Contents

Review article:

1. The olfactory origin of luteinizing hormone-releasing hormone (LHRH) neurons. A new era in reproduction physiology(DAIKOKU, S)

Original articles:

1. Multi-label image analysis of secretory cell juxtaposition in the sheep pituitary gland(ISHII, T.)
   
2. Effects of ATP and its analogues on [Ca2+]i dynamics in the rabbit corneal epithelium(KIMURA, K.)
   
3. Alteration in the expression level of calbindin D28k in the periodontal ligament of the rat molar during experimental tooth movement(YOUN, S.)
   
4. Composition of the extracellular matrix in human cricoarytenoid joint articular cartilage(PAULSEN, F)
   
5. Changes in c-Fos expression induced by noxious stimulation in the trigeminal spinal nucleus caudalis and Cl spinal neurons of rats after hyperbaric exposure.(TAKEDA, M.)
   
6. Intermittent inhibition of dentin mineralization of rat incisors under continual infusion of l-hydroxyethylidene-l, l-bisphosphonate (HEBP) using a subcutaneous mini osmotic pump(SAKAI, H.)
   
7. Scanning electron microscopic observation of apical sites of open-type paraneurons in the stomach, intestine and urethra(HASHIMOTO, Y.)
   
8. An immunocytochemical study of amelogenin proteins in the developing tooth enamel of the gar-pike, Lepisosteus oculatus (Holostei, Actinopterygii)(ISHIYAMA, M.)
   
9. Perineuronal nets of proteoglycans in the adult mouse brain are digested by conagenase(MURAKAMI, T.)
   

Summary

Title
The Olfactory Origin of Luteinizing Hormone-Releasing Hormone (LHRH) Neurons. A New Era in Reproduction Physiology

Author
Shigeo DAIKOKU Prof. Emeritus, The University of Tokushima, Tokushima, Japan

Address
Prof. Emeritus, The University of Tokushima, Tokushima, Japan

Summary
This paper reviews those studies which conceived the concept that the brain LHRH-synthesizing neurons originate in the nasal placode. LHRH isolated from mammalian hypothalamus in 1971 was first shown immunohistochemically two years later in the hypothalamic neurons which project processes to the median eminence, to release it into the portal capillaries in the guinea pig. At an early stage of development, the LHRH cells were found in the nasal placode but not in the hypothalamus as shown in in vivo and in vitro developmental studies. The cells arising in the brain were delayed. This discrepancy was solved in 1989-1990 by findings that the cells derived in the placode at an early stage left the site and migrated to the forebrain vesicles along the placode-derived terminal and vomeronasal nerve fibers, both of which were found to express immunoreactive cell adhesion molecules. The neurons, after reaching the surface of the forebrain vesicles, entered into the brain by the guidance of the cell adhesion molecule-positive fibers, and came to be distributed not only in the hypothalamus but also in the telencephalon cortex, midbrain, limbic brain, and main and accessory olfactory bulbs. The attention to these heterogeneties led to discussion of the possible neurobiological significance of this peculiar peripheral neurogenesis from an evolutionary viewpoint.

Prof. Shigeo DAIKOKU
Nakamachi 3-4-4-801
Musashino, Tokyo
180-0006 Japan
Tel & Fax: +81-422-55-4830

Title
Multi-label Image Analysis of Secretory Cell Juxtaposition in the Sheep Pituitary Gland

Author
Takashi ISHII and Hajime MIYAMOTO

Address
Laboratory of Anatomy and Cell Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Summary
An image analysis system, assigned dilrerent pseudocolors to dilrerent types of immunolabeled cells, allowed us to make a montage from two images of the respective types of cells. This system was therefore used for simultaneous identification of two or more types of immunolabeled cells in the sheep anterior pituitary. Morphometry - including a neighboring proportion defined as the probability of a cell type adj oining other cell types-was performed. We also conducted a simulation of an artificial cell mass with an image analyzer to evaluate the elrects of cell populations on the neighboring proportion. Simulation analysis showed that the predominant cell type tended to have a higher neighboring proportion, while rarer cell types had lower proportions according to their small population density. In the sheep pituitary gland, the neighboring proportions against PRL-, GH-immunolabeled cells were high (about 65% and 55%, respectively), according to their large populations. The neighboring proportion of LHfiimmunolabeled cells to the same type of cells was lower (11%) than that against other types of cells. It was thus suggested that LH cells were scattered throughout the anterior lobe. The neighboring proportion of ACTHimmunolabeled cells to the same type of cells was somewhat higher, but that of ACTH cells to PRL cells was low (52%). Accordingly, this cell type was often distributed in clusters. These quantitative results confirmed the topographical characteristics of secretory cells deduced from visual observation. In addition, a low topographical alnity between PRL and ACTH cells was indicated.

