Cell Tiss. Res. 162, 61--73 (1975) 9 by Springer-Verlag 1975
The Primate Median Eminence I. C o r r e l a t i v e S c a n n i n g - T r a n s m i s s i o n E l e c t r o n Microscopy * D. E. Scott**, Gerda Krobiseh-Dudley, W. K. P a u l l ***, G. P. Kozlowski, a n d J. R i b a s The Departments of Anatomy, University of Rochester, School of Medicine and Dentistry, Rochester, New York Vermont College of Medicine, Burlington, Vermont Colorado State University, Fort Collins, Colorado, and The Armed Forces Institute of Pathology, Washington, D. C., USA Received May 9, 1975
Summary. A combined scanning/transmissionelectron microscopic (SEM/TEM) technique was used to analyze the third cerebral ventricle and underlying tissue of the median eminence of 6 mature rhesus monkeys. The same sample of the ventricular wall was subjected to both SEN and TEM. This technique demonstrates two basic subpopulations of supraependymal ceils on the surface of the supraoptic, infundibular and mammillary recesses. Type 1 cells are definitely neuron-like in their surface configuration and internal fine structural organization. Type 2 cells are more similar to histiocytes and are not as numerous as type 1 cells. The functional capacity of type 1 cells is discussed in the context of their potential role as a neuronal network that may serve as a short, loop autoregulatory mechanism controlling the synthesis of releasing hormones or biogenic amines. Key words: Scanning-transmission electron microscopy - - Median eminence - - Third ventricle - - Supraependymal neurons - - Supraependymal histioeytes.
Introduction The e v o l u t i o n of our knowledge of the m a m m a l i a n c i r c u m v e n t r i c u l a r organ system, r e c e n t l y a n a l y g i z e d as the " S e v e n W i n d o w s of the B r a i n " (Knigge, 1975), has essentially been due to i n t e n s i v e research over the last 20 years o n the m e d i a n e m i n e n c e of the n e u r o h y p o p h y s e a l system. This bonafide n e u r o e n d o c r i n e transducer has served as ~r superb model for the u n d e r s t a n d i n g of other c i r c u m v e n t r i c u l a r organs which share m a n y c o m m o n features with respect to their n e u r o n a l , glial a n d vascular organization. The " w i n d o w " hypothesis confronts the issue t h a t these highly specialized, a n c i e n t regions of the v e r t e b r a t e b r a i n m a y serve to integrate the blood, b r a i n a n d cerebrospinaI fluid (CSF). The p r e s e n t invest i g a t i o n is a n a t t e m p t to e x p a n d our d a t a base a b o u t the p r i m a t e m e d i a n eminence as revealed b y the use of c o m b i n e d s c a n n i n g / t r a n s m i s s i o n electron microscopy u p o n the same tissue samples of the p r i m a t e h y p o t h a l a m u s .
Send o[[print request8 to: Prof. D. E. Scott, Department of Anatomy, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA. * Supported by USPHS Program Project Grant NS 11642, HD 08867 ** Career Development Awardee KO4-GM-70001 *** NSF Inst. 73-159
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Material and Methods Six mature male rhesus monkeys (t~lacaca mutatta) were utilized in this investigation. A 20 Ga s~rile stainless steel cannula was stereotaxically implanted into the lateral cerebral ventricle and secured to the surrounding calvarium with screws and dental acrylic. A sterile stylette was inserted into the cannula and was secured with a knurled screw top cap. Animals were allowed to survive one year and weekly tests for cannula patency and CSF sterility were conducted. Subsequently, monkeys were heparinized (t/2 cc 5000:1 Lipoheparin) and anesthetized with either ke~amine hydrochloride or sodium pentobarbital. The stylette was removed and the cannula was connected to a perfusion system containing Karnovsky's (1965) fixative pH 7.3 at. an osmolality of 800 m osmls. The eisterna magna was exposed and a ventriculocisternal perfusion of fixative was initiated at a rate of 2 cc/min. Simultaneously a thoracotomy was performed and a perfusion through the left cardiac ventricle was carried out. Following fixation with 600 ml of fixative the hardened brains were carefully dissected away from the calvaria and certain circumventricular organs were removed, blocked and postfixed in 1% osmium tetroxide for 2 hours. Tissues were dehydrated in ascending acetone washes into freon TF and subsequently freon 13. Tissues were critically point dried in a Bomar SPC 50 and shadow casted with 5 cm 8 mil gold in a Denton vacuum evaporator. Specimens were mounted on aluminum stubs and analyzed with Jeol Jsm-u3 and Jsm-35 scanning electron microscopes. Following evaluation with scanning electron microscopy (SEM) tissues were removed from the stubs, reinfiltrated with 50, 75 and 100% acetone and embedded in Spurr low viscosity resin and polymerized at 60~ for 24 hours. Blocks of various portions of previously scanned circumventricular organs were analyzed with routine light microscopy (LM) and transmission electron microscopy (TEM).
