine interaction. II. The ventricular system. 2nd [nt. Symp., Shizuoka 1974 (K.M. Knigge, D.E. Scott, H. Kobayashi, S. 1shii, eds.), pp. 306-312. Basel: Karger 1975 Kawakami,. M., Kimura, F., Konno, T.: Possible role of the medial basal prechiasmatic area in the release of LH and prolactin in rats. Endocrin. jap. 20, 335 344 (1973) Kawakami, M., Sakuma, Y.: Electrophysiological evidences for possible participation of periventricular neurons in anterior pituitary regulation. Brain Res. 101, 79-94 (1976) Kendall, J.W., Seaich, J.L., Allen, J.P., Vanderlaan, W.P.: Pituitary-CSF relationships in man. In: Brain-endocrine interaction. II. The ventricular system. 2nd Int. Syrup., Shizuoka 1974 (K.M. Knigge, D.E. Scott, H. Kobayashi, S. Ishii, eds.), pp. 313-323. Basel: Karger 1975 Knigge, K.M.: Opening remarks. In: Brain-endocrine interaction. II. The ventricular system. 2nd Int. Syrup., Shizuoka 1974 (K.M. Knigge, D.E. Scott, H. Kobayashi, S. Ishii, eds.), pp. 1-2. Basel: Karger 1975 Knigge, K.M., Scott, D.E., Kobayashi, H., lshii, S.: Brain-endocrine interaction. 1I. The ventricular system in neuroendocrine mechanisms. 2nd tat. Syrup., Shizuoka 1974. Basel: Karger I975 Knigge, K M . , Silverman, A.J.: Transport capacity of the median eminence. In: Brain-endocrine interaction. I. Median eminence: structure and function. Int. Symp., Munich 197l (K.M. Knigge, D.E. Scott, A. Weindl, eds.), pp. 350-363. Basel: Karger 1972 Kobayashi, H.: Absorption of cerebrospinal fluid by ependymal cells of the median eminence. In: Brain-endocrine interaction. II. The ventricular system. 2nd Int. Syrup., Shizuoka 1974 (K.M. Knigge, D.E. Scott, H. Kobayashi, S. Ishii, eds.), pp. 109-122. Basel: Karger 1975 Kobayashi, H., Waba, M., Uemura, H., Ueck, M.: Uptake of peroxidase from the third ventricle by ependymal cells of the median eminence. Z. Zellforsch. 127, 545 551 (1972) Kordon, C.: R61e des monoamines dans les regulations ad6nohypophysaires. In: Neuroendocrinologie (C. Kordon et J. Benoit, eds.), pp. 78-91. Paris: C.N.R.S. 1970 Larra, F., Droz, B.: Techniques radioautographiques et leur application g l'6tude du renouvellement des constituants cellulaires. J. Microscopie 9, 845-880 (1970) Le Beux, Y.J.: An ultrastructural study of the neurosecretory cells of the medial vascular prechiasmatic gtand. II. Nerve endings. Z. Zellforsch. 127, 439 461 (1972) Linfoot, J.A., Garcia, J.F., We/, W., Fink, R., Satin, R., Barn, J.L., Lawrence, J.H.: Human growth hormone in cerebrospinal fluid. J. clin. Endocr. 31,230-232 (1970) Merlis, J.K.: The effect of changes in the calcium content of the cerebrospinal fluid on spinal reflex activity in the dog. Amer. J. Physiol. 131, 67-72 (1940) Mikami, S. : Ultrastructure of the organum vasculosum of the lamina terminalis of the Japanese quail, Coturnix coturnixjaponica. Cell Tiss. Res. 172, 22%243 (1976) Mikami, S., Kawamura, K., Oksche, A., Farner, D.S.: The fine structure of the hypothalamic secretory neurons of the White-crowned Sparrow, Zonotrichia leucophrys gambelii. II. Magnocellular and parvocellular nuclei of the rostral hypothalamus. Cell Tiss. Res. 165, 415 434 (1976) Nakai, Y., Naito, N.: Uptake and bidirectional transport of peroxidase injected into the blood and cerebrospinal fluid by ependymal cells of the median eminence. In: Brain-endocrine interaction. 1I. The ventricular system. 2nd Int. Syrup., Shizuoka 1974 (K.M. Knigge, D.E. Scott, H. Kobayashi, S. lshii, eds.), pp. 94-108. Basel: Karger 1975 Nozaki, M.: Tanycyte absorption affected by the hypothalamic deafferentation in Japanese quail, Coturnix coturnixjaponica. Cell Tiss. Res. 163, 433-443 (i975)
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Oksche, A.: Circumventricular structures and pituitary functions. Proceedings of the Fourth International Congress of Endocrinology, Washington, D.C., 1972, pp. 73-79. Amsterdam: Excerpta Medica 1973 PeUetier, G., Leclerc, R., Dube, D.: Immunohistochemical localization of hypothalamic hormones. J. Histochem. Cytochem. 24, 864-871 (1976) Reese, T.S., Brightman, M.W.: Similarity in structure and permeability to peroxidase of epithelia overlying fenestrated cerebral capillanes. Anat. Rec. 160, 414 (1968) R6hlich, P., Wenger, T.: Elektronenmlkroskopische Untersuchungen am Organon vasculosum laminae terminalis der Ratte. Z. Zellforsch. 