Anat. Embryol. 153, 295-304 (1978)
Anatomy and Embryology 9 by Springer-Verlag 1978
A Liquor Contacting Area in the Pineal Recess of the Golden Hamster (Mesocricetus auvatus)* Maria Hewing Anatomisches Institut der Universitfit Bonn, Nuf3allee 10, D-5300 Bonn, Federal Republic of Germany
Summary. The wall of the third ventricle in the pineal recess of the golden hamster (Mesocricetus auratus) has been investigated by light and electron microscopy. Deep in the pineal recess, where the ependymal lining is thin and non-ciliated, clusters of pinealocytes protrude into the ventricular lumen. They force the ependyma apart so that their surface is directly exposed to the CSF, while basal processes extend towards the hypependymal pineal tissue. It is assumed that these cells may secrete melatonin into the CSF which is known to contain varying amounts of this hormone.
Key words: Supraependymal cells - Third ventricle - Deep pineal - Golden Hamster.
Introduction In the golden hamster (Mesocricetus auratus) the pineal system consists of a superficial and a deep pineal (Sheridan and Reiter, 1970a). These two parts, which in the adult are connected by a thin stalk, arise out of a c o m m o n anlage, situated between the habenular and posterior commissures, at the end of the fetal period. During the early weeks post partum, the greater part of the tissue becomes displaced away from the diencephalon and assumes a position under the skull near the confluens sinuum. But a smaller part of the tissue remains between the commissures and surrounds the pineal recess, thus retaining its topographical relationship with the third ventricle (Hewing, 1976). Whereas many authors consider the bloodstream as the only available pathway for transmission of melatonin or other substances from the pineal organ (Kappers, 1971; Quay, 1974), there are some indications that also the cerebrospinal fluid (CSF) might serve as a transport medium for pineal substances (Anton-Tay and Wurtman, 1969; Kamberi et al., 1971). Recently, the * Supported by the Deutsche Forschungsgemeinschaft
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existence o f melatonin in the CSF o f m a n (Smith et al., 1976) and calves (Hed: lund et al., 1977) has been reported. The aim o f this investigation was to find out whether in the deep pineal o f the golden hamster, m o r p h o l o g i c a l pecularities can be observed which might indicate a close functional relationship between the pineal tissue and the CSF.' Preliminary observations were already reported (Hewing, 1978).
Material and Methods 26 adult male golden hamsters, obtained from the Zoohandlung Koch, Holzminden, were used in the investigation. The animals were anaesthesized with pentobarbitone sodium, and perfusion fixation was performed with a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 rn phosphate buffer. 20 rain after termination of the perfusion, the skull was opened and the pineal system dissected out 'en block '. The specimens were immersed in the same fixative for 1 h, then washed in 0.1 m phosphate buffer and postfixed in 1% OsO4 (in 0.2 m phosphate buffer containing 0.1 m sucrose) for 4 h. After dehydratation in ethanol the specimens were embedded through propylene oxide into Epon mixture (Luft, 1961). Sectioning was done on a Reichert OmU2 ultramicrotome. Semithin sections were stained with a mixture of toluidineblue and pyronine G (Ito and Winchester, 1963). Ultrathin sections were contrasted with uranyl acetate and lead citrate (Reynolds, 1963) and examined in a Philips 301 electron microscope.
