Scanning electron microscopy of rat maturation ameloblasts

Summary Post-secretory, maturation-phase ameloblasts were studied by scanning electron microscopy of freeze-fractured or dry-dissected rat incisors. These cells are in contact with the enamel which they secreted at an earlier time and which undergoes a process of continuing mineralization. The lateral intercellular compartment between maturation ameloblasts is sometimes continuous with the intercellular space of the papillary layer of the enamel organ, but often closed by basal ring contacts which correspond to terminal bars seen in transmission electron microscopy. The distal poles of the cells sometimes possess striated borders. Lateral cell surfaces may show longitudinal gutter-like depressions between ridges from which numerous intercellular connections arise; or a maze of lateral folds and ridges; or they may have mostly microvillous surface projections bordering a minimal intercellular space compartment. Preliminary correlations of groupings of basal, lateral and distal cell features indicate that basal-closed plus distal striated border cells may show every type of lateral surface. Cells without a striated border, whether open or closed basally, have ridge or maze lateral surfaces bordering a wide intercellular compartment. Basal-open plus striated border cells have microvillous or maze-like surfaces. These combinations of features are encountered a few times along the length of the maturation zone of individual incisors and suggest the existence of cyclical changes in the type of activity of maturation ameloblasts.     

Planar cell polarity protein localization in the secretory ameloblasts of rat incisors

The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis.     

Endocytotic pathways at the ruffled borders of rat maturation ameloblasts

Using horseradish peroxidase (HRP) as a soluble protein tracer, electron microscopic studies were carried out in order to analyze endocytosis in the ruffle-ended ameloblasts of rat incisors. Accumulated HRP was initially incorporated from the ruffled border into the cytoplasm by means of pinocytic vacuoles ( pinosomes ) and pinocytotic coated vesicles. The majority of the HRP was taken up by the large number of pinosomes , which then formed large endocytotic vacuoles by fusing either with each other or with preexisting endocytotic vacuoles. As time passed HRP accumulated, not in the pinosomes and ruffled border but in the endocytotic vacuoles and multivesicular bodies. Frequent connections between HRP-labeled coated vesicles and these cytoplasmic bodies indicate that these vesicles serve as an HRP carrier. These findings strongly suggest that ruffle-ended ameloblasts actively absorb soluble proteins from the enamel matrix during enamel maturation.     

Endocytotic pathways at the ruffled borders of rat maturation ameloblasts

Summary Using horseradish peroxidase (HRP) as a soluble protein tracer, electron microscopic studies were carried out in order to analyze endocytosis in the ruffle-ended ameloblasts of rat incisors. Accumulated HRP was initially incorporated from the ruffled border into the cytoplasm by means of pinocytotic vacuoles (pinosomes) and pinocytotic coated vesicles. The majority of the HRP was taken up by the large number of pinosomes, which then formed large endocytotic vacuoles by fusing either with each other or with preexisting endocytotic vacuoles. As time passed HRP accumulated, not in the pinosomes and ruffled border but in the endocytotic vacuoles and multivesicular bodies. Frequent connections between HRP-labeled coated vesicles and these cytoplasmic bodies indicate that these vesicles serve as an HRP carrier. These findings strongly suggest that ruffle-ended ameloblasts actively absorb soluble proteins from the enamel matrix during enamel maturation.     

Electron microscopic radioautographic study on the incorporation of 3H-proline by mouse decidual cells

A qualitative radioautographic analysis showed that mature decidual cells of the mouse are able to incorporate 3H-proline. After 1 hour silver grains are concentrated mainly over these cells although some of them can be found over collagen fibrils. After 2,6 and 24 hours there is a progressive increase of silver grains on the extracellular space most of them concentrated over thick collagen fibrils. These results strongly indicate that mature decidual cells of the mouse produce collagen and that they conserve a behavior of the fibroblast from which they originated.     

Tubular lysosomes in ruffle-ended ameloblasts associated with enamel maturation in rat incisor

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope. Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced Kalpha1,2 x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens. The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Summary Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope.Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced K _{1, 2}, x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens.The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

A radioautographic study of the transport of 125I-labeled serum lipoproteins in rat aorta