102, 483-506 (1969) Saavedra, J.M., Brownstein, M.J., Kizer, J.S., Palkovits, M.: Biogenic amines and related enzymes in the circumventricular organs of the rat. Brain Res. 107, 412-417 (1976) Scott, D.E., Krobisch-Dudley, G.: Ultrastructural analysis of the mammalian median eminence. Morphological correlates of transependyma] transport. In: Brain-endocrine interaction. JI. The ventricutar system. 2nd Int. Symp., Shizuoka 1974 (K.M. Knigge, D.E. Scott, H. Kobayashi, S. Ishii, eds.), pp. 29-39. Basel: Karger 1975 S~tal6, G., Vigh, S., Schally, A.V., Arimura, A., Flerk6, B.: Immunohistological study of the origin of LH-RH-containing nerve fibers of the rat hypothalamus. Brain Res. 103, 597-602 (1976) Shaskan, E.G., Snyder, S.H.: Kinetics of serotonin accumulation into slices from rat brain: relationship to catecholamine uptake. J. Pharmacol. exp. Ther. 175, 404-418 (1970) Taxi, J., Droz, B. : Radioautographic study of the accumulation of some biogenic amines in the autonomic nervous system. In: Cellular dynamics of the neuron (S.M. Barondes, ed.), pp. 175-190. New York: Academic Press 1969 Uemura, H., Asai, T., Nozaki, M., Kobayashi, H.: Ependymal absorption of luteinizing hormonereleasing hormone injected into the third ventricle of the rat. Cell Tiss. Res. 160, 443-452 (1975) Vors, M,C.: Etude histologique et ultrastructurale de la posthypophyse du Canard. Th+se Sp6c. Physiol., MontpeUier (1970) Weindl, A.: Electron microscopic observations on the organum vasculosum of the lamina terminalis after i.v. injection of horseradish-peroxidase. Neurology (Minneap.) 19, 295 (1969) Weindl, A.: Neuroendocrine aspects of circumventricular organs. In: Ganong and Martini, Frontiers in neuroendocrinology, pp. 1-32. London: Oxford University Press 1973 Weindl, A., Joynt, R.J.: The median eminence as a circumventricular organ. In: Brain-endocrine interaction. I. Median eminence: structure and function. Int. Symp., Munich 1971 (K.M. Knigge, D.E. Scott, A. Weindl, eds.), pp. 280-297. Basel: Karger 1972 Weindl, A., Schinko, I.: Vascular and ventricular neurosecretion in the organum vasculosum of the lamina terminalis of the golden hamster. In: Brain-endocrine interaction. II. The ventricular system. 2nd Int. Symp., Shizuoka 1974 (K.M. Knigge, D.E. Scott, H. Kobayashi, S. Ishii, eds.), pp. 190-203. Basel: Karger 1975 Weindl, A., Schwink, A., Wetzstein, R.: Der Feinbau des Gef~il3organs der Lamina terminalis beim Kaninchen. I. Die GefiiBe. Z. Zellforsch. 79, 1-48 (1967) Weindl, A , Schwink, A., Wetzstein, R.: Der Feinbau des GefaBorgans der Lamina terminalis beim Kaninchen. II. Das neuronale und gliale Gewebe. Z. Zellforsch. 85, 552-600 (1968) Weiner, R.I., Terkel~ L~ Btake, C.A., Schal~y, A.V., Sawyer, C.A.: Changes in serum luteinizing hormone following intraventricular and intravenous injections of luteinizing hormone-releasing hormone in the rat. Neuroendocrinol. 10, 261-272 (1972) Wenger, T.: Ultrastructural changes in the vascular organ of the lamina terminalis following ovariectomy and hypophysectomy in the rat. Brain Res. 101, 95-102 (1976) Wislocki, G.B., King, L.S.: The permeability of the hypophysis and hypothalamus to vital dyes, with a study of the hypophyseal vascular supply. Amer. J. Anat. 58, 421-472 (1936) Zimmerman, E.A., Lobo Antunes, J.: Organization of the hypothalamic-pituitary system: current concepts from immunohistochemical studies. J. Histochem. Cytochem. 24, 807-815 (1976) Zimmerman, E.A., Scott, D.E., Kozlowski, G.P.: Localization of neurophysin, vasopressin and gonadotropin releasing hormone (GnRh) in the hypothalamus by immunoperoxidase technique. Endocrinol. 95, 1-8 (1974)
Accepted April 25, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
The Organum vasculosum laminae terminalis A Cytophysiological Study in the Duck, Anas platyrhynchos * O. Bosler Laboratoire de Neuroendocrinologie, E.R.A. 85 du C.N.R.S., Universit6 des Sciences de Montpellier, France (Professor I. Assenmacher, Director), and D6partement de Neurobiologie Cellulaire, I.N.P., C.N.R.S., 31 chemin 3.-Aiguier, Marseille, France (Dr. A. Calas, Director)