Results
Light Microscopy The deep pineal constitutes that tissue surrounding the pineal recess, which is a median dorsal evagination of the third ventricle located between the habenular and posterior commissures. In a median sagittal section (Fig. la), the pineal recess has the f o r m o f a semicircle o f a b o u t 0.35 m m diameter. Within the pineal recess the e p e n d y m a is not uniform. At the entrance, the ependymal cells are cuboidal to low c o l u m n a r in shape; those which cover the caudal part o f the posterior commissure bear long kinocilia. Deep in the recess, the ventricular wall is ahnost devoid of kinocilia and has a peculiar structure (Fig. l a ) : it is c o m p o s e d of endothelial-like cells, whose processes form very n a r r o w stripes separating the deep pineal f r o m the ventricular lumen. Between the flat ependymal cells, tanycytes can be observed that give off long basal processes coursing in a direction perpendicular to the ventricular surface [Fig. 1 c). Some of these processes can be followed deep into the pineal tissue. In the centre of this peculiar ependymal area lies a region where no ependymal cells could be detected at the ventricular surface. In this region, cells which can be discriminated f r o m the dark-stained ependymal cells p r o t r u d e into the CSF (Fig. 1 b, c). These lighter cells have r o u n d or oval nuclei and large pale pericarya; they resemble the p a r e n c h y m a l cells o f the deep pineal. These cells, arranged in pallisade fashion (Fig. l b ) or in layers (Fig. lc), f o r m a p r o t u b e r a n t structure. In the apical part o f the protuberance, vacuoles are a regular feature; at its base, dark cells and cell processes are f o u n d (Fig. 1 b,
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Fig. 1. a Sagittal section through the region of the deep pineal in the golden hamster. 1 deep pineal, 2 pineal stalk, 3 commissura habenularum, 4 commissura posterior, V pineal recess, b-e Higher magnification of the area indicated in Figure l a, showing protuberances of different size. The continuity of the thin ependymal layer becomes interrupted. (/~). T tanycytes. Magn. a x 80, b x 480, cx480
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Fig. 2. Low magnification of the lateral part of the zone of extrusion. V ventricular lumen, Pi ceil of the protuberance, p processes of protruding cells, E ependymal cell within the zone of extrusion, )~bundle of unmyelinated axons. Magn. x 7200
c). Examination of this area in serial sections revealed it to be a nearly round zone in which the dark cells form an irregular grid through which the light cells project into the ventricular lumen. The diameter of this grid-like zone of extrusion varies from 40 lain to 150 gin; the diameter of the protuberance is usually larger by 10 g m to 30 ~tm. This special structure could be observed in 25 of the 26 animals investigated.
Electron Microscopy A. Fine Structure of the Zone of Extrusion. The ependymal lining of the pineal recess, whose cells are joined by junctional complexes (maculae adhaerentes and gap junctions), is interrupted at the border of the protrusive area (Fig. 2). The zone of extrusion is predominantly occupied by the processes of the protruding cells, but between these, cells and cell processes are found which correspond to the dark cells seen at light microscopic level (Fig. 2). In nuclear shape, dispersion of chromatin and density of cytoplasm the latter resemble ependymal cells. They are scanty in the centre of the zone of extrusion and become more frequent towards the periphery. The majority of the dark cell processes course parallel to the ependymal lining; a small number accompanies the processes
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Fig. 3. Low magnification of a cell of the protuberance, showing tytically arranged organelles which characterize a pinealocyte. V ventricular lumen, SR smooth endoplasmic reticulum in the apical part of the pericaryon, E ependymal cell. Magn. x 9000. Inset: Synaptic ribbons below the ventricular surface of a protruding cell. Magn. x 18,000
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of the protruding cells towards the ventricular lumen for a short distance. In close relationship to the dark cells, small bundles of unmyelinated axons are found (Fig. 2). The axons exhibit evenly distributed microtubules and microfilaments, and small mitochondria possessing a dense matrix. Their terminals contain vesicles whose diameters range from 40nm to 65rim; some have a dense core measuring less than 20 nm. Larger dense core vesicles (60-100 nm) are occasionally observed. No synaptic contacts could be revealed either with the ependymal cells or with the protruding cells. Axonal profiles having identical features were observed also underneath the ependymal cells, laterally from the zone of extrusion, or within the pineal tissue. B. Fine Structure of the Protuberance. The ultrastructue of the protruding cells is markedly similar to that already described for the pinealocytes in the deep pineal (Sheridan and Reiter, 1970 b), thus confirming that these cells are pinealocytes: they are characterized by a round or oval nucleus with a conspicuous nucleolus and peripheral concentrations of chromatin, and by a large pericaryon of low density containing an extensive endoplasmic reticulum mainly of the smooth variety, slender and elongated mitochondria with a lucent matrix, a well-developed Golgi apparatus and large dense bodies that resemble lysosomal inclusions (Fig. 3). Microtubules, scattered throughout the cytoplasm, become concentrated at points where processes emerge from the cell body; they are found characteristically parallel to the long axis of the processes. The terminals exhibit clear and dense corevesicles measuring between 60 and 120 nm. Additionally, synaptic ribbons, not yet described in the deep pineal of the golden hamster but a typical component of the mammalian pinealocyte (Kappers, 1969, 1971), were observed in the protruding cells (Fig. 3) as well as in the pinealocytes within the deep pineal. They were found lying singly or in groups of two, three or more. Smaller groups were observed in the pericarya as well as in the processes, whereas groups of more than three were found only in the pericarya. Most ribbons were located centrally in the cytoplasm; only few had intimate contact with the cell membrane. The protruding pinealocytes are orientated so that their pericarya are towards the ventricular lumen. Within the pericarya, the smooth endoplasmic reticulum is concentrated in the apical cytoplasm, and synaptic ribbons are preferentially located in the same area, whereas the other cell organelles are aggregated at the opposite side of the nucleus (Fig. 3). A thick process emerges basally from the cell body and passes through the zone of extrusion to enter the hypependymal pineal tissue. The processes of the most peripherally lying cells whose pericarya operculate the marginal ependymal cells, run parallel to the ependymal surface until they reach the zone of extrusion where they turn basally. Only few processes could be observed originating from the apical part of the pericarya; terminals of pinealocyte processes are occasionally seen within the protuberance or at its surface. The pericarya usually lie next to each other with an intercellular cleft of about 200 ~. Specialized contacts could not be discerned, but juxtaposed small patches of moderate dense cytoplasmic material could be observed (Fig. 4a).