Summary The transport of ¹²⁵I-labeled serum lipoproteins through the aortic endothelium was studied by radioautography. Rat aorta and heart was perfused in vitro with a medium containing human very low density (VLDL), low density (LDL), high density lipoprotein (HDL), delipidated HDL apolipoprotein or rat HDL. In all lipoproteins more than 95% of the radioactivity was TCA precipitable and lipid radioactivity was from 2–4% in HDL, 4–6% in LDL, 7–15% in VLDL. After 18–60 min of perfusion and wash with unlabeled medium, most of the aortic radioactivity was TCA precipitable and the percent of lipid counts was similar to that in the original lipoprotein. Following perfusion with VLDL, LDL, or HDL the radioautographic reaction was seen over the endothelium, the subendothelial space and the inner media, and was separated by an unlabeled zone from the reaction present over the adventitia. Uniform labeling of the entire wall was found after perfusion with HDL apolipoprotein. The presence of silver grains over endothelial cells in regions rich in plasmalemmal vesicles suggested that these organelles participate in the transport of the labeled lipoprotein, as was shown for lactoperoxidase (Stein and Stein, 1972). The present data indicate that cholesterol may enter the aortic wall as a constituent of lipoprotein particles. Since an HDL particle carries less than 1/20 of the cholesterol present in a LDL particle, it seems that the lower susceptibility of the female to atheromatosis might be related to the higher ratio of HDL to LDL particles in the female serum.The excellent technical help of Miss R. Ben-Moshe, Mrs. A. Mandeles, Mr. G. Hollander and Mrs. Y. Dabach is gratefully acknowledged. This study was supported in part by grants from National Institute of Health (No. 06-101-1), United States Public Health Service; Delegation Generale a la Recherche Scientifique et Technique of the French Government and from the Ministry of Health, the Government of Israel.     

Fine structure of rat incisor ameloblasts in transition between enamel secretion and maturation stages

The transitional region (including the end of the secretion zone, the beginning of the maturation zone, and the transition zone between them) was studied in the lower incisor enamel organ of adult rats with the electron microscope. Towards the end of the secretion zone, the surface invaginations of the ameloblasts diminish, the enamel surface becomes smooth and a dense, granulated material appears in the extracellular spaces between ameloblasts. The presence of a 60 Å wide gap between enamel and ameloblasts, and a 100 Å thick enamel border are thought to indicate the end point of enamel secretion and the beginning of the transition zone. Ameloblasts begin to shorten very close to this point. Subsequently, the following events occur: (1) the ameloblast cell membrane lifts away from the enamel to form a 450 Å wide gap; (2) before completion of this gap, a granular material appears in vesicles within the ameloblast apex, possibly to be secreted into the gap; (3) half-desmosomes are formed at the apical cell membrane; (4) the extracellular dense material passes into the spaces between the papillary cells; finally, (5) a coarse-textured material, thought to be enamel constituents being resorbed, appears in the gap, indicating the end of the transition zone. Only following this is the 450 Å gap completed. The attachment sites of both apical and basal terminal bars remain intact throughout the transition zone. The length of the transition zone is about 170 μ. The morphologic features of the transition zone overlap each other and persist for various distances into the maturation zone.     

A correlated scanning and transmission electron microscopic study of maturation ameloblasts in developing molar teeth of rats

Summary Maturation ameloblasts of developing molar teeth of the rat were studied by both scanning and transmission electron microscopy. After fixation, teeth were frozen and split. One face of the fractured tooth was used for SEM, the other for TEM.It was found that in some regions proximal junctional complexes separate the interameloblast space from the intercellular space of the papillary layer. Thereby an intercellular ameloblastic compartment is delineated which in some specimens contains a substance interpreted to be colloidal. Elsewhere the proximal junctions of ameloblasts are not present and free communication between the extracellular spaces is evident. The apical pole of ameloblasts varies in structure. Over some areas there is a distinct distal border zone with membranous infoldings which in some regions resembles a striated or ruffled border, but in other regions the membranes show whorl configurations. The distal border zone also contains granules with flocculent material. Elsewhere the ameloblasts display no distal border zone and the cells show a smooth membrane (except for pinocytotic vesicles and hemidesmosomes) facing the enamel surface. The lateral surface of ameloblasts exhibits a variety of surface configurations similar to but not as pronounced as those reported previously in rat incisor maturation ameloblasts.The authors wish to thank Pauletta Sanders and Helen Ruane for technical assistance. This project was supported in part by USPHS NIH Grant DE04059-03 and by the Medical Research Council of Great Britain     

A radioautographic and immunocytochemical study of the GABA systems of the habenula complex in the rat

