Contents

Review article:

1. Morphogenesis of the Hypothalamus and Hypophysis : Their Association, Dissociation and Reassociation before and after "Rathke" (Kawamura, K. and S. Kikuyama)

Original articles:

1. Immunoelectron Microscopic Identification of Lysozyme-Expressing Cells in Human Labial Salivary Glands (Miyazaki, T., T. Fujiki, Y. Inoue and K.Takano)
   
2. Identification of Arterial and Venous Segments of Blood Vessels Using Alkaline Phosphatase Staining of Ink/Gelatin Injected Tissues (Ushiki, T. and K. Abe)
   
3. Localization of Cytoplasmic Free Calcium Ions in PC12 Cells with Varicose Fibers (Nakanishi, S., J. Ichikawa and Y. Endo)
   
4. MAP2 and GAP-43 Expression in Normal and Weaver Mouse Cerebellum : Correlative Immunohistochemical and in Situ Hybridization Studies (Triarhou, L. C., C. Sola, J. M. Palacios and G. Mengod)
   
5. Light and Electron Microscopic Detection of Anionic Sites in the Rat Choroid Plexus (Taguchi, T., A. Ohtsuka and T. Murakami)
   
6. Electron Microscopic Characterization of Filiform Papillae in the Normal Human Tongue (Toyoda, M., S. Sakita, M. Kagoura and M. Morohashi)
   
7. Acellular Calcified Columns in the Normal Growth Plate of Mouse Vertebrae (Yokozeki, K., K. Abe, S. Watanabe, K. Suda and K. Kaneda)
   
8. Lymphatic Drainage of the Cerebrospinal Fluid from Monkey Spinal Meninges with Special Reference to the Distribution of the Epidural Lumphatics (Miura, M., S. Kato and M. von Ludinghausen)

Summary

Title
Morphogenesis of the Hypothalamus and Hypophysis : Their Association, Dissociation and Reassociation before and after "Rathke"

Author
Kosuke KAWAMURA and Sakae KIKUYAMA

Summary
Recent progress in the experimental morphology of the development of amphibian pituitary gland is reviewed. A series of transplantation experiments were carried out using wild-type embryos of the toad as a donor and albino embryos as a recipient. Melanin granules in the wild-type cells allowed tracing of the developmental fate of the grafts as a visible cellmarker. These studies have demonstrated that the pituitary gland is not a stomodeal derivative, as it has long been believed to be under the name of "Rathke's pouch". The adenohypophysis is of neural origin. The anterior part of the neural ridge (ANR) in the neuroectoderm of the open neurula gives rise to the whole adenohypophysis, i. e., pars distatis, pars intermedia and pars tuberalis. The presumptive hypothalamus is apposed caudally to the pituitary primordium. A part of the ANR contributes neurons to the preoptic hypothalamus even after closure of the neural tube. The anlagen of the olfactory system, which include the nasal epithelia and the olfactory bulbs, are situated on both sides of the pituitary primordium in the neural ridge. In both hypothalamic-hypophyseal and olfactory systems, the peripheral and central parts derive from closely affiliated cell populations, suggesting their clonal relationships. Development of thehypophysis and hypothalamus is interdependent. On one hand, a connection with the embryonichypothalamus is essential for the pituitary proopiomelanocortin cells to develop. On the other hand, neither the hypothalamic median eminence nor its axonal supply develops without the presence of the pituitary primordium. Novel aspects of the ontogeny and phylogeny of these organs are discussed with special reference to the role of the neural ridge in the generation of a spectrum of chemoreceptive organs.

