Ameloblast Activity Research Articles

Neutron scanning reveals unexpected complexity in the enamel thickness of an herbivorous Jurassic reptile.

Eilenodontines are one of the oldest radiation of herbivorous lepidosaurs (snakes, lizards and tuatara) characterized by batteries of wide teeth with thick enamel that bear mammal-like wear facets. Unlike most reptiles, eilenodontines have limited tooth replacement, making dental longevity particularly important to them. We use both X-ray and neutron computed tomography to examine a fossil tooth from the eilenodontine Eilenodon (Late Jurassic, USA). Of the two approaches, neutron tomography was more successful and facilitated measurements of enamel thickness and distribution. We find the enamel thickness to be regionally variable, thin near the cusp tip (0.10 mm) but thicker around the base (0.15–0.30 mm) and notably greater than that of other rhynchocephalians such as the extant Sphenodon (0.08–0.14 mm). The thick enamel in Eilenodon would permit greater loading, extend tooth lifespan and facilitate the establishment of wear facets that have sharp edges for orally processing plant material such as horsetails (Equisetum). The shape of the enamel dentine junction indicates that tooth development in Eilenodon and Sphenodon involved similar folding of the epithelium but different ameloblast activity.

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants.

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Excessive fluoride inducing calcium overload and apoptosis of ameloblasts

To study the effect of excessive fluoride on calcium overload and apoptosis in cultured rat ameloblasts in vitro. Logarithmic-phase ameloblasts (HAT-7) were treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mmol · L(-1) sodium fluoride (NaF) solution. Cell activities were detected by using a Cell Counting Kit 8 (CCK-8) assay after 48 h of treatment. The effect of fluoride on cell apoptosis was analyzed by using flow cytometry. Excessive fluoride-induced calcium concentration and calreticulin expression changes in ameloblasts were detected by using laser scanning confocal microscopy, Western blot analysis, and real-time quantitative polymerase chain reaction. NaF inhibited ameloblast activity at 1.6, 3.2, and 6.4 mmol · L(-1) (dose-dependent) after 48 h of induction. The Ca2+ fluorescence intensity of HAT-7 cells incubated with 1.6 and 3.2 mmol · L(-1) NaF was higher than that in the control group. The fluoride-induced early-stage apoptosis of ameloblasts after 48 h of induction and the early-stage apoptosis rate was positively correlated with fluoride concentration. Calreticulin mRNA expression in HAT-7 cells was higher than that in the control group after 48 h of incubation with 0.8, 1.2, and 1.6 mmol · L(-1) NaF. Excessive fluoride-induced calcium overload in ameloblasts and further caused endoplasmic reticulum stress-mediated apoptosis.

Ameloblasts Express Type I Collagen during Amelogenesis

Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while post-metamorphic teeth possess enamel only. In first-generation teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution.

Microstructural defects and enamel hypoplasia in teeth of Toxodon Owen, 1837 from the Pleistocene of Southern Brazil

Enamel hypoplasia is characterized by reduction in the enamel thickness, resulting from a disruption of ameloblast activity due to systemic physiological stress. The euhypsodont teeth of Toxodon, a notoungulate from the Pleistocene of South America, often exhibit signs of enamel hypoplasia, in the form of continuous grooves or a series of pits where the enamel is thinner than in normal areas. These defects alternate with areas of normal enamel, and sometimes more than one form of enamel hypoplasia is present on the same tooth. This study analysed teeth of Toxodon from the Pleistocene Touro Passo Formation and the coastal plain of State of Rio Grande do Sul, Southern Brazil. Six types of enamel hypoplasia were observed. Upper teeth present mainly superficial grooves on the buccal surface, and the defects are less severe than those observed in the lower teeth. In the lower incisors, deep grooves with mesiodistal rows of pits were observed, showing clearly cyclical changes, which to a lesser degree, exist in all teeth. These changes are likely related to the continuous growth of euhypsodont teeth. Seven specimens were analysed under scanning electron microscopy and optical microscopy, which showed the occurrence of microstructural changes associated with the macroscopic enamel defects. Enamel underlying in the vicinity of hypoplastic defects was aprismatic and associated with prominent pathologic striae. These pathological findings might indicate that toxodonts were exposed to some stressing conditions or that their teeth were more easily abraded due to a change in diet items, related to shifting climatic conditions.