Summary. The Organum vasculosum laminae terminalis (OVLT) of the duck is lined innerly by specialized ependymal cells (tanycytes) and outwardly by a well-developed superficial vascular network, the capillaries of which often show a fenestrated endothelium. The OVLT also includes glial cells, internal non-fenestrated capillaries, bundles of fine nerve fibers and three groups of axonal swellings. One type contains granulations of 1000-1400 A. in diameter as well as 300-500/~ clear vesicles. The second type exhibits granulations and dense core vesicles of 500-800/~ in diameter along with small electron-lucent vesicles having diameters of 300-400/~. In the third type, exclusively clear vesicles 300-600 A in diameter are found. Asymmetrical synapses on dendrites and neuronal perikarya are found at every level of the organ. In the most external zone, the interposition of tanycyte endings sometimes allows neurosecretory axons to reach the parenchymal basement membrane (basal lamina). When tritiated molecules (amino acids or monoamines) are administered either in vitro by incubation or in vivo by intraventricular injections, radioautographic grains are observed over the tanycyte perikarya. Although this labeling is observed at every time point following the administration of the tracers, within three minutes only 3H-GABA appears to be concentrated in the cytoplasmic processes of the tanycytes. 3H-noradrenaline and 3H-serotonin are taken up and retained by some axons of the second type described above. Noradrenergic fibers are primarily localized in the inner zone of the OVLT where they display axodendritic synaptic contacts. Serotonergic fibers appear sparsely distributed in the OVLT but are more numerous in the lateral edges of the organ where synaptic differentiations on dendrites or on dendritic spines are also observed. Send offprint requests to: Dr. Olivier Bosler, Laboratoire de Neuroendocrinologie, Universit6 des
Sciences, Place Eug+ne-Bataillon, F-34060 Montpellier, Cedex, France * Supported by the D6partement de Biologie du C.E.A., and the I.N.S.E.R.M. (C.R.A.T., 74.1.438.45)
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It is concluded that the duck OVLT probably displays a neuroendocrine activity. Uptake and selective transport of exogenous molecules by tanycytes are also suggested by the present radioautographic observations. Finally, monoaminergic innervation is discussed at the OVLT level with special reference to the occurrence of serotonergic synapses. Key words: Organum vasculosum laminae terminalis - Radioautography Tanycytes - Monoaminergic innervation - Domestic mallard.
Introduction
The ventricular walls of the vertebrate brain include specialized regions which appear to constitute privileged sites of exchange between nervous tissue, blood and cerebrospinal fluid (CSF) (Oksche, 1973; Knigge et al., 1975). One of these highly differentiated structures is the Organum vasculosum laminae terminalis (OVLT) originally named the supraoptic crest (Wislocki and King, 1936). Morphological observations have suggested a neurosecretory activity in the OVLT as in other "circumventricular organs" (Hofer, 1958; Weindl, 1973), in mammals (Weindl et al., 1967, 1968; R6hlich and Wenger, 1969; Le Beux, 1972; Weindl and Schinko, 1975) and in birds (Mikami et al., 1976; Mikami, 1976). Moreover, recent studies with complementary techniques have suggested the possible intervention of this organ in pituitary gonadotropic function in mammals (Barry et al., 1973; Zimmerman et al., 1974; S6t/tld et al., 1976; Kawakami et al., 1973; Kawakami and Sakuma, 1976; Brownstein et al., 1976; Wenger, 1976). A suitable model system for the study of such a regulation has been the bird, especially the photosensitive species such as the duck (Benoit and Assenmacher, 1955). Following the works of Benoit and Assenmacher (1955) and of Calas (1974) on the median eminence (ME) of the duck and those of Vors (1970) and Alonso (1973) on the posthypophysis of the duck, a cytophysiological approach was employed for the study of the OVLT in the same avian family. Initially, this organ is considered from a histological and ultrastructural point of view. Secondly, the intra-organ distribution of exogenous radioactive molecules is studied since previous studies have demonstrated that specialized ependymal cells, the tanycytes (Horstmann, 1954), are capable of transferring substances across circumventricular organs (Horstmann, 1954; Knigge et al., 1975; Reese and Brightman, 1968; Weindl, 1969). In the present study, tritiated monoamines or amino acids were administered either in vivo by stereotaxic intraventricular injections or in vitro by incubation of the isolated organ. Since the cerebral monoamines are probably involved not only in hypothalamic neuroendocrine mechanisms (Kordon, 1970) but also in tanycyte function in the ME (Calas, 1975; Nozaki, 1975), the localization and possible function of monoaminergic innervation at the OVLT level is discussed.