Observations
Scanning Electron Microscopy T h e dorsal t h a l a m i c walt of the t h i r d ventricle of all 6 male macaques disp l a y e d a dense p o p u l a t i o n of k n o b - t i p p e d cilia which obscured u n d e r l y i n g substructure. A v e n t r a l progression toward the i n f u n d i b u l a r recess revealed a decrease i n cilial d e n s i t y a n d the emergence of u n d e r l y i n g microvilli. The floor of the i n f u n d i b u l a r recess of all a n i m a l s d e m o n s t r a t e d a significantly large populat i o n of supra,ependymal cells (Fig. 1A). Based on t h e i r surface u l t r a s t r u c t u r e a n d configuration several t y p e s could be i n i t i a l l y delineated. Bipolar cells with r e t i c u l a t e d surfaces were most a b u n d a n t (Figs. 1 C, 2A). However, stellate ceils with n u m e r o u s r a d i a t i n g processes were also observed (Fig. 1 B). The processes of these two cell types course horizontally over the u n d e r l y i n g v e n t r i c u l a r surface a n d e x h i b i t m a n y branches a n d ramifications. Collaterals or w h a t could be described as delicate rootlets of these branches often course between e p e n d y m a l microvilli of the v e n t r i c u l a r surface a n d p e n e t r a t e the u n d e r l y i n g p a r e n c h y m a (Fig. 1D). A t h i r d cell type, d i s t i n c t l y different from the first two described, can f r e q u e n t l y be observed. These cells posses microvilli a n d folds on their surfaces a n d often d e m o n s t r a t e long, f l a t t e n e d p a l m a t e processes with fluted, ruffled m a r g i n s (Figs. 1 C, 5A). F r e q u e n t l y these cells formed satellite relationships with t h e former two tivpes (Figs. 1 B, 1 C). These three types were n o t confined to t h e i n f u n d i b u l a r recess b u t could be observed as well i n the supraoptic, the m a m m i l l a r y recess, a n d occasionally on the surface of the o r g a n u m vasculosum. However, t h e y were rarely observed u p o n the v e n t r i c u l a r surfaces of other circum-
Fig. 1. A. Low magnification SEM of infundibular recess of m a t u r e , normal male rhesus m o n k e y . The ventricular floor is s t u d d e d with n u m e r o u s s u p r a e p e n d y m a l cells (arrows). x 1000. B. S u p r a e p e n d y m a l cell (SC) on floor of infundibular recess is stellate in its configuration a n d possesses n u m e r o u s branching processes which radiate over surface of ventricular l u m e n in a neuron-like fashion, x 1600. C. Bipolar neuron-like cell (B) with a reticulated surface is a d j a c e n t to a distinctly different t y p e of cell (arrows) t h a t exhibits a fluted surface with n u m e r o u s foldings or villi. This cell t y p e possesses long p a l m a t e processes (P). x 6000. D. High magnification SEM of s u p r a e p e n d y m a l neuron-like cell (SC) on surface of infundi. bular recess. Note n u m e r o u s collaterals or rootlets (arrows) which appear to penetrate into the underlying p a r e n c h y m a of t h e ventricular floor which is p u n c t u a t e d b y n u m e r o u s linear a n d clavate microvilli. X 10000
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ventricular organs. The greatest density of supraependymal cells was observed in the infundibular recess. Frequently large concentrations of spheroidal bodies, 2-18 ~ in diameter, were seen along the ventrolateral thalamic wall and floor of the infundibular recess (Fig. 4A). The surfaces of these round bleb-like formations were variegated, demonstrating either very smooth or pitted surfaces. The population density of these structures in certain areas was sufficient to obscure underlying ventricular substructure.
Light and Transmission Electron Microscopy Preparations of diencephalon and median eminence (infundibular recess) previously analyzed with SEM revealed a remarkable degree of ultrastructural preservation. Light microscopic cross sections through the infundibular recess amply demonstrated well preserved tanycytes with distinct ventrally coursing shafts and numerous apical blebs. At the ultrastructural level, both tanycytes and non-tanycytic cuboidal-ependymal cells exhibited an excellent degree of preservation despite previous critical point drying and shadow casting with gold. The ultra-architectural integrity was such that even the organization of delicate microvilli was well retained along with easily identifiable organelles such as Golgi cisternae, rough endoplasmic retieulum, mitochondria and polysomes (Figs. 3A, 3 B). A distinct gold coating (approximately 70 A thick) could be detected over the ventricular surfaces of ependymal cells (Figs. 3C, D). However, this coating from previous shadow casting for SEM was neither consistent nor uniform and over certain areas of ependymal surfaces was incomplete. TEM of areas where dense spheroidal populations were noted previously with SEM revealed distinct ependymal protoplasmic protrusions or "blebs" which extended away from the ependymal surface into the lumen of the third ventricle. These blebs were usually attached to underlying ependyma (both cuboidal and tanycytic) by an isthmus of cytoplasm (Fig. 4B). Other spherules appeared to be free in the ventricular lumen and were apparently undergoing degTadation at the time of fixation (Fig. 4C). Amorphous colloidal material of differing density was the characteristic hallmark of these cytoplasmic modifications. I n regions where numerous Type 1 and 2 supraependymal cells were noted, semi-thick sections revealed cells on the surface of the ventricular lumen which possessed dark, Nissl filled cytoplasm and lightly stained nuclei. TEM analysis of alternate sections from such areas demonstrated cells upon the ventricular lumen with many neuronal characteristics. These included well developed Golgi
Fig. 2A. SEM of bipolar supraependymal cell (SC) on ventricular surface of infundibular recess. Small branches from main processes leave at right angles to the axis of the cell. Polygonal structures on ventricular floor adjacent to bipolar cell are regarded as artifact, x 2200 B. TEM of supraependymal cell (SC) in direct contact with the ventricular lumen (V). It p o s s e s s e s numerous neuronal characteristics including extensive Golgi eisternae (G), dense core vesicles (DCV) and a synaptie-like process (S) that terminates upon its plasmalemma. This latter structure is filled with numerous, small dense core and microvesicles. E ependymal cell. x 9 630 5
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Fig. 3. (A) Low magnification transmission electron microgram of infundibular wall from hypothalamus previously critically point dried and analyzed with SEM. Cuboidal ependymal cells (E) of this region are well preserved and possess solely microvilli. N nuclei, N U nucleolus, V third ventricle. X 3200. (B) Higher magnification TEM of floor of infundibular recess of normal male rhesus monkey. Despite critical point drying, shadow casting and SEM, the ultrastructural organization of organelles and inclusions in these ependymal cells is well
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cisternae, rough endoplasmic reticulum, clear coated and dense core vesicles as well as axon terminals filled with microvesicles and small dense core vesicles which terminated upon their ventricular surfaces forming apparent synaptic relationships (Fig. 2B). Ultrastructural detail beneath the scanned ventricular floor is preserved equally well, and in some cases, preservation is superior. Arcuate neurons similar in appearance to those cells observed in contact with the ventricular lumen possess easily identifiable substructures and exhibit axosomatic synapses. The palisade zone of critically point dried rhesus median eminence exhibits shafts of tanycytic ependyma with numerous microtubules and filaments. Interspersed between these specialized ependymal cells are longitudinally sectioned axons which exhibit numerous easily identifiable dense core vesicles. The contact zone of the rhesus monkey is equally well preserved despite critical point drying and possesses all the elements characterized in previous investigations in other mammalian species which have employed solely TEM techniques alone. These include numerous tuberoinfundibular axon terminals filled with abundant dense core vesicles, perivascular glial cells and typically fenestrated portal vessels upon which these elements terminate (Fig. 6). Discussion