Excess Fluoride Interferes with Chloride-channel-dependent Endocytosis in Ameloblasts

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Both CF and dental fluorosis result in protein retention in mature enamel. We hypothesized that excess fluoride might cause protein retention by interfering with CFTR function, resulting in abnormal expression of proteases and pathological endocytosis. Millimolar concentrations of fluoride reduced uptake of Emdogain, an enamel matrix derivative, in ameloblast-like PABSo-E cells, while stimulating an acidic intracellular environment at the same time. When CFTR function was inhibited by either an siRNA or a chloride channel inhibitor, CFTRinh-172, fluoride’s effect on Emdogain uptake was partially blocked. Treatment of cells with CFTR siRNA down-regulated expression of proteases MMP20 and KLK4 and increased intracellular pH. We conclude that excess fluoride inhibits endocytic activity of ameloblasts through the CFTR chloride channel or other chloride channels. The intracellular pH might be the key mechanism by which abnormal proteolytic activity and defective endocytosis cause the residual protein observed in enamel of patients with CF and dental fluorosis.

Stress Sensitization in Schizophrenia

It is well known that environmental factors, such as early life events, perinatal damage, and urbanicity, which interact with multiple genes, induces persistent sensitization to stress possibly through an imbalance in interactions between dopaminergic and glutamatergic systems. This stress sensitization may be critical in the development or relapse of schizophrenia. The neural correlates of a negative mood might be impaired, resulting in stress sensitization and difficulties in social adjustment (Dr. Habel). Urbanicity is associated with later schizophrenia. Metabolic stress induces stress sensitization via dysregulation of dopaminergic and/or noradrenergic systems in activated HVA and cortical response (Dr. Marcelis). The glutamatergic regulation activates HPA axis in stress response (Dr. Zelena). Ameloblast activity in human molar's enamel slowed by exposure to stress, and the segment of enamel rods is smaller, making a particular dark line. Stress sensitization may be induced at the age of 10.5 to 11.5 years resulting from severe emotional stress at the age of 10.5 to 11.5 years (Dr. Yui). It has been reported that volume reductions in the amygdala, hippocampus, superior temporal gyrus, and anterior parietal cortex common to both patient groups may represent the vulnerability to schizophrenia, while volume loss of the prefrontal cortex, posterior parietal cortex, cingulate, insula, and fusiform cortex preferentially observed in schizophrenia may be critical for overt manifestation of psychosis (Dr. Suzuki).

Gene Expression and Localization of Insulin-like Growth Factors and Their Receptors throughout Amelogenesis in Rat Incisors

Insulin-like growth factors (IGFs) are expressed in many tissues and control cell differentiation, proliferation, and apoptosis. In teeth, the temporo-spatial pattern of expression IGFs and their receptors has not been fully characterized. The purpose of this study was to obtain a comprehensive profile of their expression throughout the life cycle of ameloblasts, using the continuously erupting rat incisor model. Upper incisors of young male rats were fixed by perfusion, decalcified, and embedded in paraffin. Sections were processed for in situ hybridization and immunohistochemistry. mRNA and protein expression profiles IGF-I, IGF-II, IGF-IR, and IGF-IIR mRNA were essentially identical. At the apical loop of the incisor, very strong signals were seen in the outer enamel epithelium while the inner enamel epithelium showed a moderate reaction. In the region of ameloblasts facing pulp, inner enamel epithelium cells were still moderately reactive while signals over the outer enamel epithelium were slightly reduced. In the region of ameloblasts facing dentin and the initial portion of the secretory zone, signals in ameloblasts were weak while those over the outer enamel epithelium were strong. In the region of postsecretory transition, signals in both ameloblasts and papillary layer cells gradually increased. In maturation proper, signals in ameloblasts appeared as alternating bands of strong and weak reactivities, which corresponded to the regions of ruffle-ended and smooth-ended ameloblasts, respectively. Papillary layer cells also showed alternations in signal intensity that matched those in ameloblasts. These results suggest that the IGF family may act as an autocrine/paracrine system that influences not only cell differentiation but also the physiological activity of ameloblasts.