Material and Methods Thirty adult male Pekin ducks, Anas platyrhynchos, were used in this study. They were reared in natural temperature and photoperiodicity conditions and were sacrificed throughout the year.
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Morphological Techniques The animals were perfused intravascularly under Numbutal anesthesia first with a physiological saline solution and then with an appropriate fixative. For light microscopic examination the brains were fixed with Bouin-Hollande fluid, embedded in paraffin, sagitally sectioned at 8 Ilm, then stained with Cresyl violet. For electron microscopic investigation large pieces containing the OVLT were excised from the brains after perfusion and immersed in 3.64~ glutaraldehyde in 0.01 M phosphate buffer, pH 7.4. Carefully dissected blocks were post-fixed with 2 ~ osmium tetroxide in the same buffer, dehydrated in ethanol and embedded in Epon. The polymerized pieces were sectioned with a Reichert OmU2 ultramicrotome. Control semithin sections were stained with toluidine blue for light microscopic examination. The ultrathin sections of the OVLT were contrasted with a 5 ~ alcoholic solution of uranyl acetate and then with lead citrate, and were observed with a JEM-100 B electron microscope.
Radioautography The two radioactive monoamines used in this study were 3H-noradrenaline (3H-1,1' NA, specific activity (SA): 20Ci/mmole, C.E.A. Saclay, France) and 3H-serotonin (5-hydroxytryptamine, 3H-5 HT, G, S.A.: 5-14 Ci/mmole, Radiochemical Centre Amersham, England). Six hours before administering the tracers, the animals were intraperitoneally injected with nialamide, 200 mg/kg (generously supplied by Pfizer). The other tritiated molecules used were: 3H-L-histidine (3H-2,5 His, SA: 20 Ci/mmole), 3H-L-proline (3H-5 Pro, SA: 20 Ci/mmole) and 3H-~-aminobutyric acid (3H-2,3 GABA, SA: 43 Ci/mmole) from C.E.A. Saclay, France. For in vivo experiments, the tracers were dried under nitrogen flow, then reconstituted in 50 ~tl of artificial CSF (Merlis, 1940) to obtain a final concentration of 10-5 M monoamine or amino acid. They were then stereotaxically introduced into the third cerebral ventricle. The brains were fixed after 15 or 30min for monoamines and after 10 or 90min for amino acids by intravascular perfusion with glutaraldehyde as described above. For the in vitro experiments, the animals were sacrificed by decapitation and the OVLT, rapidly dissected out, was incubated for 30 rain at 37~ in oxygenated Tyrode solution containing the tritiated monoamine at a concentration of 10-5 M to 10 -7 M. For incubation with tritiated amino acids, a shorter time was employed (3min) and the concentration of the amino acid was 10 -6 M. After rinsing in Tyrode solution the pieces were fixed with glutaraldehyde. Following the in vivo and in vitro administration of the tracers, the specimens were prepared for electron microscopy as described above. For radioautography semithin sections were affixed to glass slides and coated by dipping in Ilford Emulsion K5 diluted 1:1. These light microscopic radioautographs were developed in Kodak D I 9 after 8 to 20 days of exposure and were then stained with toluidine blue. For high resolution radioautography, ultrathin sections on a celloidin film deposited on glass slides were stained with uranyl acetate and lead citrate and lightly vaporized with carbon. After dipping in Ilford emulsion L4 diluted 1:4 the radioautographs were exposed for 20 to 60 days at which time they were processed in Kodak Microdol X or in Phenidon and collected on grids. After the celloidin membranes had been thinned by isoamyl acetate, the grids were observed with a Jem 100-B electron microscope.
ResuRs A. Light Microscopic Examination ( F i g . 1 A a n d B) W h e n o b s e r v e d i n a s a g i t t a l p l a n e , t h e lamina terminal& o f t h e d u c k s t r e t c h e s obliquely from the optic chiasma to the anterior commissure and borders on the p r e o p t i c r e c e s s o f t h e t h i r d v e n t r i c l e . O n t h e o u t e r side, it is l i m i t e d b y t h e r i c h v a s c u l a r s u p p l y o f t h e c&terna praechiasmatica. T h e O V L T is t h a t v e r y t h i n p a r t o f t h e lamina terminal& w h i c h e x h i b i t s s p e c i a l i z e d e p e n d y m a l cells, t h e t a n y c y t e s ( H o r s t m a n n , 1954).