From these data it becomes abundantly clear that the combined SEM/TEM analysis on the same sample of ventricular wall and underlying median eminence provides an excellent opportunity to expand the size of the ultrastructural sample while still preserving the fine structural integrity of the tissue. These data also necessitate a re-examination of the ependymal blebhing phenomenon referred to as "ependymosecretion". Previous literature (Knowles and Kumar, 1969; Coates, 1973) has linked this phenomenon with estrus related events and/or alterations in ambient levels of gonadal steroids. Knowles and K u m a r (1969) have further proposed that, based on ultrastructural alterations of their apical (ventricular) configurations, tanycytes m a y function as feedback receptors for gonadal steroids. In view of the fact that only mature male monkeys were utilized in this investigation, animals which ostensibly possess tonic levels of gonadal steroids, it would appear that this phenomenon may have nothing to do with gonadal steroid feedback or, if so, it may not he a purely estrus related event. Furthermore, it is not restricted to tanycytes alone but is freqently observed on the surfaces of
preserved. G Golgi eisternae, E R rough endoplasmic reticulum, M mitochondria, N nucleus, M V microvilli, V ventrieular lumen, x 9 630. (C) High magnification TEM of ventricular wall of normal male rhesus monkey. In this preparation from a region adjacent to :Fig. 3B, the distinct gold coat from shadow casting {arrows) is apparent upon the luminal surfaces of microvilli. E ependymal cytoplasm, x 14000. (D) TEM of infundibular recess of normal male monkey. The gold coat (arrows) from previous shadow casting is not always consistent nor even and is missing in various regions. This inconsistency may be due to incomplete coating during shadow casting or separation during technical processing for TEM. E ependymal cell, N nucleus, x 12100
Fig. 4. (A) Low magnification SEM of ventral wall of rhesus t h i r d ventricle. Large numbers of ependymal blebs (B) appear as pleomorphic spherules which project pendulously int~) the ventricular lumen. C cilia. • (B) T E M of same region of ventricnlar wall as viewed in :Fig. 4A. These cytoplasmic excrescences (B) appear to be filled with homogeneous colloidal cytoplasm a n d in the early stages of their development are connected to the perikarya of underlying ependyma b y a t h i n neck or isthmus of cytoplasm. These blebs are not confined to true tanycytes b u t as evidenced here are also characteristic of cuboidal ciliated ependymal cells t h a t line the third ventricle of the rhesus monkey. C cilia, M V microvilli, V t h i r d ventricle. • 6600. (C) T E M of ventricular wall demonstrating ependymal blebs (B) in a terminal s t a t e of degradation. The gold coating (arrows) from shadow casting is still apparent. However, t h e plasmatemma in several regions appears to have broken down a n d the homogeneous colloidal material of these cpendymal blebs is in direct contact with the ventricular lumen (V). C cilia, E ependymal cell. •
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Fig. 5. (A) SEM of supraependymal cell (SC) on ventricular floor in the supraoptic recess of the rhesus monkey t h i r d cerebral ventricle. This cell type is distinctly different from those previously described a n d possesses flat palmate processes with fluted, ruffled edges (arrows). Compare with Figs. 1 ~ n d 2. C cilia, RBC, red blood cell. • 3 220. (B) TEM of same region of ventricle as seen in Fig. 5A. Cell type (H) observed is slightly elevated above ventricular surface in the lumen (V) and possesses numerous lamellapodia (arrows) a n d dense granular cytoplasm. Based on their fine structural configuration this cell type is interpreted to be histiocytic in nature. N nucleus. • 9 630
:Fig. 6. T E M of palisade-contact zone of median eminence of normal male rhesus monkey. Despite previous critical point drying and analysis with SEM, the ultrastructural preservation of elements here is remarkably good. Tuberoinfundibular axon preterminats and ter~ minals (A) with a b u n d a n t dense core vesicles (DCV) predominate and are observed to terminate upon the perivascular space (PS) of an adjacent portal vessel (PV). I n the upper q u a d r a n t of the micrograph a large Herring Body (HB) can be observed filled with large vesicles, m a n y of which are pale or lucent. The reduction in density of these magnocellular inclusions m a y be due to elution of their contents from technical processing. N nucleus, PG perivaseular glial cell. x 10800
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ciliated cuboidal ependymal cells. The identity and chemical nature of the content of these ependymal blebs remains enigmatic. However, because of their amorphouscolloidal appearance, they could represent an apical secretion product with a specific target organ influence. On the other hand, these apical ependymal "blebs", common to most m a m m a l i a n species studied to date, m a y simply be the byproduct(s) of metabolism and thus "blebbing" m a y be an inherent process in all ependymae t h a t line the cerebral ventricular system. This latter hypothesis is not unreasonable in view of the fact t h a t the central nervous system lacks a true lymphatic drainage system. Thus, the CSF m a y not only serve to transport biologically active molecules (Heller et al., 1968; Heller, 1969; Feldberg and Meyers, 1960; Vorherr, 1968; Linfoot etal., 1970; Herren, 1969; Joseph etal., 1973; Knigge and Joseph, 1974; Joseph, 1975), it m a y in addition be a mechanism for the removal of cellular metabolites from ependymal cells and the adjacent periventrieular neuropil. The presence of supraependymal cells or CSF contacting elements and/or processes in the mammalian ventricle is not a finding original to this investigation and has been reported in earlier light, SEM and TEM investigations b y Bleier (1972), Vigh and Vigh-Teehmann (1970, 1971), MeKenna and Rosenbluth (1974), Paull etal. (1974, 1975), Leonhardt and Lindner (1967), Wittkowski (1968), Westergaard (1970), Clementi and Marini (1972), Scott et al. (1973, 1974a, b) and Allan and Low (1974). These investigations coupled with the elegant S E E investigations by Coates (1973) have confirmed the presence and legitimacy of such elements beyond any doubt. However, what is new to this investigation is our capacity to identify with greater precision the nature of snpraependymal celltypes. From combined SEM/TEM it would appear t h a t two basic types of cells can be found upon the ventricular surface: Type 1, bipolar and multipolar neuron-like cells, and Type 2, histiocytic-like cells with some apparent capacity of motility based on the possession of long palmate processes with fluted, ruffled edges (Revel,1974). Macrophages have been reported in the cerebral-ventricular system of other mammals and m a y simply serve as a mechanism to rid the ventricular CSF of the products of normal cell metabolism or debris from cell death and turnover (Hosoya and Fujita, 1973). The well established concept of liquor (CSF) contact neuron-like cells is further confirmed by these data and raises the issue of the existence of a system or network of supraependymal cells t h a t m a y have a precise neurological function. Within the context of such a paradigm, two concepts come to mind. (1) Either these neuron-like cells or their processes secrete biologically active hormones into the CSF, or (2) they m a y serve as a sensory network which assesses altering concentrations of physiologically active molecules in the CSF, and simply transmit excitatory or inhibiting information back to the very cell groups which arc responsible for the synthesis, storage and release of bioactive molecules such as releasing hormones or biogenic amines. Thus, supraependymal neuron-like cells observed in abundance in the third ventricle and parenthetically in close proximity to suspected hypophysiotrophic pools of parvicellular neurons (Halaw et al., 1962) m a y function as an ultra-short loop autoregulatory feedback mechanism which m a y assist in the exquisite control of the peripheral endocrine milieu and m a y be but one component of the highly complex circumventricular brain.