Fig.4a-c. Spatial relationship of protuberant pinealocytes, a In the apical region special junctions of neighbouring cells are omitted. V ventricle, ,~ small juxtaposed patches of dense cytoplasm, * coated vesicles. Magn. x 44,000. b In the upper part the intercellular space becomes markedly enlarged. V ventricle. Magn. x 15,900. e In deeper parts and just above the zone of extrusion the pinealocytes form tongue-like interdigitations. Magn. x 18,000
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In addition, fiat membranous saccules strung symmetrically along neighbouring cell membranes are a regular feature. A few micra from the ventricular surface of the protuberance, the intercellular cleft may be found widened to form round or oval distensions (Fig. 4b) which correspond to the vacuoles seen at light microscopic level. They range in size from 2 to 6 gm in diameter and often contain a flocculent material of moderate electron density. Coated vesicles indicating an exchange of products between the cytoplasm and the vacuole occur, but are not restricted to the distensions; they can also be found fused with the regular intercellular cleft, or, at the surface of the proturbance, with the ventricular lumen (Fig. 4a). Just above the zone of extrusion, irregularly formed distensions of the intercellular space occur between the basal processes of the protruding pinealocytes. At this level, the processes are connected with each other by small lateral offshoots containing a few sacs of endoplasmic reticulum, some free ribosomes and both clear and dense core vesicles. In a cross section through the zone of extrusion parallel to the ependymal lining (Fig. 4c), conglomerates of about 20 such tongue-like off-shoots were found, closely packed and occasionally showing maculae adhaerentes.
Discussion The results of this study show that the deep pineal of the golden hamster has a particular form of contact with the third ventricle. The contact zone is characterized by a thin ependyma devoid of cilia, and by a special region of pineal tissue protruding into the CSF. Whereas similar bouquet-like structures observed in the basal hypothalamus of the rabbit elevate the ependyma, the ependymal cells forming a cell-cap over the bouquet (Leonhardt, 1968), other structures in diverse regions of the ventricular system project into the CSF without covering ependyma (for lit. see Fleischhauer, 1972; Rodriguez, 1976). The protruding cells in the pineal recess of the golden hamster belong to the latter group; they appear to prise the ependyma apart so that their surface gains immediate contact with the CSF. The supraependymaI pinealocytes possess many of the ultrastructural characteristics often associated with synthetic activity. The conjecture that these are degenerating cells being sequestered into the ventricular lumen can be excluded. Moreover, the protruding cells are regularly rooted by basal processes in the tissue of the deep pineal. The arrangement of the cells, and also the polarity exhibited in the distribution of the cell organelles, suggest that the cells might be regarded as a specialized link between the CSF and the pineal system. From our morphological studies, it could be speculated that, since these cells are literally bathed in the CSF they might absorb substances from the CSF or might act as sensors of CSF concentration of various chemicals. Alternatively, they might secrete into the CSF. The assumption that the protruding cells secrete substances into the CSF would be strongly supported by the following physiological findings : The greater