Dr. Takashi ISHII
Laboratory of Anatomy and Cell Biology Graduate School of Agriculture
Kyoto University
Kyoto, 606-8502 Japan
Tel: +81-75-753-6334
Fax: +81-75-753-6345

Title
Effects of ATP and Its Analogues on [Ca²⁺]_i Dynamics in the Rabbit Corneal Epithelium

Author
Katsura KIMURA, Tomoko NISHIMURA and Yoh-ichi SATOH

Address
Departments of Histology and Ophthalmology, Iwate Medical University School of Medicine, Morioka, Japan

Summary
Adenosine-5'-triphosphate (ATP) plays a pivotal role in various tissues as an extracellular transmitter. ATP released from nerve endings and/or damaged cells may elicit reactions in adj acent cells. To identify such reactions, we investigated the dynamics of the intracellular calcium ion concentrations ([Ca²⁺]_i) in the rabbit corneal epithelium during ATP-stimulation. Intact epithelial sheets isolated from corneal tissue were loaded with Fura-2, and [Ca²⁺]_i dynamics in each cell layer were analyzed using a digital imaging system (Argus 50/CA). Normal architecture was preserved, suggesting that functional integrity remained intact. Perfusion with HEPES-bufrered Ringer's solution containing ATP (10yM) and uridine-5'-triphosphate (UTP ; 10 yM) caused a biphasic [Ca²⁺]_i increase in the superficial layer that manifested itself as a rapid initial spike followed by a long-lasting plateau phase. Adenosine-5'-diphate (10 yM) elevated the [Ca²⁺]_i level, but induced only the initial spike, which was smaller than those induced by ATP and UTP. Adenosine (10 yM) did not elicit any [Ca²⁺]_i changes in the epithelial cells. Suramin (10 pM ; a P2 receptor antagonist) blocked the ATP-induced [Ca²⁺]_i increase, whereas the P2X receptor agonists, a, li-methylene ATP (10 yM), 2-methylthio ATP (10 yM) and Benzoylbenzoyl ATP (10 yM), did not elicit any increases in [Ca²⁺]_i. In the basal cell layer, ATP-induced [Ca²⁺]_i dynamics were biphasic, while oscillatory fluctuations of [Ca²⁺]_i were induced in the wing cells of the mid layer of the corneal epithelium by ATP stimulation. Ca²⁺ oscillations were sometimes synchronized among adj acent wing cells, but these waves did not propagate to other cell layers. These results suggest that extracellular ATP elicits a [Ca²⁺]_i increase mainly via P2Y receptors. In addition, synchronized Ca²⁺ oscillation in the wing cell layer indicates that intracellular events may spread to neighboring cells within the layer.

Prof. Yoh-ichi SATOH
Department of Anatomy 2 (Histology)
Iwate Medical University School of Medicine 19-1 Uchimaru, Morioka
020-8505 Japan
Tel: +81-19-651-5111
Fax: +81-19-651-5605
E-mail :yisatoh@iwate-med.ac.jp

Title
Alteration in the Expression Level of Calbindin D28k in the Periodontal Ligament of the Rat Molar during Experimental Tooth Movement

Author
Suk Hyun YOUN, Takeyasu MAED2, Kojiro KURISU and Satoshi WAKISAKA

Address
Department of Oral Anatomy and Developmental Biologyl, Osaka University Faculty of Dentistry, Osaka; and Department of Oral Amatomy 2, Niigata University School of Dentistry, Niigata, Japan