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Fig. 1. A Sagittal section of the duck OVLT. Cresyl violet stain. POR preoptic recess; OC optic chiasma; SP superficial plexus; N nerve ceil bodies, x 100. B Semithin section of the duck OVLT. Internal zone. Toluidine blue stain. Ep ependyma; N neuron; GL glial cell; IC internal capillary. x 1200
The p e r i k a r y a of these cells display a typical elongated a n d regularly shaped n u c l e u s a n d constitute a n i n t e r n a l m o n o c e l l u l a r layer. A m o r e diversified sube p e n d y m a l layer c o n t a i n s n u m e r o u s r o u n d e d glial cells, as well as several n e u r o n s , nerve fibers a n d small capillaries. I n the thicker external zone, these O V L T comp o n e n t s are separated by cytoplasmic processes of tanycytes which extend toward the vascular network.
Fig. 2 A - D . Internal (A, B, C) and external zone of the duck OVLT. A OVLT tanycytes joined by apical junctional complexes (arrows) show microvilli (MV) and protrusions (P) into the lumen of the 3rd ventricle (3rd V) and cytoplasmic processes (TP). G Golgi apparatus; M mitochondria; L lysosome. • 6500. B Cytoplasmic branched process of a tanycyte rich in microfilaments. M mitochondria. • 19,000. C Non-fenestrated internal capillary surrounded by a narrow perivascular space (PS). • 4200. D A glial cell body (GL) abuts on the peripheral basement m e m b r a n e (B) lining the perivascular space (PS) of an external fenestrated capillary (arrows). P tanycytic or glial processes; FB fibroblastic processes. • 10,000
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Fig. 3. Subependymalperikaryonof the duck OVLT. Note the preferentiallocation of neurosecretory granules (NG) within the Golgi area (G). RER rough endoplasmic reticulurn; M mitochondria; Nnucleus. x 17,000
B. Electron Microseopic Examination 1. Ependyma. In the vicinity of the OVLT (lateral, rostral and caudal edges) typical ependymal cells are observed. The ependymal tanycytes, however, contain nuclei which are more elliptical and have a more irregular size distribution (Fig. 2 A). Their apical membranes, presenting some bulbous extensions and a few microvilli into the third ventricle, are generally devoid of cilia. In their most apical part, the interdigitated lateral membranes display zonulae adhaerentes and desmosomes but the identification of tight junctions above these junctional complexes is impossible without high molecular weight tracer experiments (Brightmann and Reese, 1969). The cytoplasmic organelles are confined to the supranuclear region of the cells and include numerous ribosomes which are either free or bound to fragments o f endoplasmic reticulum, a well developed Golgi apparatus, mitochondria, some inclusions (lysosomes, lipofuscin) and many micro filaments. Below the somatic region, the ependymal tanycyte consists of a long and branched process (Fig. 2 B) projecting from the soma through the neuropil and abutting with foot-like endings on the peripheral basal lamina (Fig. 4B). These processes, composed of many
Fig. 4 A - F . Innervation of the duck OVLT. A Internal zone. Bundles of small unmyelinated axons (*) are isolated from each other by the cytoplasmic processes (P) of tanycytes and glial cells (GL). x 5000. B External zone. A type a neurosecretory axon reaches the parenchymal basement membrane (arrow). A x a other axons of the first type; P S perivascular space; GL glial cell. x 8000. C Two nerve fibers of type b display asymmetrical synaptic contacts with the same dendrite (D 1). One of them also constitutes a synaptic afference on another dendritic profile (D2). Arrows indicate dense core vesicles. • 23,000. D Asymmetrical synaptic contact between a type c axon and a dendrite (arrow). M mitochondria, x 23,000. E A neuronal perikaryon receives a synaptic afference of type b (arrow). • 16,000. F A synaptic differentiation (arrow) is also observed on a dendrite belonging to a neuron of the organ (N). • 12,000
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microfilaments, contain longitudinally oriented mitochondria and some dense inclusions, more frequently observed in their endings. 2. Neurons. The OVLT of the duck contains some neuronal perikarya 8-10 ~tm
in diameter. They are scattered in the subependymal and external regions and appear as large cells with a clear cytoplasm and nucleoplasm and an irregularly shaped nucleus. The endoplasmic reticulum is predominantly granular and a welldeveloped Golgi apparatus is also present. Free ribosomes, large and circular mitochondria, multivesicular bodies, lysosomal bodies and lipid droplets are seen throughout the cytoplasm. Neurosecretory granules, 1000-2000 A in diameter, can sometimes be observed, primarily in the Golgi area of the cell (Fig. 3). 3. Glial Cells. Glial cells are found scattered throughout the entire organ, being
more numerous in the subependymal layer. Glial and ependymal cells have a similar appearance, but the former present a more rounded cell body and nucleus. In addition, glial cells seem to give rise to cytoplasmic processes toward the superficial region of the OVLT, where a glial cell body can occasionally be seen to contact the parenchymal basal lamina (Fig. 2 D). 4. Nerve Fibers. The OVLT of the duck exhibits a peculiar arrangement of different
types of nerve fibers. Numerous bundles of small axons (0.1 to 0.5 ~tm in diameter), generally unmyelinated and containing microtubules, run in a rostro-caudal direction in the thickness of the organ (Fig. 4A). Some larger axonal sections containing vesicles with dense material and/or clear vesicles are also found throughout the OVLT. They can be classified into three principal groups (Fig. 4): a) This category includes axons containing granulations of about 1000-2000/~ in diameter (the electron dense material occupies the whole vesicle) and some clear vesicles (300-500 A in diameter). b) Granulations 500-800 A, in diameter as well as other vesicles with a smaller dense core are observed in the numerous axons of this category. Some electron lucent vesicles (300400 A) are either intermingled with them or confined to a restricted area, namely the vicinity of the axonal membrane. c) This group includes the smallest number of axons which contain only clear vesicles 300 to 600 A in diameter.