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u p t a k e o f H 3 - m e l a t o n i n by the r a t b r a i n when this s u b s t a n c e is a d m i n i s t e r e d i n t r a v e n t r i c u l a r l y as o p p o s e d to i n t r a v e n o u s l y has been a c c e p t e d as evidence for the n o r m a l release o f pineal m e l a t o n i n into the C S F ( A n t o n - T a y a n d W u r t m a n , 1969), a n d the finding t h a t the injection o f m e l a t o n i n into the ventricle inhibits o v u l a t i o n , whereas m e l a t o n i n a d m i n i s t e r e d p e r i p h e r a l l y does n o t (Collu et al., 1971), suggest t h a t the C S F m i g h t be i n v o l v e d in the t r a n s p o r t o f e n d o g e n o u s l y released p i n e a l m e l a t o n i n . Recently, m e l a t o n i n has been m e a s u r e d in the C S F o f calves ( H e d l u n d al., 1977). The average m e l a t o n i n c o n c e n t r a t i o n in the C S F was f o u n d to be twice as high as in the b l o o d p l a s m a d u r i n g the day. D u r i n g the night, m e l a t o n i n levels in the C S F i n c r e a s e d 17-fold while o n l y six-fold in the p l a s m a . T h e m e c h a n i s m s for the entry o f m e l a t o n i n into the C S F discussed in the l i t e r a t u r e a s s u m e Secretion via the c h o r o i d plexus, which is t h o u g h t to t a k e u p the h o r m o n e f r o m either the general c i r c u l a t i o n (Mess et al., 1975) o r r e t r o g r a d e v e n o u s flow in the s u p e r i o r sagittal sinus (Quay, 1973; R e i t e r et al., 1975). H o w e v e r , the findings o b t a i n e d in the p r e s e n t i n v e s t i g a t i o n offer some evidence for a n o t h e r , m o r e direct r o u t e o f entry, n a m e l y secretion o f the h o r m o n e by p i n e a l o c y t e s p r o t r u d i n g into the C S F .
References Anton-Tay, F., Wurtman, R.J.: Regional uptake of H3-melatonin from blood or cerebrospinal fluid by rat brain. Nature (Loud.) 221, 474-475 (1969) Collu, R., Fraschini, F., Martini, L.: Blockade of ovulation by melatonin. Experientia 27, 844-845 (1971) Fleischhauer, K.: Ependyma and subependymal layer. In: Bourne, G,H. (ed): The structure and function of nervous tissue, Vol. VI. New York and London: Academic Press 1972 Hedlund, L., Lischko, M.M., Rollag, M.D., Niswender, G.D.: Melatonin: Daily cycle in plasma and cerebrospinal fluid of calves. Science 195, 686-687 (1977) Hewing, M.: Die postnatale Entwicklung der Epiphysis cerebri beim Goldhamster. Verb. Anat. Ges., 70, 85-92 (1976) Hewing, M. : Zur Struktur einer Liquorkontaktzone im Recesses pinealis des Goldhamsters. Verh. Anat. Ges., 72. Vers. 1978 (in press) Ito, S., Winchester, R.J.: The fine structure of the gastric mucosa in the bat. J. Cell Biol. 16, 541-577 (1963) Kamberi, I.A., Mical, R.S., Porter, J.C.: Effects of melatonin and serotonin on the release of FSH and prolactin. Endocrinology 88, 1288-1293 (1971). Kappers, J.A. : The mammalian pineal organ. J. neuro-visc. Ret., Suppl. 9, 140 184 (1969) Kappers, J.A.: The pineal organ: An introduction. In: Wolstenholme, G.E.W. and Knight, J. (eds): The pineal gland, a Ciba Foundation symposium. Edingburgh and London: Churchill Livingstone 1971 Leonhardt, H. : Bukettf6rmige Strukturen im Ependym des Regio hypothalamica des III. Yentrikels beim Kaninchen. Z. Zellforsch. 88, 297-307 (1968) Luft, J.H.: Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414 (1961) Mess, B., Trentini, G.P., Kovacs, L., DeCaetani, C.F. : Melatonin, cerebrospinal fluid, pineal gland interrelationships. In: Knigge, K.M., Scott, D.E., Kobayashi, H., and Ishii, S. (eds): BrainEndocrine interaction II. The ventricular system in neuroendocrine mechanisms. Basel: Karger 1975 Quay, W.B.: Pineal Chemistry in cellular and physiological mechanisms. Springield, Illinois: Charles C. Thomas 1974
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Reiter, R.J., Vaughan, M.K., Blask, D.E. : Possible role of the cerebrospinal fluid in the transport o F pineal hormones in mammals. In: Knigge, K.M., Scott, D.E., Kobayashi, H., and Ishii, S. (eds): Brain-Endocrine interaction II. The ventricular system in neuroendocrine mechanisms. Basel: Karger 1975 Reynolds, E.S. : The use of lead citrate at high pH as an electron -opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Rodriguez, E.M.: Review. The cerebrospinal fluid as a pathway in neuroendocrine integration. J. Endocr. 71, 407-443 (1976) Sheridan, M.N., Reiter, R.J.: Observations on the pineal system in the hamster. 1. Relation of the superficial and deep pineal to the epithalamus. J. Morph. 131, 153-162 (1970a) Sheridan, M.N., Reiter, R.J.: Observations on the pineal system in the hamster. II. Fine structure of the deep pineal. J. Morph., 131, 163-178 (1970b) Smith, I., Mullen, P.E., Snedden, W., Wilson, B.W. : Absolute identification of melatonin in human plasma and cerebrospinal fluid. Nature (Lond.) 260, 718-719 (1976)