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References Allan, D. J., Low, F. N. : The ependymal surface of the lateral ventricle of the dog as revealed by scanning electron microscopy. Amer. J. Anat. 187, 483489 (1973) Bleier, R. : Structural relationship of ependymal cells and their processes within the hypothalamus. In: Brain-Endocrine Interaction. Median Eminence: Structure and Function, (Knigge, Scott and Weindl, eds.), p. 306-318. Basel: Karger 1972 Clementi, F., Marini, D. : The surface fine structure of the walls of cerebral ventricles and of choroid plexus in cat. Z. Zellforsch. 123, 81-95 (1972) Coates, P. W. : Supraependymal cells : light and transmission electron microscopic determination. Brain Res. 57, 502-507 (1973) Feldberg, W., Meyers, R. D. : The appearance fo 5-hydroxytryptamine and an unidentified lipid acid in effluent form perfused cerebral ventricles. J. Physiol. (Lond.) 184, 837-855 (1966) Halaw B., Pupp, L., Uhlarik, S. : Hypophysiotrophic area in the hypothalamus. J. Endocr. 25, 147-154 (1962) I-Idler, H. : Neurohypophyseal hormones in the cerebrospinal fluid. In: Zirkumventrikul~re Organe und Liquor, (Sterba, ed.), p. 232-235. Jena: Fisher 1969 Heller, H., Hasan, S . H . , Saifi, A. 0.: Antidiuretic activity in the cerebrospinal fluid. J. Endocr. 41, 273-280 (1968) Horton, E. W. : The hypothesis on physiological rates of prostaglandins. Physiol. Rev. 49, 122 161 (1969) Hosoya, u Fujita, T. : Scanning electron microscopic observation of intraventricular macrophages (Kolmer cells) in the rat brain. Arch. Histol. Jap. 3~, 133-140 (1973) Joseph, S. A., Scott, D. E., Vaala, S. S., Knigge, K. M., Krobisch-Dudley, G. : Localization and content of thyrotrophin releasing factor (TRF) in median eminence of hypothalamus. Acta endocr. (Kbh.) 74, 215-225 (1973) Joseph, S.A., Sorrentino, S., Sundberg, D. K.: Releasing hormones in the cerebrospinal fluid;in: Knigge, Scott, Kobayashi and Ishfi, Brain-Endocrine Interaction. The ventricular system in neuroendocrine mechanisms. Basel: Karger, in press (1975) Karnovsky, M. J. : A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A-138A (1965) Knigge, K. M.: Opening remarks; in: Knigge, Scott, Kobayashi and Ishii, Brain-Endocrine Interaction II. The ventricular system in neuroendocrine mechanisms. Basel: Karger, in press (1975) Knigge, K. M., Joseph, S. A. : Thyrotrophin releasing factor (TRF) in CSF of third ventricle of rat brain. Acta endocr. (Kbh.) 76, 209-213 (1974) Leonhardt, H., Lindner, E. : Marklose Nervenfasern im III. und IV. Ventrikel des Kaninchenund Katzengehirns. Z. Zellforsch. 78, 1-18 (1967) Linfoot, J . A . , Garcia, J. F., Wei, W., Fink, R., Satin, R., Born, J. L., Lawrence, J . H . : Human growth hormone levels in cerebrospinal fluid. J. clin. Endocr. 81, 230-232 (1970) McKenna, O., Rosenbluth, J.: Cytological evidence for catecholamine-containing sensory cells bordering the ventricle of the toad hypothalamus. J. comp. Neurol. 154, 133-148 (1974) Paull, W. K., Scott, D. E. : Cerebral ventrieular surfaces; in: Hyat, Principles and Techniques of Scanning Electron Microscopy, in press (1975) Paull, W. K., Scott, D. E., Boldosser, W. G. : A cluster of supraependymal neurons located within the infundibular recess of the rat third ventricle. Amer. J. Anat. 140, 129-133 (1974) Revel, J. P. : Scanning electron microscopy and freeze cleaving of cell surfaces in developing systems. In: Symp. Cell Surface Topography and Properties of the Membranes. Proc. Amer. Assoc. Anat. (1974) Scott, D. E. : The ultrastructural correlates of circumventricular organ function. I. The median eminence as a neuroendocrine transducer; in: Kumar, Neuroendocrine Regulation of Fertility. New Delhi, India, in press (1974) Scott, D. E., Kozlowski, G.P., Sheridan, M. N. : Scanning electron microscopy in the ultrastructural analysis of the mammalian cerebral ventricular system. Int. Rev. Cytol. 36, 349-388 (1974)
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Scott, D. E., Krobisch-Dudley, G.: Ultrastructural analysis of mammalian median eminence. I. Morphologic correlates of transependymal transport; in: Knigge, Scott, Kobayashi and Ishii, Brain-Endocrine Interaction, The vcntricular system in neuroendocrine mechanisms. Basel: Karger, in press (1975) Vigh-Teichmann, I., Vigh, B., Aros, B.: Liquorkontaktneurone im Nucleus infundibularis des Kiikens. Z. Zellforsch. 112, 188-200 (1971) Vigh-Teichmann, I., Vigh, B., Koritshnszky, S., Aros, B. : Liquorkontaktneurone im Nucleus infundibularis. Z. Zellforsch. 108, 17-34 (1970) Vorherr, H., Bradbury, M . W . B . , Hoghoughi, M., Kleeman, C.R.: Antidiuretic hormone in cerebrospinal fluid during endogenous and exogenous changes in its blood level. Endocrinology 88, 246-250 (1968) Westergaard, E. : The lateral cerebral ventricles and the ventricular walls. An anatomical, histological and electron microscopic investigation on mice, rats, hamsters, guinea pigs and rabbits. Thesis: Andelsbogfrykkeriet: Odense 1-216 (1970) Wittkowski, W. : Elektronenmikroskopische Studien zur intraventrikuli~ren Neurosekretion in den Recessus infundibularis der Maus. Z. Zellforsch. 92, 207-216 (1968)
The Primate Median Eminence I. C o r r e l a t i v e S c a n n i n g - T r a n s m i s s i o n E l e c t r o n Microscopy * D. E. Scott**, Gerda Krobiseh-Dudley, W. K. P a u l l ***, G. P. Kozlowski, a n d J. R i b a s The Departments of Anatomy, University of Rochester, School of Medicine and Dentistry, Rochester, New York Vermont College of Medicine, Burlington, Vermont Colorado State University, Fort Collins, Colorado, and The Armed Forces Institute of Pathology, Washington, D. C., USA Received May 9, 1975
Summary. A combined scanning/transmissionelectron microscopic (SEM/TEM) technique was used to analyze the third cerebral ventricle and underlying tissue of the median eminence of 6 mature rhesus monkeys. The same sample of the ventricular wall was subjected to both SEN and TEM. This technique demonstrates two basic subpopulations of supraependymal ceils on the surface of the supraoptic, infundibular and mammillary recesses. Type 1 cells are definitely neuron-like in their surface configuration and internal fine structural organization. Type 2 cells are more similar to histiocytes and are not as numerous as type 1 cells. The functional capacity of type 1 cells is discussed in the context of their potential role as a neuronal network that may serve as a short, loop autoregulatory mechanism controlling the synthesis of releasing hormones or biogenic amines. Key words: Scanning-transmission electron microscopy - - Median eminence - - Third ventricle - - Supraependymal neurons - - Supraependymal histioeytes.