Contact
Dr. Kosuke KAWAMURA
Department of Bilogy
Waseda University School of Education
1-6-1 Nishi-Waseda, Shinjuku-ku, Tokyo
169-8050 Japan
Tel: +81 3 5286 1517
Fax: +81 3 3207 9694

Title
Immunoelectron Microscopic Identification of Lysozyme-Expressing Cells in Human Labial Salivary Glands

Author
Toshihiro MIYAZAKI, Toru FUJIK12, Yasuhisa INOUE and Kunio TAKANO

Address
Department of Oral Histology* and First Department of Oral and Maxillofacial Surgery', Nagasaki University School of Dentistry, Nagasaki, Japan

Summary
Although human labial gland secretions contain serous components such as the bactericidal enzyme, lysozyme, the presence of serous cells in this gland has yet to be clearly visualized under the electron microscope. The present study identifies lysozyme-expressing cells of the labial glands using microwave-fixed, Epon-Araldite-embedded specimens, which showed excellent preservation of both ultrastructural detail and antigenicity for post-embedding immunogold labeling of lysozyme. Ultrastructurally, all of the secretory cells of the glands appeared to be a mucous-type and have a serial maturation relationship, consistent with a previous report by TANDLER et al. (1969a) : their secretory granules were electron-lucent and exhibited reactivity for mucus staining by the periodic acid-thiocarbo-hydrazide-silver proteinate (PA-TCH-SP) method. We classified them into two immature types (1, II) and two mature types (1, II). Their distinctive features were the following : 1) relatively small (0.5-1 micrometer) secretory granules and well-developed basal rough endoplasmic reticulum for the immature types; 2) larger (1-2pm) secretory granules and well-developed Golgi apparatus, which showed intense PA-TCH-SP reactivity in the 2-3 trans-cisterns, for the mature types; 3) few secretory granules in the immature type I; and 4) the darkest appearance for the mature type II. Immunogold labeling with anti-lysozyme showed specific labeling of the two immature-type cells, in which gold particles were found mainly over the secretory granules and Golgi apparatus, and moderately over the rough endoplasmic reticulum. In the secretory granules, the labeling was distributed throughout the contents and was present even if they showed strong PA-TCH-SP reactivity; in the Golgi area, it was seen over the stacked cisternae, trans-Golgi networks, and condensing vacuoles. No specific labeling was seen in
the mature-type cells or in the duct cells. These immature- and mature-type cells were almost equivalent to the "serous demilune or acinus" and "mucous tubule" cells, respectively, at the light-microscopic level. These results indicate that the traditional "immature mucoustype cells" of the human labial glands produce lysozyme and should be classified as seromucous cells.

Contact
Dr. Toshihiro MIYAZAKI
Department of Oral Histology
Nagasaki University School of Dentistry
1-7-1, Sakamoto, Nagasaki
852-8588 Japan
Tel: +81 95 849 7631
Fax: +81 95 849 7633
E-mail: miyazaki@net.nagasaki-u.ac.jp

Title
Identification of Arterial and Venous Segments of Blood Vessels Using Alkaline Phosphatase Staining of Ink/Gelatin Injected Tissues

Author
Tatsuo USHIKI and Kazuhiro ABE2

Address
Department of Anatomy, Niigata University School of Medicine, Niigata; and Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan

Summary
The present study describes a method for discriminating between the arterial and venous segments of blood vessels in mouse tissues and organs using alkaline phosphatase (ALPase) staining of ink/gelatin injected tissues. Anesthetized mice were injected through the left ventricle with blue ink/gelatin, and various organs and tissues were removed from the body and fixed by immersion in 10% formalin. Sections 50-100 micrometer thick were incubated for ALPase in a medium containing naphthol
AS-BI phosphate and fast red TR by the azocoupling method. In such specimens as the brain and skeletal muscles, ALPase activity was found in arterioles and capillaries on the arterial side, whereas it was absent in capillaries on the venous side and in venules. In the liver, only branches of the hepatic artery were positive. ALPase activity was absent in the vessels of the lung except for a positive reaction in branches of the bronchial arteries. These findings indicate that the ALPase activity is confined to the arterioles and arterial segments of the capillaries in the systemic circuit. Thus, ALPase staining of ink/gelatin injected specimens is a useful method for differentiating the arterial and venous segments of the micro-vascular bed in various organs and tissues in mice.