Radioautography of [³H]GABA accumulation and immunocytochemistry of glutamate decarboxylase have been used to study anatomically and morphologically the GABA system of the rat habenular (Hb) complex. Radioautographic visualisation of GABA specific neurons show a very high innervation of the complex including both stria medullaris (SM), the habenular commissure and the periventricular thalamic fibers (FPVT). A massive labeled fiber system in the SM appears to divide into two branches when it reaches the Hb nuclei: a part of fibers continue their course dorsally to the nuclei up to the habenular commissure; other fibers enter the Hb lateralis or run along the ventral Hb medialis at the level of FPVT. The staining is markedly diminished in the entire complex in response to SM lesions. In the Hb lateralis, the radioautographic-positive reaction is mainly bound to labeled fibers or axonal varicosities. However GAD immunocytochemistry reveals some GAD-positive cell bodies in the ventro-median portion of the nucleus. In the Hb medialis the radioautographic and immunocytochemical staining is observed in the neuropile between the unlabeled large cell bodies. In the subependymal layer bundles of processes are strongly labeled and form a continual strain behind the unlabeled ependymocytes. Three types of reactive terminals have been differentiated based on size and shape of vesicles. Some of them are exclusively characterized by clear round vesicles and probably have their origin in the septum. Others contain clear vesicles and some large dense-cored vesicles and disappear after mesencephalic Raphe lesions or 5,7-dihydroxytryptamine treatment. They could correspond to terminals of raphe neurons with a double potentiality GABA and 5HT. The last exhibit mainly a dense population of large dark-cored granules similar to the ones found in neurosecretory nerve endings. However numerous fibers morphologically similar to the reactive fibers are unlabeled.     

A radioautographic study of collagen synthesis by earthworm epidermis

Secreation by the epidermis of two oligochaetes (Eisenia and Enchytraeus) was investigated radioautographically following administration of 3H-proline, 3H-tryptophan or Na2(35)SO4. Regionally epidermal columnar cells of Enchytraeus synthesize the overlying, probably collagenous, cuticle. Eisenia epidermis does not recordably synthesize the cuticle until after wounding (first eight segments removed). By two days postoperative the epidermal columnar cells of Eisenia synthesize the collagenous cuticle and, later in regeneration, the epidermis may simultaneously synthesize the different collagen of the underlying basement lamella. The epidermis of Enchytraeus, but not of Eisenia, synthesizes some sulfated material associated with the cuticle surface.     

Effects of acute doses of sodium fluoride on the morphology and the detectable calcium associated with secretory ameloblasts in rat incisors

Fluoride in high concentrations is known to have an adverse effect on the formation of enamel. The effect of a single injection of two concentrations of sodium fluoride on inner enamel secretory ameloblasts was investigated morphologically by electron microscopy and functionally by assessing the location and relative amount of available calcium, using the potassium pyroantimonate method. The results showed that acute doses of fluoride interfere with the normal function of secretory ameloblasts. The increase in the population of lysosome-like structures observed after fluoride administration is suggestive of defects in the synthetic pathway. Concomitant with the effect of fluoride on secretory ameloblasts is an inhibition of enamel formation, resulting in incomplete enamel rods and leaving large remnants of Tomes' processes buried in the enamel. The distribution of the calcium pyroantimonate deposits found tends to support the concept of calcium traveling between the cells to the enamel. Acute doses of fluoride also reduce the amount of calcium available for complexing with pyroantimonate in the intercellular region.     

Localized infusion of tunicamycin in rat hemimandibles: alteration of the basal lamina associated with maturation stage ameloblasts.

At the beginning of the maturation stage of amelogenesis, ameloblasts deposit a basal lamina (BL) at the interface between their apical surface and maturing enamel. This structure is rich in glycoconjugates and is proposed to exhibit adhesive and/or filtering functions. To clarify its role, we have applied a recently developed surgical window model to locally administer tunicamycin (TM), an antibiotic that interferes with N-glycosylation, in the rat hemimandible using an osmotic minipump. Male Wistar rats were infused with either TM or saline as a control. Lectin-gold cytochemistry was performed to reveal glycoconjugates in the BL. Immunogold labeling of enamel proteins and albumin was carried out to verify whether depletion of N-linked sugars in the BL affects the content and distribution of endogenous and exogenous proteins in the enamel layer. Under the influence of the drug, the BL became irregular and exhibited alterations in structural organization and composition. The number of Helix pomatia agglutinin binding sites was not significantly affected but their distribution was altered. The labeling density of wheat germ agglutinin over the BL was slightly reduced. Immunoreactivity for enamel proteins showed only a small decrease, but that of albumin, both between ameloblasts and within the enamel layer, increased significantly. No structural alterations were observed in the contralateral incisor and in other sampled tissues and organs. These results demonstrate that it is possible to achieve a localized administration of TM without systemic side effects and lend support to the proposal that the BL represents a specialized structure with filtering functions.(J Histochem Cytochem 49:165-176, 2001)