Received January 20, 1978
Anatomy and Embryology 9 by Springer-Verlag 1978
A Liquor Contacting Area in the Pineal Recess of the Golden Hamster (Mesocricetus auvatus)* Maria Hewing Anatomisches Institut der Universitfit Bonn, Nuf3allee 10, D-5300 Bonn, Federal Republic of Germany
Summary. The wall of the third ventricle in the pineal recess of the golden hamster (Mesocricetus auratus) has been investigated by light and electron microscopy. Deep in the pineal recess, where the ependymal lining is thin and non-ciliated, clusters of pinealocytes protrude into the ventricular lumen. They force the ependyma apart so that their surface is directly exposed to the CSF, while basal processes extend towards the hypependymal pineal tissue. It is assumed that these cells may secrete melatonin into the CSF which is known to contain varying amounts of this hormone.
Key words: Supraependymal cells - Third ventricle - Deep pineal - Golden Hamster.
Introduction In the golden hamster (Mesocricetus auratus) the pineal system consists of a superficial and a deep pineal (Sheridan and Reiter, 1970a). These two parts, which in the adult are connected by a thin stalk, arise out of a c o m m o n anlage, situated between the habenular and posterior commissures, at the end of the fetal period. During the early weeks post partum, the greater part of the tissue becomes displaced away from the diencephalon and assumes a position under the skull near the confluens sinuum. But a smaller part of the tissue remains between the commissures and surrounds the pineal recess, thus retaining its topographical relationship with the third ventricle (Hewing, 1976). Whereas many authors consider the bloodstream as the only available pathway for transmission of melatonin or other substances from the pineal organ (Kappers, 1971; Quay, 1974), there are some indications that also the cerebrospinal fluid (CSF) might serve as a transport medium for pineal substances (Anton-Tay and Wurtman, 1969; Kamberi et al., 1971). Recently, the * Supported by the Deutsche Forschungsgemeinschaft
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existence o f melatonin in the CSF o f m a n (Smith et al., 1976) and calves (Hed: lund et al., 1977) has been reported. The aim o f this investigation was to find out whether in the deep pineal o f the golden hamster, m o r p h o l o g i c a l pecularities can be observed which might indicate a close functional relationship between the pineal tissue and the CSF.' Preliminary observations were already reported (Hewing, 1978).
Material and Methods 26 adult male golden hamsters, obtained from the Zoohandlung Koch, Holzminden, were used in the investigation. The animals were anaesthesized with pentobarbitone sodium, and perfusion fixation was performed with a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 rn phosphate buffer. 20 rain after termination of the perfusion, the skull was opened and the pineal system dissected out 'en block '. The specimens were immersed in the same fixative for 1 h, then washed in 0.1 m phosphate buffer and postfixed in 1% OsO4 (in 0.2 m phosphate buffer containing 0.1 m sucrose) for 4 h. After dehydratation in ethanol the specimens were embedded through propylene oxide into Epon mixture (Luft, 1961). Sectioning was done on a Reichert OmU2 ultramicrotome. Semithin sections were stained with a mixture of toluidineblue and pyronine G (Ito and Winchester, 1963). Ultrathin sections were contrasted with uranyl acetate and lead citrate (Reynolds, 1963) and examined in a Philips 301 electron microscope.