Contact
Prof. Tatsuo USHIKI
Department of Anatomy
Niigata University School of Medicine
Asahimachi-dori, Niigata
951-8510 Japan
Tel.: +81 25 227 2058
Fax: +81 25 224 1767

Title
Localization of Cytoplasmic Free Calcium Ions in PC12 Cells with Varicose Fibers

Author
Setsuko NAKANISHI, Jun ICHIKAWA' and Yasuhisa ENDO'

Address
Pharmaceutical Frontier Research Laboratories, Japan Tobacco Inc. , Yokohama; and Department of Applied Biology', Kyoto Institute of Technology, Kyoto, Japan

Summary
Differentiated PC12 cells with varicose fibers were used as a model of sympathetic neurons to demonstrate the intracellular localization of cytoplasmic free calcium ions. Changes in the concentration of cytoplasmic free calcium ions were analyzed at individual varicosities upon stimulation with acetylcholine by laser scanning confocal microscopy. Transient increases in cytoplasmic free calcium ion concentration were localized in the varicosities and recognized in both the absence and presence of extracellular Ca2+. Immunocytochemical analysis of intracellular calcium channels, l, 4, 5-trisphosphate receptors and ryanodine receptors, by electron microscopy demonstrated that immunoreactive sites were mainly localized in large dense core vesicles in the varicosities and neurites. These results suggest that the exocytosis of large dense core vesicles is regulated by an increase in cytoplasmic free calcium ion concentration from an intracelluler Ca2+ store.

Contact
Dr. Setsuko NAKANISHI
Pharmaceutical Frontier Research Laboratories
Japan Tobacco Inc.
1-13-2, Fukuura, Kanazawa-ku, Yokohama
236-0004 Japan
Tel: +81 45 786 7694
Fax: +81 45 786 7692

Title
MAP2 and GAP-43 Expression in Normal and Weaver Mouse Cerebellum : Correlative Immunohistochemical and in Situ Hybridization Studies

Author
Lazaros C. TRIARHOUl, Carme SOLA2, Jos6 M. PALACIOS3,4 and Guadalupe MENGOD3

Address
Department of Pathology and Laboratory Medicine and Program in Medical Neurobiologyl, Indiana University School of Medicine, Indianapolis, U. S. A.; Department of Pharmacology and Toxicology', Conseio Superior de Investigaciones Cientificas, Barcelona; Department of Neurochemistry', Instituto de Investigaciones Biom6dicas de Barcelona, Consejo Superior de Investigaciones Cientificas, Barcelona; and Research Centre+, Almirall Prodesfarma, S.A., Barcelona, Spain



Summary
MAP2 is a major microtubule-associated brain protein, selectively localized in dendrites; growth-associated phosphoprotein GAP-43 is a neuronspecific protein associated with axonal outgrowth. In adult cerebellum, both of these proteins and their corresponding RNA transcripts are most strongly expressed by granule cells. Using immunocytochemistry with antibodies and in situ hybridization histochemistry with [3'P] labeled oligonucleotide probes, we examined the cellular localization of MAP2, GAP-43 and their mRNAs in the cerebellum of control and weaver (wv/ wv) mutant mice, which exhibit massive granule cell death. In wild-type (+/+) mice, MAP2 immunoreac-tivity was seen in neuronal somata and dendrites of the granule cell layer; GAP-43 immunoreactivity was present in molecular layer, corresponding to the distribution of parallel fibres. Transcripts encoding MAP2 and GAP-43 were localized in the layer of the granule cell somata. In heterozygous weaver mice (wv/+), which feature an intermediate degree of granule cell loss, MAP2 immunoreactivity was localized in the granular layer, and the pattern of GAP-43 immunostaining was also similar to +/+ , the only difference being a thinner molecular layer. Heterozygotes had an anatomical pattern of MAP2 and GAP-43 mRNA hybridization qualitatively similar to that of the wild-type with some deviations in signal intensity. In homozygous weaver mutants (wv/wv), MAP2 immunoreactivity was extremely weak in the area beneath Purkinje cells and a certain GAP-43 immunoreactivity was seen in the upper part of cerebellar cortex. Hybridization signals for MAP2 and GAP-43 mRNAs were minimal. The reported alterations in regional pattern of MAP2 and GAP-43 expression in mutant mice offer a molecular correlate of dendritic and axonal protein gene transcription pertinent to the neuropathological manifestations of certain forms of heredodegenerative ataxia.