Original article scanning electron microscopic observations of surface prismless enamel formed by minute crystals in some human permanent teeth.

It might have been recognized that surface prismless enamel with no prism sheaths or boundaries is formed by needle-shaped crystals in parallel arrangements and shows almost the same crystal size as the underlying prismatic enamel. This study found that some island- and band-shaped prismless enamel in third molars and dome-shaped prismless areas in the region adjacent to the partial hypoplastic enamel of a premolar were formed by minute crystals compared with the underlying prismatic enamel when observing afibrillar cementum and cementicle-like structures by scanning electron microscopy. Their minute crystals became smaller in size towards the natural enamel surface. In the band- and dome-shaped prismless enamel, the minute crystals abruptly changed to the usual-sized crystals in the underlying prismatic enamel, although the minute crystals in the dome-shaped prismless areas tended to show random arrangements. The band-shaped prismless layers might be similar to afibrillar cementum, but shallow Tomes' process pits were present in the natural surface and no appositional laminations were observed in the fractured surface. The minute crystal formation in such prismless regions might be caused by the remarkable decline of ameloblast activity immediately after the disappearance of Tomes' processes producing prism structures surrounded by prism sheaths during the final stage of amelogenesis.

Development of larval and transformed teeth in Ambystoma mexicanum (Urodela, Amphibia): an ultrastructural study

Odontogenesis of early larval non-pedicellate teeth, late larval teeth with a more or less distinct dividing zone and fully transformed pedicellate teeth in Ambystoma mexicanum (Urodela) was studied to obtain insights into the development of differently structured teeth in lower vertebrates. Using transmission electron microscopy we investigated five developmental stages: (1) papilla; (2) bell stage (secretion of the matrix begins); (3) primordium (mineralization and activity of ameloblasts starts); (4) replacement tooth (young, old); and (5) established, functional tooth. Development of the differently structured teeth is largely identical in the first three stages. Mineralization takes place in apico-basal direction up to the (prospective) pedicel (early and some late larvae) or up to the zone that divides the late larval and transformed tooth in pedicel and dentine shaft (pedicellate condition). Mineralization starts directly at the collagen and by means of matrix vesicles. First odontoblasts develop small processes that extend to the basal lamina of the inner epithelial layer of the enamel organ. The processes are small and lack organelles in early larval teeth, but become larger, arborescent, and contain some organelles in late larval and transformed teeth. The processes are surrounded by unmineralized matrix (predentine). Odontoblasts at the basis of the teeth, at the pedicel, and in the zone of division do not develop significant cytoplasmic processes that extend into the matrix. Cells of the inner enamel epithelium differentiate to ameloblasts that secrete the enamel. In the early larval tooth they show an extensive basal labyrinth that becomes regressive when the enamel layer is completed. In late larval and transformed teeth, however, a large cavity arises between the basal ruffled border of ameloblasts and their basal lamina. This cavity appears to mediate amelogenesis. A small apical zone in early, but not in late larval teeth directly below the thin enamel layer consists of enameloid and is free of dentine channels.

Thermodynamic and Kinetic Interpretation of Enamel Mineralization: Octacalcium Phosphate (OCP) as an Inevitable Intermediate Phase.