Introduction The e v o l u t i o n of our knowledge of the m a m m a l i a n c i r c u m v e n t r i c u l a r organ system, r e c e n t l y a n a l y g i z e d as the " S e v e n W i n d o w s of the B r a i n " (Knigge, 1975), has essentially been due to i n t e n s i v e research over the last 20 years o n the m e d i a n e m i n e n c e of the n e u r o h y p o p h y s e a l system. This bonafide n e u r o e n d o c r i n e transducer has served as ~r superb model for the u n d e r s t a n d i n g of other c i r c u m v e n t r i c u l a r organs which share m a n y c o m m o n features with respect to their n e u r o n a l , glial a n d vascular organization. The " w i n d o w " hypothesis confronts the issue t h a t these highly specialized, a n c i e n t regions of the v e r t e b r a t e b r a i n m a y serve to integrate the blood, b r a i n a n d cerebrospinaI fluid (CSF). The p r e s e n t invest i g a t i o n is a n a t t e m p t to e x p a n d our d a t a base a b o u t the p r i m a t e m e d i a n eminence as revealed b y the use of c o m b i n e d s c a n n i n g / t r a n s m i s s i o n electron microscopy u p o n the same tissue samples of the p r i m a t e h y p o t h a l a m u s .
Send o[[print request8 to: Prof. D. E. Scott, Department of Anatomy, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA. * Supported by USPHS Program Project Grant NS 11642, HD 08867 ** Career Development Awardee KO4-GM-70001 *** NSF Inst. 73-159
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Material and Methods Six mature male rhesus monkeys (t~lacaca mutatta) were utilized in this investigation. A 20 Ga s~rile stainless steel cannula was stereotaxically implanted into the lateral cerebral ventricle and secured to the surrounding calvarium with screws and dental acrylic. A sterile stylette was inserted into the cannula and was secured with a knurled screw top cap. Animals were allowed to survive one year and weekly tests for cannula patency and CSF sterility were conducted. Subsequently, monkeys were heparinized (t/2 cc 5000:1 Lipoheparin) and anesthetized with either ke~amine hydrochloride or sodium pentobarbital. The stylette was removed and the cannula was connected to a perfusion system containing Karnovsky's (1965) fixative pH 7.3 at. an osmolality of 800 m osmls. The eisterna magna was exposed and a ventriculocisternal perfusion of fixative was initiated at a rate of 2 cc/min. Simultaneously a thoracotomy was performed and a perfusion through the left cardiac ventricle was carried out. Following fixation with 600 ml of fixative the hardened brains were carefully dissected away from the calvaria and certain circumventricular organs were removed, blocked and postfixed in 1% osmium tetroxide for 2 hours. Tissues were dehydrated in ascending acetone washes into freon TF and subsequently freon 13. Tissues were critically point dried in a Bomar SPC 50 and shadow casted with 5 cm 8 mil gold in a Denton vacuum evaporator. Specimens were mounted on aluminum stubs and analyzed with Jeol Jsm-u3 and Jsm-35 scanning electron microscopes. Following evaluation with scanning electron microscopy (SEM) tissues were removed from the stubs, reinfiltrated with 50, 75 and 100% acetone and embedded in Spurr low viscosity resin and polymerized at 60~ for 24 hours. Blocks of various portions of previously scanned circumventricular organs were analyzed with routine light microscopy (LM) and transmission electron microscopy (TEM).