Summary
The present immunohistochemical study was designed to investigate changes in the distribution and expression level of calbindin D28k in the periodontal ligament during experimental tooth movement in the rat molar to clarify the physiological role of this protein in the ligament. In normal animals, calbindin D28k - like immun o rea ctivity appe a r ed spa r s e ly i n spindle-shaped cells in the alveolar half of the periodontal ligament. Electron microscopic observations showed that these immunoreactive cells were characterized by well-developed rough-surfaced endoplasmic reticulum and phagosomes-which often contained collagen fibers-suggesting that these cells could be categorized as periodontal fibroblasts. Twelve hours following the onset of the experimental tooth movement, cells positive for calbindin D28k increased in number in the periodontal ligament, especially in the alveolar half of the pressured side. Immunoelectron microscopy showed that the calbindin D28k-immunopositive cells had morphological features similar to those of fibroblasts in the normal ligament, and that these cells occasionally made contact with immunonegative macrophage-like cells. Immunopositive cells gradually decreased in number, and the distribution of the cells and intensity of the immunoreactivity returned to normal levels by 14 days following the induction of the experimental tooth movement. The present results suggest that calbindin D28k plays an important role in the homeostasis and cytoprotection of fibroblasts in the periodontal ligament at the initial phase of experimental tooth movement.

Dr. Satoshi WAKISAKA
Department of Oral Anatomy
and Developmental Biology
Osaka University Faculty of Dentistry l-8, Yamadaoka, Suita, Osaka
565-0871 Japan
Tel : +81-6-6879-2872
Fax: +81-6-6879-2875
E-mail: wakisaka@dent.osaka-u.ac.jp

Title
Composition of the Extracellular Matrix in Human Cricoarytenoid Joint Articular Cartilage

Author
Friedrich PAULSEN and Bernhard TILLMANN

Address
Department of Anatomy, Christian-Albrechts-University of Kiel, Kiel, Germany

Summary
The extracellular matrix of the human cricoarytenoid joint articular cartilage is involved in dilrerent pathological changes. Interestingly, in contrast to the limb joints, the extracellular matrix composition of the healthy cricoarytenoid joint articular cartilage has not yet been elucidated except by some light microscopical investigations. The present study investigates the extracellular matrix components of the cricoarytenoid joint articular cartilage by means of light microscopy, immunohistochemistry, transmission electron microscopy and scanning electron microscopy and compares them with the limb joints for a better understanding of their involvement in joint disease.
Chondrocytes near the joint surface of the cricoid and arytenoid cartilage dilrer from chondrocytes of deeper cartilage layers. The extracellular matrix of the articular cartilage contains chondroitin-4-sulfate, chondroitin-6-sulfate and keratansulfate as well as collagen types II, III, VI, IX and XI. Type-III-collagen shows a special distribution throughout the joint cartilage. In deeper cartilage layers, type-III-collagen occurs only pericellularly; in higher cartilage layers type-III-collagen is also located territorially and interterritorialy in small amounts. Scanning and transmission electron microscopy have revealed the articular surface of the cricoid and arytenoid cartilage to consist of a network of irregularly organized collagen fibrils, which are lined by a layer of electron dense material. The network coats subjacent collagen bundles which descend obliquely downward and intermingle at right angles in the middle part of the articular cartilage with collagen bundles of the deeper cartilage zones.
The articular cartilage surface shows structural characteristics which dilrer from the underlying cartilage. The superficial electron dense layer possibly plays a role in the lubrication of the articular cartilage surface. The alignment of the fibrillar structures in the articular cartilage of the cricoarytenoid joint varies from those
of the limb joints based on the diirerent strain occurring during arytenoid movement. Nevertheless, the human cricoarytenoid joint articular cartilage can be compared with the joints of the limbs despite its extracellular matrix composition and its involvement in joint pathology.
Evidence of type III collagen in the outermost layer of the articular cartilage of the cricoarytenoid joint presents a peculiarity, which has yet not be demonstrated in the articular cartilage of limb joints.

Dr. Friedrich PAULSEN
Department of Anatomy
Christian-Albrechts-University of Kiel Olshausenstraße 40
D-24098 Kiel
Germany
Tel : +49 431 880 2597
Fax: +49 431 880 1557
E-mail : fpaulsen@anat.uni-kiel.de

Title
Changes in c-Fos Expression Induced by Noxious Stimulation in the Trigeminal Spinal Nucleus Caudalis and C1 Spinal Neurons of Rats after Hyperbaric Exposure

Author
Mamoru TAKEDA, Takeshi TANIMOTO, Mizuho IKEDA, Toshimi NISHIKAWA, Naomi KAWANISHI, Motohiko MOHRI, Tsuyoshi SHIMIZU and Shigeji MATSUMOTO