Contact
Lazaros C. TRIARHOUl, M.D., Ph. D.
Department of Pathology
and Laboratory Medicine
Indiana University School of Medicine
635 Barnhill Drive, Mailstop A-128
Indianapolis, IN 46202-5120, U.S.A.
Tel: +1 317 274-7254
Fax: +1 317 278-2018

Title
Light and Electron Microscopic Detection of Anionic Sites in the Rat Choroid Plexus

Author
Takehito TAGUCHI. Aiji OHTSUKA and Takuro MURAKAMI

Address
Department of Anatomy, Okayama University School of Medicine, Okayama, Japan

Summary
Electron microscopy of ultrathin sections stained with cationic iron colloid revealed that, in the choroid plexus of the rat brain ventricles, the luminal surface and fenestral diaphragm of the capillary endothelium as well as the basement membranes of the endothelium and epithelium are strongly anionic or intensely negatively charged. The iron colloid reaction to these anionic sites was erased by treatment with hyaluronidase or digestions with chondroitinase ABC/ heparitinase/keratanase. These results indicate that sulfated proteoglycans provide such anionic sites of the choroidal capillaries. Discussion suggested that the negative charge on the luminal surface of the capillary endothelium prevents the adhesion of blood cells to capillary walls and also prevents endothelial adhesion by their repelling each other. It was further discussed that the negatively charged endothelial fenestrae and basement membranes may act as a charge barrier to inhibit the passage of anionic molecules.

Contact
Dr. Takehito TAGUCHI
Human Morphology Section
Department of Anatomy
Okayama University School of Medicine
2-5-1 Shikata-cho, Okayama
700-8558 Japan

Title
Electron Microscopic Characterization of Filiform Papillae in the Normal Human Tongue

Author
Masahiko TOYODA, Shigeaki SAKITA, Masayori KAGOURA and Masaaki MOROHASHI

Address
Department of Dermatology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan

Summary
The fine structure of filiform papillae on the normal human tongue was examined level by level, from the basal layer to the surface, in specimens taken from the dorsal side of the lingual body. Human lingual epithelia showed three distinct regions: epithelia on the anterior and on the posterior sides of filiform papillae and an interpapillary epithelium. While the basal and the squamous cell layers were similar throughout these three regions, differences were noted in the granular and the horny layers. The interpapillary epithelium actually lacked both the granular and the horny layers. The epithelium on the anterior side of filiform papillae was characterized by alternating layers of granular cells and of cornified cells. Granular cells possessed three types of keratohyaline-like granules within their cytoplasm: uniformly electron dense, relatively less electron dense, and a heterogeneous type. While the number of the keratohyaline-like granules was remarkably diminished in the epithelium on the posterior side of filiform papillae, a considerable amount of tonofibrils was present in the cytoplasm. In the uppermost portion of the anterior side of filiform papillae, coherence between adjacent epithelial cells depended mainly on the interlocking of cytoplasmic villi and poorly developed desmosomes on villi. On the other hand, epithelial cells on the posterior side of filiform papillae appeared to be more tightly adhesive compared with those on the anterior side. This was due to focal thickening of the plasma membrane and to desmosomes at the interface between the granular and cornified cells, and to the formation of a marginal band and increased intercellular cement presumably derived from lamellar bodies in the horny layer. These findings demonstrate distinct differences between the anterior and the posterior sides of filiform papillae in the human tongue with respect to keratinization patterns, structures associated with cell-to-cell adhesion and the strength of cellular cohesion in the uppermost portion, and the turnover of corni-fied cells. These differences may contribute to the formation of the unique external configurations of filiform papillae.