Fig. 5 A - E . Light microscopic (A-B) and high resolution (C-E) radioautographs of duck OVLT 30min after intraventricular injections of tritiated monoamines. A 3H-NA. Sagittal section. The reaction is more intense over the inner zone (In Z) of the OVLT. Note the prominent labeling of ependyma and the occurrence of clusters of silver grains over the subependymal zone (arrows). Ex Z external zone. • 300. B 3H-5HT. Frontal section. Scattered silver grains have no preferential localization. Dense accumulations are more numerous over the lateral edges of the lamina terminalis. O V organum vasculosum. • 200. C 3H-NA. A large varicosity of the subependymal zone (type b) exhibits an intense labeling. Non-labeled fibers belonging to the same axonal group are also present. T tanycyte nucleus. TP tanycyte process. • 12,000. D 3H-NA. Only one type b fiber is labeled and displays an axodendritic synaptic contact. TP tanycyte process. • 18,000. E 3H-5HT. A synaptic contact is observed lateral to the OVLT between a labeled type b axon and a dendritic spine (DS). x 28,000
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All these fibers are present at each level of the organ although the type (a) is preferentially observed in the most external zone. Certain types (b and c) occasionally establish synaptic contacts, generally of the asymmetrical type (type 1 according to Gray, 1959), with neuronal perikarya or, more frequently, with dendrites (Fig. 4C, D, E, F). In the external region, the foot-like endings oftanycyte processes sometimes allow type (a) axonal endings to abut on the outer basement membrane of the OVLT.
5. Vascularization. The duck OVLT exhibits both internal and external capillaries. Their endothelial cells are surmounted by narrow or large perivascular spaces and contain collagen fibrils and fibroblastic processes. In contrast to internal vessels, however, only the external capillaries often show a fenestrated endothelium (Fig. 2C, D).
C. Radioautographic Study of the Incorporation of Tritiated Molecules 1. Monoamines. In light microscopic radioautographs, the localization of the reaction in the OVLT is similar after an incubation in vitro and an intraventricular administration of the tracers. In addition to scattered silver grains, some intense accumulations are observed, the distribution of which differs depending on the tracer (Fig. 5 A, B). With 3H-NA the labeling is more significant over the inner zone of the OVLT and particularly over the ependyma. Clumps of silver grains are seen essentially in the subependymal zone. With 3H-5 HT, these intense accumulations of silver grains appear more evident in the lateral edges, where the organ becomes thicker; some are also present on the subependymal and outer regions. At the ultrastructural level, the intense ependymal radioautographic reaction is located primarily in the perikarya of tanycytes; the labeling of their processes is not prominent against the diffuse reaction observed in the surrounding neuropil. The aggregates of silver grains observed after light microscopic examination are found to correspond to axonal varicosities. The specific distribution for 3N-NA and 3H-5 HT is confirmed. The 3H-NA is incorporated into small or large axons which are scattered among non-labeled axons and which are contained in the second category described above (Fig. 5 C, D). Some of them establish axodendritic synaptic contacts (Fig. 5D). With 3H-5 HT, the labeled fibers are found to be exclusively small axons belonging to the same group. In addition, true asymmetrical synaptic differentiations on dendrites or on dendritic spines are also observed (Fig. 5 E). 2. Amino Acids. In addition to a significant labeling of the ependyma with 3H-His or 3H-Pro, certain neuronal cell bodies exhibit more silver grains than their surroundings. Also the radioautographic reaction is more intense on the tanycytes of the OVLT than on the lateral ependyma of the lamina terminalis, 90 min following the intraventricular administration of 3H-His (Fig. 6 A). Within three minutes of the administration of 3H-GABA in vitro the tanycytes of the OVLT exhibit a very high degree of labeling which extends to their extremities (Fig. 6 B). Glial and endothelial vascular cells are also significantly labeled.