Observations
Scanning Electron Microscopy T h e dorsal t h a l a m i c walt of the t h i r d ventricle of all 6 male macaques disp l a y e d a dense p o p u l a t i o n of k n o b - t i p p e d cilia which obscured u n d e r l y i n g substructure. A v e n t r a l progression toward the i n f u n d i b u l a r recess revealed a decrease i n cilial d e n s i t y a n d the emergence of u n d e r l y i n g microvilli. The floor of the i n f u n d i b u l a r recess of all a n i m a l s d e m o n s t r a t e d a significantly large populat i o n of supra,ependymal cells (Fig. 1A). Based on t h e i r surface u l t r a s t r u c t u r e a n d configuration several t y p e s could be i n i t i a l l y delineated. Bipolar cells with r e t i c u l a t e d surfaces were most a b u n d a n t (Figs. 1 C, 2A). However, stellate ceils with n u m e r o u s r a d i a t i n g processes were also observed (Fig. 1 B). The processes of these two cell types course horizontally over the u n d e r l y i n g v e n t r i c u l a r surface a n d e x h i b i t m a n y branches a n d ramifications. Collaterals or w h a t could be described as delicate rootlets of these branches often course between e p e n d y m a l microvilli of the v e n t r i c u l a r surface a n d p e n e t r a t e the u n d e r l y i n g p a r e n c h y m a (Fig. 1D). A t h i r d cell type, d i s t i n c t l y different from the first two described, can f r e q u e n t l y be observed. These cells posses microvilli a n d folds on their surfaces a n d often d e m o n s t r a t e long, f l a t t e n e d p a l m a t e processes with fluted, ruffled m a r g i n s (Figs. 1 C, 5A). F r e q u e n t l y these cells formed satellite relationships with t h e former two tivpes (Figs. 1 B, 1 C). These three types were n o t confined to t h e i n f u n d i b u l a r recess b u t could be observed as well i n the supraoptic, the m a m m i l l a r y recess, a n d occasionally on the surface of the o r g a n u m vasculosum. However, t h e y were rarely observed u p o n the v e n t r i c u l a r surfaces of other circum-
Fig. 1. A. Low magnification SEM of infundibular recess of m a t u r e , normal male rhesus m o n k e y . The ventricular floor is s t u d d e d with n u m e r o u s s u p r a e p e n d y m a l cells (arrows). x 1000. B. S u p r a e p e n d y m a l cell (SC) on floor of infundibular recess is stellate in its configuration a n d possesses n u m e r o u s branching processes which radiate over surface of ventricular l u m e n in a neuron-like fashion, x 1600. C. Bipolar neuron-like cell (B) with a reticulated surface is a d j a c e n t to a distinctly different t y p e of cell (arrows) t h a t exhibits a fluted surface with n u m e r o u s foldings or villi. This cell t y p e possesses long p a l m a t e processes (P). x 6000. D. High magnification SEM of s u p r a e p e n d y m a l neuron-like cell (SC) on surface of infundi. bular recess. Note n u m e r o u s collaterals or rootlets (arrows) which appear to penetrate into the underlying p a r e n c h y m a of t h e ventricular floor which is p u n c t u a t e d b y n u m e r o u s linear a n d clavate microvilli. X 10000
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ventricular organs. The greatest density of supraependymal cells was observed in the infundibular recess. Frequently large concentrations of spheroidal bodies, 2-18 ~ in diameter, were seen along the ventrolateral thalamic wall and floor of the infundibular recess (Fig. 4A). The surfaces of these round bleb-like formations were variegated, demonstrating either very smooth or pitted surfaces. The population density of these structures in certain areas was sufficient to obscure underlying ventricular substructure.
Light and Transmission Electron Microscopy Preparations of diencephalon and median eminence (infundibular recess) previously analyzed with SEM revealed a remarkable degree of ultrastructural preservation. Light microscopic cross sections through the infundibular recess amply demonstrated well preserved tanycytes with distinct ventrally coursing shafts and numerous apical blebs. At the ultrastructural level, both tanycytes and non-tanycytic cuboidal-ependymal cells exhibited an excellent degree of preservation despite previous critical point drying and shadow casting with gold. The ultra-architectural integrity was such that even the organization of delicate microvilli was well retained along with easily identifiable organelles such as Golgi cisternae, rough endoplasmic retieulum, mitochondria and polysomes (Figs. 3A, 3 B). A distinct gold coating (approximately 70 A thick) could be detected over the ventricular surfaces of ependymal cells (Figs. 3C, D). However, this coating from previous shadow casting for SEM was neither consistent nor uniform and over certain areas of ependymal surfaces was incomplete. TEM of areas where dense spheroidal populations were noted previously with SEM revealed distinct ependymal protoplasmic protrusions or "blebs" which extended away from the ependymal surface into the lumen of the third ventricle. These blebs were usually attached to underlying ependyma (both cuboidal and tanycytic) by an isthmus of cytoplasm (Fig. 4B). Other spherules appeared to be free in the ventricular lumen and were apparently undergoing degTadation at the time of fixation (Fig. 4C). Amorphous colloidal material of differing density was the characteristic hallmark of these cytoplasmic modifications. I n regions where numerous Type 1 and 2 supraependymal cells were noted, semi-thick sections revealed cells on the surface of the ventricular lumen which possessed dark, Nissl filled cytoplasm and lightly stained nuclei. TEM analysis of alternate sections from such areas demonstrated cells upon the ventricular lumen with many neuronal characteristics. These included well developed Golgi
Fig. 2A. SEM of bipolar supraependymal cell (SC) on ventricular surface of infundibular recess. Small branches from main processes leave at right angles to the axis of the cell. Polygonal structures on ventricular floor adjacent to bipolar cell are regarded as artifact, x 2200 B. TEM of supraependymal cell (SC) in direct contact with the ventricular lumen (V). It p o s s e s s e s numerous neuronal characteristics including extensive Golgi eisternae (G), dense core vesicles (DCV) and a synaptie-like process (S) that terminates upon its plasmalemma. This latter structure is filled with numerous, small dense core and microvesicles. E ependymal cell. x 9 630 5
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Fig. 3. (A) Low magnification transmission electron microgram of infundibular wall from hypothalamus previously critically point dried and analyzed with SEM. Cuboidal ependymal cells (E) of this region are well preserved and possess solely microvilli. N nuclei, N U nucleolus, V third ventricle. X 3200. (B) Higher magnification TEM of floor of infundibular recess of normal male rhesus monkey. Despite critical point drying, shadow casting and SEM, the ultrastructural organization of organelles and inclusions in these ependymal cells is well
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cisternae, rough endoplasmic reticulum, clear coated and dense core vesicles as well as axon terminals filled with microvesicles and small dense core vesicles which terminated upon their ventricular surfaces forming apparent synaptic relationships (Fig. 2B). Ultrastructural detail beneath the scanned ventricular floor is preserved equally well, and in some cases, preservation is superior. Arcuate neurons similar in appearance to those cells observed in contact with the ventricular lumen possess easily identifiable substructures and exhibit axosomatic synapses. The palisade zone of critically point dried rhesus median eminence exhibits shafts of tanycytic ependyma with numerous microtubules and filaments. Interspersed between these specialized ependymal cells are longitudinally sectioned axons which exhibit numerous easily identifiable dense core vesicles. The contact zone of the rhesus monkey is equally well preserved despite critical point drying and possesses all the elements characterized in previous investigations in other mammalian species which have employed solely TEM techniques alone. These include numerous tuberoinfundibular axon terminals filled with abundant dense core vesicles, perivascular glial cells and typically fenestrated portal vessels upon which these elements terminate (Fig. 6). Discussion