Results
Light Microscopy The deep pineal constitutes that tissue surrounding the pineal recess, which is a median dorsal evagination of the third ventricle located between the habenular and posterior commissures. In a median sagittal section (Fig. la), the pineal recess has the f o r m o f a semicircle o f a b o u t 0.35 m m diameter. Within the pineal recess the e p e n d y m a is not uniform. At the entrance, the ependymal cells are cuboidal to low c o l u m n a r in shape; those which cover the caudal part o f the posterior commissure bear long kinocilia. Deep in the recess, the ventricular wall is ahnost devoid of kinocilia and has a peculiar structure (Fig. l a ) : it is c o m p o s e d of endothelial-like cells, whose processes form very n a r r o w stripes separating the deep pineal f r o m the ventricular lumen. Between the flat ependymal cells, tanycytes can be observed that give off long basal processes coursing in a direction perpendicular to the ventricular surface [Fig. 1 c). Some of these processes can be followed deep into the pineal tissue. In the centre of this peculiar ependymal area lies a region where no ependymal cells could be detected at the ventricular surface. In this region, cells which can be discriminated f r o m the dark-stained ependymal cells p r o t r u d e into the CSF (Fig. 1 b, c). These lighter cells have r o u n d or oval nuclei and large pale pericarya; they resemble the p a r e n c h y m a l cells o f the deep pineal. These cells, arranged in pallisade fashion (Fig. l b ) or in layers (Fig. lc), f o r m a p r o t u b e r a n t structure. In the apical part o f the protuberance, vacuoles are a regular feature; at its base, dark cells and cell processes are f o u n d (Fig. 1 b,
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Fig. 1. a Sagittal section through the region of the deep pineal in the golden hamster. 1 deep pineal, 2 pineal stalk, 3 commissura habenularum, 4 commissura posterior, V pineal recess, b-e Higher magnification of the area indicated in Figure l a, showing protuberances of different size. The continuity of the thin ependymal layer becomes interrupted. (/~). T tanycytes. Magn. a x 80, b x 480, cx480
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Fig. 2. Low magnification of the lateral part of the zone of extrusion. V ventricular lumen, Pi ceil of the protuberance, p processes of protruding cells, E ependymal cell within the zone of extrusion, )~bundle of unmyelinated axons. Magn. x 7200
c). Examination of this area in serial sections revealed it to be a nearly round zone in which the dark cells form an irregular grid through which the light cells project into the ventricular lumen. The diameter of this grid-like zone of extrusion varies from 40 lain to 150 gin; the diameter of the protuberance is usually larger by 10 g m to 30 ~tm. This special structure could be observed in 25 of the 26 animals investigated.
Electron Microscopy A. Fine Structure of the Zone of Extrusion. The ependymal lining of the pineal recess, whose cells are joined by junctional complexes (maculae adhaerentes and gap junctions), is interrupted at the border of the protrusive area (Fig. 2). The zone of extrusion is predominantly occupied by the processes of the protruding cells, but between these, cells and cell processes are found which correspond to the dark cells seen at light microscopic level (Fig. 2). In nuclear shape, dispersion of chromatin and density of cytoplasm the latter resemble ependymal cells. They are scanty in the centre of the zone of extrusion and become more frequent towards the periphery. The majority of the dark cell processes course parallel to the ependymal lining; a small number accompanies the processes
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Fig. 3. Low magnification of a cell of the protuberance, showing tytically arranged organelles which characterize a pinealocyte. V ventricular lumen, SR smooth endoplasmic reticulum in the apical part of the pericaryon, E ependymal cell. Magn. x 9000. Inset: Synaptic ribbons below the ventricular surface of a protruding cell. Magn. x 18,000