Contact
Dr. Masahiko TOYODA
Department of Dermatology
Faculty of Medicine
Toyama Medical and Pharmaceutical University
2630 Sugitani, Toyama
930-0194 Japan
Tel: +81 764 34 2281
Fax: +81 764 34 5028

Title
Acellular Calcified Columns in the Normal Growth Plate of Mouse Vertebrae

Author
Kanoh YOKOZEKI, Kazuhiro ABE, Satoshi WATANABE, Kohta SUDA and Kiyoshi KANEDA

Address
Department of Orthopedic Surgery, Hokkaido University School of Medicine; and Department of Anatomy', Hokkaido University Graduate School of Medicine, Sapporo, Japan

Summary
The present study aims to demonstrate the calcified columnar structures of the growth plate of mouse vertebrae and to show their age related changes. For light microscopy, paraffin sections of decalcified lumbar spines were stained with hematoxylin-eosin or toluidine blue; methacrylate sections of undecalcified specimens were stained to detect calcium precipitate. For scanning electron microscopy, Iumbar spines treated with 5% NaCIO solution were dehydrated by acetone and metal-coated. Light microscopy of hematoxylin-eosin stained sections revealed that the acellular columnar structures appeared between the chondrocyte stacks in the growth plate. These structures were stained more densely by toluidine blue. The methacrylate-embedded sections for calcium staining showed calcium deposition in the columns. Scanning electron microscopy of NaCIO-treated specimens enabled the direct observation of the columns on both the epiphyseal and diaphyseal bone surfaces
facing the cartilaginous growth plate. Numerous projections on each surface were distributed in mirror images ; the corresponding projections were similar in size and shape, indicating that the projections bonded with each other and formed calcified columns in the cartilaginous growth plate. Longitudinal sections of the spine confirmed these findings. The calcified columns first appeared about 2 or 3 weeks of age and increased in number with time. While increasing in number, they also grew in size fusing with the neighboring ones. The proportional area of the columns occupying the surface facing the growth plate also increased with age. These findings indicate that the calcified column ultimately concerns the cessation of the bone growth.

Contact
Dr. Kanoh YOKOZEKI
Department of Orthopedic Surgery
Hokkaido University School of Medicine
Kita-15 Nishi-7, Sapporo
060-8638 Japan
Tel: 81 11 716 1161(5935)
Fax: 81 11 706 6054
E-mail: kanoh@med.hokudai.ac.jp

Title
Lymphatic Drainage of the Cerebrospinal Fluid from Monkey Spinal Meninges with Special Reference to the Distribution of the Epidural Lymphatics

Author
Masahiro MIURA, Seiji KATO and Michael von LUDlNGHAUSEN

Address
Departments of Anatomy and Human Biology, Oita Medical University, Oita, Japan; and Institute of Anatomy',  University of Wurzburg, Wurzburg, Germany

Summary
The structural organization of the epidural lymphatics and lymphatic drainage of the cerebrospinal fluid from spinal meninges was studied in Japanese monkeys (Macaca fuscata) by an enzyme-histochemical method. The spinal meninges were examined at various intervals from I to 48 h, as well as at 30 days, following an injection of ultrafine carbon particles into the subarachnoidal space (cisterna magna). Lymphatics were differentiated from blood capillaries by the 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining method (KATO et al. 1991, 1993) both in the whole-mount preparations and tissue sections. Carbon-filled collecting lymphatics and lymph nodes constantly appeared in the cervical and thoracic regions but only rarely in the lumbo-sacral region after carbon injection. Networks of 5'-Nase-positive lymphatics in the epidural connective tissues were seen in a large area on the dorsal surface around each spinal nerve root in the cervical and upper thoracic regions, especially at a level corresponding to the brachial plexus (C5-Thl). Carbon particles were often found within the 5'-Nase-positive lymphatics. In the lower thoracic and lumbo-sacral regions, on the other hand, the epidural lymphatic network covered only a small area around each spinal nerve root. These findings suggest that the epidural lymphatics are well developed on the dorsal side of the lower cervical spinal dura mater and may function as an absorptive pathway for the cerebrospinal fluid from the subarachnoidal space.

Contact
Dr. Masahiro MIURA
Departments of Anatomy
Oita Medical University
1-1 Idaigaoka, Hasama-machi, Oita
879-5593 Japan
Tel: +81 97 586 5623
Fax: +81 97 586 5623
E-mail: miura@oita-med.ac.jp