Much attention has been given to the growth mechanism of enamel mineral by which the unique features (i. e., morphology, size and alignment) of enamel crystallites in mammals are achieved. In this paper, we report our experimental and theoretical approaches to a long-debated and unsettled issue, i. e., the involvement and role of an acidic precursor in enamel mineralization. Analysis of nucleation events, which were induced in neutral media at 37°C and 1.8% partial CO2 pressure, substantiated the involvement of octacalcium phosphate (OCP) in precipitation reactions. We also developed a new crystal growth model using miniaturized columns in tandem, in which OCP itself was proved to be equally competent, in comparison with hydroxyapatite (HA), as a template for crystal precipitation and as an adsorbent for interaction with enamel proteins. Simulation of the precipitation process taking place in situ with calcium transport (its increment) into enamel fluid also allowed us to predict that OCP is more kinetically favorable than HA, if Ca ions are supplied through the cellular activity of ameloblasts. The results obtained support the theory that OCP is involved as an inevitable intermediate phase to yield large enamel carbonatoapatite during amelogenesis.

Autoradiographic investigation of the effect of 1-hydroxyethylidene-1, 1-bisphosphonate on matrix protein synthesis and secretion by secretory ameloblasts in rat incisors

Seven daily subcutaneous injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) can induce enamel hypoplasia. Several enamel-free zones were observed along the crown-analogue side of rat incisors during the secretory stage of amelogenesis. Ameloblasts related to the enamel-free zones lay directly on the abnormally non-mineralized mantle dentine, whereas the adjacent ameloblasts, which were forming the enamel matrix layer, were associated with the region where mineralization of dentine was proceeding. The further purpose of this study was to investigate the synthetic and secretory activity of these two groups of ameloblasts and to trace the fate of the radioactively labelled proteins. [ 3H]-proline was administered to Wistar rats 12 h after the last injection of HEBP. Light-microscopic autoradiography was performed. Quantitative analysis indicated that the ameloblasts of the enamel-forming zones in the drug-treated group showed a distribution pattern of silver grains similar to that of the controls. The ameloblasts of the enamel-free zones also demonstrated incorporation of [ 3H]-proline at the same level. There was some labelling over the non-mineralized mantle dentine, which was supposed to indicate the penetration of ameloblast products. From these results, it is concluded that HEBP does not affect the ameloblast activity in protein synthesis. The complete failure of enamel-layer formation in some specific regions is probably due to the failure in protein secretion and protein deposition. This study provides additional evidence that the mineralization of dentine is an essential factor in successful enamel matrix secretion and deposition.

Proton microprobe assessment of the distribution of fluoride in the enamel and dentine of developing central incisors of sheep and changes induced by daily fluoride supplements

Ten sheep were given 0.5 mg fluoride (F) and 10 sheep 0.2 mg F/kg body wt orally for periods of 1–6 months while 8 sheep received no additional F. One incisor from each sheep was sectioned longitudinally in the midline and, using the proton microprobe, multiple scans for calcium and F were made across the enamel and dentine. F was determined by proton-induced γ-ray emission and calcium by X-ray emission. Tooth length and hence the stage of ameloblast activity for each of the 28 teeth at the start of the experiment was determined using a tetracycline marker. In addition, the stage of enamel development of the eight control teeth (no dietary F) at the time of their extraction was assessed from their macroscopic appearance. Continuous changes in F levels occurred in both enamel and dentine throughout tooth development and also in the mature enamel and associated dentine after ameloblast regression. All scans for all stages of tooth development and all F treatments showed a high F concentration at the enamel surface. Early in the secretory phase, a wide-based F peak occupied the entire width of the enamel with a similar F peak in the dentine. In the control teeth, no consistent increase in F concentration occurred at the enamel surface during later development. When F supplements were started early in the maturation phase an increase in F concentration only at the enamel surface was recorded. When F supplements were also given during the secretory phase, higher F concentrations were recorded not only at the enamel surface but also for the inner enamel and dentine plateau. These findings, based on a small number of sheep, indicate that further research is needed to clarify the method and control of F uptake and to determine the changes in these processes during the different stages of tooth development.

Immunolocalization of CD44 and heparan sulfate chains on the stratum intermedium and papillary layer in the rat enamel organ.