Address
epartment of Physiology, School of Dentistry at Tokyo, Nippon Dental University, Tokyo; Japan Marine Science and Technology Center, Yokosuka; and Department of Physiology, Fukushima Medical College, Fukushima, Japan

Summary
The present study aims to test the hypothesis that hyperbaric exposure inhibits nociceptive processing in the trigeminal spinal nucleus caudalis and CT spinal neurons. We investigated the c-Fos-like immunoreactivity of the brainstem and upper cervical spinal cord (Cl region) following an injection of mustard oil (15 ill of 20%) into the nasal mucosa of pentobarbital anesthetized rats after exposure to hyperbaric (2atmospheres, 1 h) and normobaric pressures. After the hyperbaric exposure, the mean number of Fos-immunoreactive neurons in the ipsilateral laminae I-II and III-IV of the trigeminal spinal nucleus caudalis were significantly lower than those in the normobaric condition. Similarly, the mean number of c-Fos positive neurons in the superficial layer (I-II) of the ipsilateral CT segment were significantly reduced as compared with that in the normobaric condition. When treated with the vehicle alone, no significant dilrerence was detected in the numbers of c-Fos positive neurons in the trigeminal spinal nucleus caudalis and CT regions between hyperbaric and normobaric conditions. These results suggest that hyperbaric exposure may attenuate nociceptive signals from the area innervated by the trigeminal nerves at the level of both the trigeminal spinal nucleus caudalis and CT dorsal horn.

Dr. Mamoru TAKEDA
Department of Physiology
School of Dentistry at Tokyo
Nippon Dental University
1-9-20, Fujimi-cho, Chiyoda-ku Tokyo, 102-8159 Japan
Tel: +81-3-3261-8311
Fax: +81-3-3264-8399
E-mail: m-takeda@tokyo.ndu.ac.jp

Title
Intermittent Inhibition of Dentin Mineralization of Rat Incisors under Continual Infusion of 1-Hydroxyethylidene-1, 1-Bisphosphonate (HEBP) Using a Subcutaneous Mini Osmotic Pump

Author
Hideo SAKAI, Yoshiro TAKANO, Keiichi OHYA and Norimasa KUROSAKI

Address
Department of Oral Diagnosis and General Dentistry; Department of Oral Anatomy II; and Department of Oral Pharmacology, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan

Summary
The inhibitory efrect of the continual administration of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) (8 mgP/kg/day) through a mini osmotic pump on dentin mineralization was examined in relation to the diurnal rhythm of the rat and compared with that of daily injections of same amounts of HEBP known to inhibit dentin mineralization.
After daily injections of HEBP, a series of alternating rows of mineralized and non-mineralized dentin islands appeared in the newly formed portion of the crown-analogue of rat incisors. A similar phenomenon occurred under the continual administration of HEBP in rats raised either under regular environmental photofraction or constant lighting conditions. The average distance between the adjacent mineralized dentin islands was 521.0 +/- 51.3 Jim in the injected rats. After continual HEBP administration, this was 426.0 +/- 13.2 Jim and 416.5 +/- 19.4 Jim under ordinary photofraction and constant light, respectively. Although the pattern of individual mineralized dentin islands tended to become irregular in nocturnal rats, no statistical difrerence was noted between the two values. Rows of mineralized and non-mineralized dentin islands also appeared in the root analogue dentin. No sign of the intermittent inhibition of mineralization was recognized in mesodermal hard tissues other than dentin in the HEBP-afrected animals.
These data implicate the presence of intrinsic cycles in dentin mineralization at the growing end of rat incisors independent of environmental photofraction as well as the ameloblast function.

Prof. Yoshiro TAKANO
Department of Oral Anatomy II
Faculty of Dentistry
Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 Japan
E-mail: takanoy.oan2@dent.tmd.ac.jp

Title
Scanning Electron Microscopic Observation of Apical Sites of Open-Type Paraneuons in the Stomach, Intestine and Urethra

Author
Yoshiharu HASHIMOTO, Tatsuo USHIKI, Takashi UCHIDA, Junzo YAMADA and Toshihiko IWANAGA

Address
Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo; Department of Anatomy, Niigata University School of Medicine, Niigata; Department of Oral Anatomy Hiroshima University School of Dentistry, Hiroshima; and Department of Veterinary Anatomy, Obihiro University of Agricuiture and Veterinary Medicine, Obihiro, Japan