From these data it becomes abundantly clear that the combined SEM/TEM analysis on the same sample of ventricular wall and underlying median eminence provides an excellent opportunity to expand the size of the ultrastructural sample while still preserving the fine structural integrity of the tissue. These data also necessitate a re-examination of the ependymal blebhing phenomenon referred to as "ependymosecretion". Previous literature (Knowles and Kumar, 1969; Coates, 1973) has linked this phenomenon with estrus related events and/or alterations in ambient levels of gonadal steroids. Knowles and K u m a r (1969) have further proposed that, based on ultrastructural alterations of their apical (ventricular) configurations, tanycytes m a y function as feedback receptors for gonadal steroids. In view of the fact that only mature male monkeys were utilized in this investigation, animals which ostensibly possess tonic levels of gonadal steroids, it would appear that this phenomenon may have nothing to do with gonadal steroid feedback or, if so, it may not he a purely estrus related event. Furthermore, it is not restricted to tanycytes alone but is freqently observed on the surfaces of
preserved. G Golgi eisternae, E R rough endoplasmic reticulum, M mitochondria, N nucleus, M V microvilli, V ventrieular lumen, x 9 630. (C) High magnification TEM of ventricular wall of normal male rhesus monkey. In this preparation from a region adjacent to :Fig. 3B, the distinct gold coat from shadow casting {arrows) is apparent upon the luminal surfaces of microvilli. E ependymal cytoplasm, x 14000. (D) TEM of infundibular recess of normal male monkey. The gold coat (arrows) from previous shadow casting is not always consistent nor even and is missing in various regions. This inconsistency may be due to incomplete coating during shadow casting or separation during technical processing for TEM. E ependymal cell, N nucleus, x 12100
Fig. 4. (A) Low magnification SEM of ventral wall of rhesus t h i r d ventricle. Large numbers of ependymal blebs (B) appear as pleomorphic spherules which project pendulously int~) the ventricular lumen. C cilia. • (B) T E M of same region of ventricnlar wall as viewed in :Fig. 4A. These cytoplasmic excrescences (B) appear to be filled with homogeneous colloidal cytoplasm a n d in the early stages of their development are connected to the perikarya of underlying ependyma b y a t h i n neck or isthmus of cytoplasm. These blebs are not confined to true tanycytes b u t as evidenced here are also characteristic of cuboidal ciliated ependymal cells t h a t line the third ventricle of the rhesus monkey. C cilia, M V microvilli, V t h i r d ventricle. • 6600. (C) T E M of ventricular wall demonstrating ependymal blebs (B) in a terminal s t a t e of degradation. The gold coating (arrows) from shadow casting is still apparent. However, t h e plasmatemma in several regions appears to have broken down a n d the homogeneous colloidal material of these cpendymal blebs is in direct contact with the ventricular lumen (V). C cilia, E ependymal cell. •
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Fig. 5. (A) SEM of supraependymal cell (SC) on ventricular floor in the supraoptic recess of the rhesus monkey t h i r d cerebral ventricle. This cell type is distinctly different from those previously described a n d possesses flat palmate processes with fluted, ruffled edges (arrows). Compare with Figs. 1 ~ n d 2. C cilia, RBC, red blood cell. • 3 220. (B) TEM of same region of ventricle as seen in Fig. 5A. Cell type (H) observed is slightly elevated above ventricular surface in the lumen (V) and possesses numerous lamellapodia (arrows) a n d dense granular cytoplasm. Based on their fine structural configuration this cell type is interpreted to be histiocytic in nature. N nucleus. • 9 630
:Fig. 6. T E M of palisade-contact zone of median eminence of normal male rhesus monkey. Despite previous critical point drying and analysis with SEM, the ultrastructural preservation of elements here is remarkably good. Tuberoinfundibular axon preterminats and ter~ minals (A) with a b u n d a n t dense core vesicles (DCV) predominate and are observed to terminate upon the perivascular space (PS) of an adjacent portal vessel (PV). I n the upper q u a d r a n t of the micrograph a large Herring Body (HB) can be observed filled with large vesicles, m a n y of which are pale or lucent. The reduction in density of these magnocellular inclusions m a y be due to elution of their contents from technical processing. N nucleus, PG perivaseular glial cell. x 10800
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ciliated cuboidal ependymal cells. The identity and chemical nature of the content of these ependymal blebs remains enigmatic. However, because of their amorphouscolloidal appearance, they could represent an apical secretion product with a specific target organ influence. On the other hand, these apical ependymal "blebs", common to most m a m m a l i a n species studied to date, m a y simply be the byproduct(s) of metabolism and thus "blebbing" m a y be an inherent process in all ependymae t h a t line the cerebral ventricular system. This latter hypothesis is not unreasonable in view of the fact t h a t the central nervous system lacks a true lymphatic drainage system. Thus, the CSF m a y not only serve to transport biologically active molecules (Heller et al., 1968; Heller, 1969; Feldberg and Meyers, 1960; Vorherr, 1968; Linfoot etal., 1970; Herren, 1969; Joseph etal., 1973; Knigge and Joseph, 1974; Joseph, 1975), it m a y in addition be a mechanism for the removal of cellular metabolites from ependymal cells and the adjacent periventrieular neuropil. The presence of supraependymal cells or CSF contacting elements and/or processes in the mammalian ventricle is not a finding original to this investigation and has been reported in earlier light, SEM and TEM investigations b y Bleier (1972), Vigh and Vigh-Teehmann (1970, 1971), MeKenna and Rosenbluth (1974), Paull etal. (1974, 1975), Leonhardt and Lindner (1967), Wittkowski (1968), Westergaard (1970), Clementi and Marini (1972), Scott et al. (1973, 1974a, b) and Allan and Low (1974). These investigations coupled with the elegant S E E investigations by Coates (1973) have confirmed the presence and legitimacy of such elements beyond any doubt. However, what is new to this investigation is our capacity to identify with greater precision the nature of snpraependymal celltypes. From combined SEM/TEM it would appear t h a t two basic types of cells can be found upon the ventricular surface: Type 1, bipolar and multipolar neuron-like cells, and Type 2, histiocytic-like cells with some apparent capacity of motility based on the possession of long palmate processes with fluted, ruffled edges (Revel,1974). Macrophages have been reported in the cerebral-ventricular system of other mammals and m a y simply serve as a mechanism to rid the ventricular CSF of the products of normal cell metabolism or debris from cell death and turnover (Hosoya and Fujita, 1973). The well established concept of liquor (CSF) contact neuron-like cells is further confirmed by these data and raises the issue of the existence of a system or network of supraependymal cells t h a t m a y have a precise neurological function. Within the context of such a paradigm, two concepts come to mind. (1) Either these neuron-like cells or their processes secrete biologically active hormones into the CSF, or (2) they m a y serve as a sensory network which assesses altering concentrations of physiologically active molecules in the CSF, and simply transmit excitatory or inhibiting information back to the very cell groups which arc responsible for the synthesis, storage and release of bioactive molecules such as releasing hormones or biogenic amines. Thus, supraependymal neuron-like cells observed in abundance in the third ventricle and parenthetically in close proximity to suspected hypophysiotrophic pools of parvicellular neurons (Halaw et al., 1962) m a y function as an ultra-short loop autoregulatory feedback mechanism which m a y assist in the exquisite control of the peripheral endocrine milieu and m a y be but one component of the highly complex circumventricular brain.