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Fig. 6 A - C . Radioautographs of semithin (A-B) and thin (C) sections of duck O V L T after administration o f tritiated amino acids. A Intraventricular administration of 3H-His and fixation after 90 min. Frontal section. The most intense radioautographic accumulations of silver grains are seen u p o n the tanycytes of the thinner part of the lamina terminalis which constitutes the OVLT (limited by the two arrows). Note the preferential labeling of ependyma (Ep). S V superficial vessels. • 180. B and C In vitro incubation with 3H-GABA for 3 min. Frontal sections. B Ependyma (Ep), tanycyte processes (arrows) and vascular endothelium are significantly labeled. S V superficial vessels. • 180. C Note the distinct labeling of tanycyte processes (TP) at the ultrastructural level. Phenidon developer. M mitochondria, Ax a type a neurosecretory axons, x 13,500
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The light microscopic radioautographic images obtained for each tritiated molecule are confirmed at the ultrastructural level. 3H-His and 3H-Pro are primarily retained in tanycyte cell bodies, in neuronal perikarya and in dendrites. Finally, although 3H-GABA yields an intense diffuse labeling over all tissue elements, silver grains are clearly prominent on the perikarya and processes of tanycytes (Fig. 6 C).
Discussion The 0 V L T as a Potential Site o f Neurohemal Release
The occurrence of various nerve fibers containing granulations as well as dense and/or clear vesicles in the OVLT of the duck suggests a neuroendocrine activity at this level. The conditions of fixation used in the present study result in the description of three principal groups of axonal profiles. Type a neurosecretory fibers predominate in the external zone close to the superficial capillaries, the fenestrated endothelium of which provides a morphological support for the supposition of a neurohemal release. Moreover, some of these axons are sometimes in close association with the parenchymal basement membrane which lines the perivascular space. In addition, as postulated by Kobayashi (1975), the contents of axon endings which are isolated from the capillary wall by the interposition of tanycyte processes might also be released into the blood by passing between and/or through these processes. Morphological signs of neuroendocrine activity in the OVLT have also been recently reported in other avian species, the White-crowned Sparrow (Mikami et al., 1976) and the quail (Mikami, 1976), where both internal and external capillaries exhibit a fenestrated endothelium. In the duck, the different ultrastructural features of the two kinds of vessels are identical to those described in the ME (Calas and Assenmacher, 1970). In mammals, in addition to morphological data, some experimental evidence has already suggested the participation of the OVLT in neuroendocrine mechanisms. Ultrastructural changes in the OVLT were reported in the golden hamster following castration (Weindl and Schinko, 1975) and in the rat after ovariectomy or hypophysectomy (Wenger, 1976). Moreover, the electrical stimulation of the periventricular area which includes the OVLT induces a release of LH (Kawakami et al., 1973). Additional evidence that this organ may be involved in gonadotropic regulation is furnished by biochemical assays demonstrating that significant amounts of LH-RH are present in the rat OVLT (Brownstein et al., 1976). Immunocytochemical studies at the histological level have also contributed to the identification of this neurohormone in the OVLT of mammals (Barry et al., 1973; Zimmerman et al., 1974; Barry and Carette, 1975; Weindl and Schinko, 1975; S6tfil6 et al., 1976) and of the duck (Calas, 1975) where the external localization of LH-RHcontaining fibers is analogous to that of the ME (Calas et al., 1973). In addition, other neurohormones were also detected in the OVLT of mammals: somato-
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statin (Pelletier et al., 1976), vasopressin and oxytocin (Zimmerman and Lobo Antunes, 1976). If neurohormones (and other molecules) can be released into the blood at the level of external vessels, the fenestrations of endothelial cells might also allow passage of intravascular substances in the opposite direction, as already demonstrated in the rat (Weindl, 1969). Thus, the OVLT, like the other "windows" of the brain (Knigge, 1975), might be involved in hormonal feed-back circuits directed toward the central nervous system. Large molecules, however, might be intracellularly transported through the tanycytes (Nakai and Naito, 1975).