Summary
The apical region of open-type paraneurons in tubular organs functions as a receptor site for chemical information in the lumen. Electron microscopic studies have demonstrated a tuft of microvilli on the lumina! surface of cells, but failed to visualize it threedimensionally. The present scanning electron microscope (SEM) observation succeeded in viewing, from the lumina! side, open-type paraneurons distributed in epithelia of the stomach, intestine, and urethra. The pyloric antrum of avian species and the duodenum of human fetuses, the latter forming an endocrine cell colony at every villus tip, were chosen for SEM observation in order to eliminate visual obstruction by adjacent epithelial cells with developed microvilli. The lumina! surface of gut endocrine cells was consistently covered with a tuft of 80-200microvilli. Pyloric paraneurons possessed thick and stifr microvilli as compared with those of exocrine cells. The microvilli on intestinal paraneurons were more irregular in length and more loosely grouped than those composing the striated border of enterocytes. Urethral paraneurons containing serotonin were surrounded by three or four polygonal epithelial cells. Their narrow apical surface was provided with 30-100 microvilli which varied in length from cell to cell, and which were conspicuously projected above the lumina! surface of the urethra. The microvillous crown of the gut and urethral paraneurons was so prominent and constant a structure on the apical surface as to allow easy identification of open-type paraneurons under the SEM.

Prof. Toshihiko IWANAGA
Laboratory of Anatomy
Graduate School of Veterinary Medicine Hokkaido University
Kita 18 Nishi 9, Kita-ku
Sapporo, 060-0818 Japan
Tel: +81-11-706-5187
Fax: +81-11-717-7569
E-mail: tiwanaga@vetmed.hokudai.ac.jp

Title
An Immunocytochemical Study of Amelogenin Proteins in the Developing Tooth Enamel of the Gar-Pike, Lepisosteus oculatus (Holostei, Actinopterygii)

Author
Mikio ISHIYAMA, Toshihiko INAGE and Hitoyata SHIMOKAWA

Address
Department of Histology, The Nippon Dental University School of Dentistry at Niigata, Niigata; Department of Anatomy Nihon University School of Dentistry, Tokyo; Department of Oral Biochemistry, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan

Summary
Previous studies have demonstrated the morphological similarity of the enamel-like layer found in the teeth of the coelacanth, lungfish and gar-pike to the enamel of tetrapods. In order to clarify the phylogenetic continuity between both structures, tooth germs of the gar-pike were immunocytochemically studied using an anti-bovine amelogenin polyclonal antibody. Intense immunoreaction was shown over the enamel-like matrix layer. Certain cell organelles associated with the secretory pathway of the ameloblasts were recognized as immunoreactive. These results indicate that the enamel-like layer of the gar-pike is a tissue homologous with the mammalian enamel because both possess a common, amelogenin-like substance.

Prof. Takuro MURAKAMI
Department of Anatomy
Faculty of Medicine
Okayama University Medical School 2-5-1 Shikata-cho, Okayama
700-8558 Japan
Tel: +81-86-235-7088
Fax: +81-86-235-7095
E-mail : em2kai@med.okayama-u.ac.jp

Title
Perineuronal Nets of Proteoglycans in the Adult Mouse Brain are Digested by Collagenase

Author
Takuro MURAKAMI, Tetsuro MURAKAMI, W. D. SU, Aiji 0HTSUKA, Koji ABE and Yoshifumi NINOMIYA

Address
Departments of Anatomy, Neurology and Molecular Biology and Biochemistry, Faculty of Medicine, Okayama University Medical School, Okayama, Japan

Summary
As our previous study has indicated, perineuronal proteoglycans in the adult mouse brain are associatedwith some collagenousmolecules which can be stained with Gömöri's ammoniacal silver and are resistant to hyaluronidase digestion. The present study demonstrated that these molecules are thoroughly digested with collagenase,and suggests that they represent a hyaluronic acid-binding domain of the ligand proteoglycans connecting the perineuronal proteoglycans and nerve cell surface glycoproteins.

Prof. Takuro MURAKAMI
Department of Anatomy
Faculty of Medicine
Okayama University Medical School
2-5-1 Shikata-cho, Okayama
700-8558 Japan

Tel: +81-86-235-7088
Fax: +81-86-235-7095
E-mail: em2kai@med.okayama-u.ac.jp