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of the protruding cells towards the ventricular lumen for a short distance. In close relationship to the dark cells, small bundles of unmyelinated axons are found (Fig. 2). The axons exhibit evenly distributed microtubules and microfilaments, and small mitochondria possessing a dense matrix. Their terminals contain vesicles whose diameters range from 40nm to 65rim; some have a dense core measuring less than 20 nm. Larger dense core vesicles (60-100 nm) are occasionally observed. No synaptic contacts could be revealed either with the ependymal cells or with the protruding cells. Axonal profiles having identical features were observed also underneath the ependymal cells, laterally from the zone of extrusion, or within the pineal tissue. B. Fine Structure of the Protuberance. The ultrastructue of the protruding cells is markedly similar to that already described for the pinealocytes in the deep pineal (Sheridan and Reiter, 1970 b), thus confirming that these cells are pinealocytes: they are characterized by a round or oval nucleus with a conspicuous nucleolus and peripheral concentrations of chromatin, and by a large pericaryon of low density containing an extensive endoplasmic reticulum mainly of the smooth variety, slender and elongated mitochondria with a lucent matrix, a well-developed Golgi apparatus and large dense bodies that resemble lysosomal inclusions (Fig. 3). Microtubules, scattered throughout the cytoplasm, become concentrated at points where processes emerge from the cell body; they are found characteristically parallel to the long axis of the processes. The terminals exhibit clear and dense corevesicles measuring between 60 and 120 nm. Additionally, synaptic ribbons, not yet described in the deep pineal of the golden hamster but a typical component of the mammalian pinealocyte (Kappers, 1969, 1971), were observed in the protruding cells (Fig. 3) as well as in the pinealocytes within the deep pineal. They were found lying singly or in groups of two, three or more. Smaller groups were observed in the pericarya as well as in the processes, whereas groups of more than three were found only in the pericarya. Most ribbons were located centrally in the cytoplasm; only few had intimate contact with the cell membrane. The protruding pinealocytes are orientated so that their pericarya are towards the ventricular lumen. Within the pericarya, the smooth endoplasmic reticulum is concentrated in the apical cytoplasm, and synaptic ribbons are preferentially located in the same area, whereas the other cell organelles are aggregated at the opposite side of the nucleus (Fig. 3). A thick process emerges basally from the cell body and passes through the zone of extrusion to enter the hypependymal pineal tissue. The processes of the most peripherally lying cells whose pericarya operculate the marginal ependymal cells, run parallel to the ependymal surface until they reach the zone of extrusion where they turn basally. Only few processes could be observed originating from the apical part of the pericarya; terminals of pinealocyte processes are occasionally seen within the protuberance or at its surface. The pericarya usually lie next to each other with an intercellular cleft of about 200 ~. Specialized contacts could not be discerned, but juxtaposed small patches of moderate dense cytoplasmic material could be observed (Fig. 4a).
Fig.4a-c. Spatial relationship of protuberant pinealocytes, a In the apical region special junctions of neighbouring cells are omitted. V ventricle, ,~ small juxtaposed patches of dense cytoplasm, * coated vesicles. Magn. x 44,000. b In the upper part the intercellular space becomes markedly enlarged. V ventricle. Magn. x 15,900. e In deeper parts and just above the zone of extrusion the pinealocytes form tongue-like interdigitations. Magn. x 18,000
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In addition, fiat membranous saccules strung symmetrically along neighbouring cell membranes are a regular feature. A few micra from the ventricular surface of the protuberance, the intercellular cleft may be found widened to form round or oval distensions (Fig. 4b) which correspond to the vacuoles seen at light microscopic level. They range in size from 2 to 6 gm in diameter and often contain a flocculent material of moderate electron density. Coated vesicles indicating an exchange of products between the cytoplasm and the vacuole occur, but are not restricted to the distensions; they can also be found fused with the regular intercellular cleft, or, at the surface of the proturbance, with the ventricular lumen (Fig. 4a). Just above the zone of extrusion, irregularly formed distensions of the intercellular space occur between the basal processes of the protruding pinealocytes. At this level, the processes are connected with each other by small lateral offshoots containing a few sacs of endoplasmic reticulum, some free ribosomes and both clear and dense core vesicles. In a cross section through the zone of extrusion parallel to the ependymal lining (Fig. 4c), conglomerates of about 20 such tongue-like off-shoots were found, closely packed and occasionally showing maculae adhaerentes.