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References Allan, D. J., Low, F. N. : The ependymal surface of the lateral ventricle of the dog as revealed by scanning electron microscopy. Amer. J. Anat. 187, 483489 (1973) Bleier, R. : Structural relationship of ependymal cells and their processes within the hypothalamus. In: Brain-Endocrine Interaction. Median Eminence: Structure and Function, (Knigge, Scott and Weindl, eds.), p. 306-318. Basel: Karger 1972 Clementi, F., Marini, D. : The surface fine structure of the walls of cerebral ventricles and of choroid plexus in cat. Z. Zellforsch. 123, 81-95 (1972) Coates, P. W. : Supraependymal cells : light and transmission electron microscopic determination. Brain Res. 57, 502-507 (1973) Feldberg, W., Meyers, R. D. : The appearance fo 5-hydroxytryptamine and an unidentified lipid acid in effluent form perfused cerebral ventricles. J. Physiol. (Lond.) 184, 837-855 (1966) Halaw B., Pupp, L., Uhlarik, S. : Hypophysiotrophic area in the hypothalamus. J. Endocr. 25, 147-154 (1962) I-Idler, H. : Neurohypophyseal hormones in the cerebrospinal fluid. In: Zirkumventrikul~re Organe und Liquor, (Sterba, ed.), p. 232-235. Jena: Fisher 1969 Heller, H., Hasan, S . H . , Saifi, A. 0.: Antidiuretic activity in the cerebrospinal fluid. J. Endocr. 41, 273-280 (1968) Horton, E. W. : The hypothesis on physiological rates of prostaglandins. Physiol. Rev. 49, 122 161 (1969) Hosoya, u Fujita, T. : Scanning electron microscopic observation of intraventricular macrophages (Kolmer cells) in the rat brain. Arch. Histol. Jap. 3~, 133-140 (1973) Joseph, S. A., Scott, D. E., Vaala, S. S., Knigge, K. M., Krobisch-Dudley, G. : Localization and content of thyrotrophin releasing factor (TRF) in median eminence of hypothalamus. Acta endocr. (Kbh.) 74, 215-225 (1973) Joseph, S.A., Sorrentino, S., Sundberg, D. K.: Releasing hormones in the cerebrospinal fluid;in: Knigge, Scott, Kobayashi and Ishfi, Brain-Endocrine Interaction. The ventricular system in neuroendocrine mechanisms. Basel: Karger, in press (1975) Karnovsky, M. J. : A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A-138A (1965) Knigge, K. M.: Opening remarks; in: Knigge, Scott, Kobayashi and Ishii, Brain-Endocrine Interaction II. The ventricular system in neuroendocrine mechanisms. Basel: Karger, in press (1975) Knigge, K. M., Joseph, S. A. : Thyrotrophin releasing factor (TRF) in CSF of third ventricle of rat brain. Acta endocr. (Kbh.) 76, 209-213 (1974) Leonhardt, H., Lindner, E. : Marklose Nervenfasern im III. und IV. Ventrikel des Kaninchenund Katzengehirns. Z. Zellforsch. 78, 1-18 (1967) Linfoot, J . A . , Garcia, J. F., Wei, W., Fink, R., Satin, R., Born, J. L., Lawrence, J . H . : Human growth hormone levels in cerebrospinal fluid. J. clin. Endocr. 81, 230-232 (1970) McKenna, O., Rosenbluth, J.: Cytological evidence for catecholamine-containing sensory cells bordering the ventricle of the toad hypothalamus. J. comp. Neurol. 154, 133-148 (1974) Paull, W. K., Scott, D. E. : Cerebral ventrieular surfaces; in: Hyat, Principles and Techniques of Scanning Electron Microscopy, in press (1975) Paull, W. K., Scott, D. E., Boldosser, W. G. : A cluster of supraependymal neurons located within the infundibular recess of the rat third ventricle. Amer. J. Anat. 140, 129-133 (1974) Revel, J. P. : Scanning electron microscopy and freeze cleaving of cell surfaces in developing systems. In: Symp. Cell Surface Topography and Properties of the Membranes. Proc. Amer. Assoc. Anat. (1974) Scott, D. E. : The ultrastructural correlates of circumventricular organ function. I. The median eminence as a neuroendocrine transducer; in: Kumar, Neuroendocrine Regulation of Fertility. New Delhi, India, in press (1974) Scott, D. E., Kozlowski, G.P., Sheridan, M. N. : Scanning electron microscopy in the ultrastructural analysis of the mammalian cerebral ventricular system. Int. Rev. Cytol. 36, 349-388 (1974)
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Scott, D. E., Krobisch-Dudley, G.: Ultrastructural analysis of mammalian median eminence. I. Morphologic correlates of transependymal transport; in: Knigge, Scott, Kobayashi and Ishii, Brain-Endocrine Interaction, The vcntricular system in neuroendocrine mechanisms. Basel: Karger, in press (1975) Vigh-Teichmann, I., Vigh, B., Aros, B.: Liquorkontaktneurone im Nucleus infundibularis des Kiikens. Z. Zellforsch. 112, 188-200 (1971) Vigh-Teichmann, I., Vigh, B., Koritshnszky, S., Aros, B. : Liquorkontaktneurone im Nucleus infundibularis. Z. Zellforsch. 108, 17-34 (1970) Vorherr, H., Bradbury, M . W . B . , Hoghoughi, M., Kleeman, C.R.: Antidiuretic hormone in cerebrospinal fluid during endogenous and exogenous changes in its blood level. Endocrinology 88, 246-250 (1968) Westergaard, E. : The lateral cerebral ventricles and the ventricular walls. An anatomical, histological and electron microscopic investigation on mice, rats, hamsters, guinea pigs and rabbits. Thesis: Andelsbogfrykkeriet: Odense 1-216 (1970) Wittkowski, W. : Elektronenmikroskopische Studien zur intraventrikuli~ren Neurosekretion in den Recessus infundibularis der Maus. Z. Zellforsch. 92, 207-216 (1968)