The Function o f Tanycytes
In the last few years, the tanycytes have been considered only as an anatomical (Weindl and Joynt, 1972) or a functional link (Knigge and Silverman, 1972; Kobayashi et al., 1972; Knigge et al., 1975) between CSF and blood in the ME. From this latter point of view, the tanycytes of the ME would have the ability to take up and transfer certain substances such as ~-amino-butyric acid and thyroxine (Knigge and Silverman, 1972), peroxidase (Kobayashi et al., 1972), LH-RH (Uemura et al., 1975) and dopamine (Scott and Krobisch-Dudley, 1975). The specialized ependymal cells of the duck OVLT exhibit the same morphological features as ME tanycytes; possible functional similarities may then be discussed in relation to our radioautographic study of the incorporation of tritiated monoamines and amino acids. In the present experiments, including the use of nialamide to inhibit the major (3H-NA) or the sole (3H-5 HT) way of tritiated monoamine catabolism (monoamine oxidase), the diffuse radioautographic distribution of silver grains is to be related essentially to free 3H-NA or 3H-5HT retained by the glutaraldehyde (Descarries and Droz, 1970). Our radioautographic technique is also reliable for the detection of the sites of uptake and/or retention of tritiated amino acids (Larra and Droz, 1970). Free radiolabeled amino acids may be visualized by using glutaraldehyde as a fixative. Thus, tritiated monoamines and amino acids are shown to penetrate the duck OVLT after their administration in vitro or in vivo. The pronounced radioautographic reaction generally observed on the ependyma suggests an uptake of these radiolabeled molecules by the perikarya of tanycytes. Our present observations show, however, that although 3H-His labels the OVLT intensely and more specifically its ependyma, only 3H-GABA appears to have been clearly accumulated in the tanycyte processes. On the other hand, since data concerning a possible release of this 3H-GABA at a site different from that of its uptake are lacking, we cannot clearly relate the ubiquitous labeling of tanycyte cytoplasm to a functional transependymal transfer. If such a transport does exist in the duck OVLT, it would be very selective, as already suggested by the analogous incorporation of 3H-His (and not 3H-Pro) in the ME (Calas, 1975). Moreover, our present results along with those of Calas suggest that a functional difference probably exists between ME and OVLT tanycytes. Transependymal transport as a potential neuroendocrine route in the ME is
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supported both by the identification of several factors in the CSF, namely adenohypophyseal hormones (Linfoot et al., 1970; Kendall et al., 1975), neurohormones (Heller, 1969; Joseph et al., 1975) and neuromediators (Denker and H~iggendal, 1969) and by the effect of intraventricularly injected LH-RH on serum LH level (Weiner et al., 1972; Ben-Jonathan et al., 1974; Uemura et al., 1975) and TRH on serum TSH level (Gordon et al., 1972). The OVLT probably participates in neuroendocrine regulations according to the accepted concept of axonal transfer, storage and excretion of hypothalamic neurohormones: it might also be involved in another, transependymal, alternative route.
The Monoaminergic Innervation of the 0 VLT If the diffuse radioautographic distribution of silver grains is to be related essentially to free 3H-NA or 3H-5 HT in the detection of tritiated monoamines, then the intense sites of accumulation reveal the structures which have concentrated the tracers (Descarries and Droz, 1970), i.e. monoaminergic fibers (Taxi and Droz, 1969). Although these fibers belong to the same group (type b which also includes non-monoaminergic axons), some facts argue for a selective uptake of 3H-NA and 3H-5HT by distinct noradrenergic and serotonergic fibers, including the different topographic distribution of sequestering axons after the administration of either 3H-NA or 3H-5 HT. In addition, the identity of the radioautographic reaction after both in vivo experiments and in vitro incubations in Tyrode medium containing 10 7 M 3H-5HT also eliminates the possibility of a non-specific uptake by catecholaminergic axons (Shaskan and Snyder, 1970). According to Descarries et al. (1976), the specificity of the in vivo uptake of 3H-5 HT injected at a higher concentration might be accounted for by an intraventricular and intratissual dilution of the tracer. Concerning the labeling of catecholaminergic fibers by 3H-NA, the reactive axons of the duck OVLT are most probably noradrenergic since under these experimental conditions dopaminergic fibers cannot be distinctly detected by radioautography (Calas et al., 1976). Thus, the internal localization of noradrenergic fibers appears to be quite similar to that of the ME of the duck (Calas, 1973). In contrast, most of the indolaminergic fibers are more laterally distributed and do not appear to innervate specifically the OVLT. However, the occurrence of true serotonergic synapses (in addition to noradrenergic ones) on dendrites and dendritic spines at the lamina terminalis level argues for a possible direct intervention of serotonergic fibers on neurosecretory neurons. Non-monoaminergic synapses are also present throughout the organ. Based on light microscopic radioautographic observations, the monoaminergic innervation of the OVLT does not appear to be significant. This observation agrees with biochemical results of Saavedra et al. (1976) who found moderate amounts of noradrenaline and serotonin in the rat OVLT. In contrast, cholinergic structures are probably more numerous than monoaminergic structures, as assessed by the detection of high levels of choline acetyltransferase activity (Saavedra et al., 1976).
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Conclusion The OVLT of the duck appears to be a neurovascular structure which is similar to the ME not only by its constitutive elements but also by its noteworthy abilities of exchange between blood, CSF and nervous tissue. Such exchange mechanisms most probably involve a neurohemal release of molecules from neurosecretory axons. Moreover, because of their close relationship with the CSF and the external perivascular space, the OVLT tanycytes appear to be capable of taking up and selectively translocating certain substances between the ventricular and the vascular pole of the organ. Concerning its monoaminergic innervation, the occurrence of noradrenergic and serotonergic synapses supports a direct, although discrete, participation of these monoamines in neuroendocrine mechanisms at the OVLT level. In addition, this "window" of the brain appears to be a good model for the study of the synaptic control of serotonin on neurosecretory neurons.
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Accepted April 25, 1977