Discussion The results of this study show that the deep pineal of the golden hamster has a particular form of contact with the third ventricle. The contact zone is characterized by a thin ependyma devoid of cilia, and by a special region of pineal tissue protruding into the CSF. Whereas similar bouquet-like structures observed in the basal hypothalamus of the rabbit elevate the ependyma, the ependymal cells forming a cell-cap over the bouquet (Leonhardt, 1968), other structures in diverse regions of the ventricular system project into the CSF without covering ependyma (for lit. see Fleischhauer, 1972; Rodriguez, 1976). The protruding cells in the pineal recess of the golden hamster belong to the latter group; they appear to prise the ependyma apart so that their surface gains immediate contact with the CSF. The supraependymaI pinealocytes possess many of the ultrastructural characteristics often associated with synthetic activity. The conjecture that these are degenerating cells being sequestered into the ventricular lumen can be excluded. Moreover, the protruding cells are regularly rooted by basal processes in the tissue of the deep pineal. The arrangement of the cells, and also the polarity exhibited in the distribution of the cell organelles, suggest that the cells might be regarded as a specialized link between the CSF and the pineal system. From our morphological studies, it could be speculated that, since these cells are literally bathed in the CSF they might absorb substances from the CSF or might act as sensors of CSF concentration of various chemicals. Alternatively, they might secrete into the CSF. The assumption that the protruding cells secrete substances into the CSF would be strongly supported by the following physiological findings : The greater
Liquor Contacting Area in Golden Hamster
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u p t a k e o f H 3 - m e l a t o n i n by the r a t b r a i n when this s u b s t a n c e is a d m i n i s t e r e d i n t r a v e n t r i c u l a r l y as o p p o s e d to i n t r a v e n o u s l y has been a c c e p t e d as evidence for the n o r m a l release o f pineal m e l a t o n i n into the C S F ( A n t o n - T a y a n d W u r t m a n , 1969), a n d the finding t h a t the injection o f m e l a t o n i n into the ventricle inhibits o v u l a t i o n , whereas m e l a t o n i n a d m i n i s t e r e d p e r i p h e r a l l y does n o t (Collu et al., 1971), suggest t h a t the C S F m i g h t be i n v o l v e d in the t r a n s p o r t o f e n d o g e n o u s l y released p i n e a l m e l a t o n i n . Recently, m e l a t o n i n has been m e a s u r e d in the C S F o f calves ( H e d l u n d al., 1977). The average m e l a t o n i n c o n c e n t r a t i o n in the C S F was f o u n d to be twice as high as in the b l o o d p l a s m a d u r i n g the day. D u r i n g the night, m e l a t o n i n levels in the C S F i n c r e a s e d 17-fold while o n l y six-fold in the p l a s m a . T h e m e c h a n i s m s for the entry o f m e l a t o n i n into the C S F discussed in the l i t e r a t u r e a s s u m e Secretion via the c h o r o i d plexus, which is t h o u g h t to t a k e u p the h o r m o n e f r o m either the general c i r c u l a t i o n (Mess et al., 1975) o r r e t r o g r a d e v e n o u s flow in the s u p e r i o r sagittal sinus (Quay, 1973; R e i t e r et al., 1975). H o w e v e r , the findings o b t a i n e d in the p r e s e n t i n v e s t i g a t i o n offer some evidence for a n o t h e r , m o r e direct r o u t e o f entry, n a m e l y secretion o f the h o r m o n e by p i n e a l o c y t e s p r o t r u d i n g into the C S F .
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Reiter, R.J., Vaughan, M.K., Blask, D.E. : Possible role of the cerebrospinal fluid in the transport o F pineal hormones in mammals. In: Knigge, K.M., Scott, D.E., Kobayashi, H., and Ishii, S. (eds): Brain-Endocrine interaction II. The ventricular system in neuroendocrine mechanisms. Basel: Karger 1975 Reynolds, E.S. : The use of lead citrate at high pH as an electron -opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Rodriguez, E.M.: Review. The cerebrospinal fluid as a pathway in neuroendocrine integration. J. Endocr. 71, 407-443 (1976) Sheridan, M.N., Reiter, R.J.: Observations on the pineal system in the hamster. 1. Relation of the superficial and deep pineal to the epithalamus. J. Morph. 131, 153-162 (1970a) Sheridan, M.N., Reiter, R.J.: Observations on the pineal system in the hamster. II. Fine structure of the deep pineal. J. Morph., 131, 163-178 (1970b) Smith, I., Mullen, P.E., Snedden, W., Wilson, B.W. : Absolute identification of melatonin in human plasma and cerebrospinal fluid. Nature (Lond.) 260, 718-719 (1976)
Received January 20, 1978