We studied the immunohistochemical localization of CD44 and heparan sulfate (HS) chains in rat enamel organ by confocal laser scanning microscopy and transmission electron microscopy. We also investigated the binding sites of basic fibroblast growth factor (bFGF), one of the heparin-binding growth factors (HBGF), on Microslicer-sections to clarify its role in the cell-cell interaction of HS. At the differentiation stage of ameloblast, weak immunoreactivity for CD44 was detected on the plasma membrane of the inner enamel epithelium, external enamel epithelium and the cells adjacent to the inner enamel epithelium. In accordance with the differentiation of preameloblasts into secretory ameloblasts, this immunoreactivity increased in stratum intermedium cells. At the secretory stage, stratum intermedium cells showed the most intense immunoreactivity in the enamel organ. At the maturation stage, strong immunoreactivity was seen on papillary layer cells. On the other hand, the lateral plasma membrane of ruffle-ended (RA) and smooth-ended ameloblast (SA) showed weak reactivity. No immunoreactivity was detected on the ruffled border of RA and the distal plasma membrane of SA. Immunolocalization of HS chains was similar to that of CD44. The binding activity of bFGF was also intense on stratum intermedium cells and papillary layer cells. These findings suggest that: 1) stratum intermedium cells and papillary layer cells express CD44 and HS chains in accordance with their differentiation; 2) HS chains on the plasma membrane of these cells may regulate calcium transport by their negative charge; and 3) HS chains on the stratum intermedium and papillary layer may play an important role in the differentiation and activity of ameloblasts by preserving HBGF.

Developmental defects of enamel--historical and present-day perspectives of their pathogenesis.

Developmental defects of enamel are visible deviations from the normal translucent appearance of tooth enamel resulting from enamel organ dysfunction. In the past, information about the activities of the ameloblasts has determined the terminology used to describe the lesions. Advances in our understanding of the complicated secretory and maturation phases of amelogenesis have required a re-appraisal of the concepts of defect formation. The phase of ameloblast activity, the duration of the disturbance, and its severity leading to temporary or permanent inactivity of the cells determine the appearance of the three common types of lesions--hypoplasia, and diffuse and demarcated opacities.

Secretory Activity as A Function of the Development and Maturation of Ameloblasts

The biosynthetic and secretory activity of rat incisor ameloblasts was studied by grain count analysis of radioautographs at various times following a single injection of either 3H-methionine, 3H-leucine, or 3H-glycine. Experiments were also carried out with leupeptin, a thiol and serine proteinase inhibitor which blocks degradation of proteins within lysosomes. The results from this study indicate that the biosynthetic and secretory activities of ameloblasts increase steadily as the cells differentiate (presecretory stage) and start to form the enamel layer (secretory stage). Secretory activity reaches a peak when the ameloblasts form about one-third of the eventual thickness of the enamel, and remains at this high level until shortly before they start to form the outer and final layers of enamel. Secretory activity then drops rapidly as the cells undergo postsecretory transition, and declines slowly thereafter as the shortened ameloblasts modulate continuously along the surface of the maturing enamel. Ameloblasts appear to biosynthesize more proteins than are secreted. The excess proteins are degraded rapidly in lysosomes and the amino acids reutilized for production of new exportable and/or structural proteins.

The macroscopic and scanning electron-microscopic appearance and microhardness of the enamel, and the related histological changes in the enamel organ of erupting sheep incisors resulting from a prolonged low daily dose of fluoride

The hypothesis that diffuse opacities in enamel result from a chronic, mild disturbance to ameloblast activities was tested using fluoride. Three sheep (HF) were dosed orally with 0.5, and 3 (LF) with 0.2 mg fluoride/kg body weight daily for 6 months. A control sheep (C) received no additional fluoride. The 7 sheep were killed at or close to the time of emergence of their permanent central incisors. One tooth from each sheep was sectioned longitudinally. The enamel related to the secretory (S) and maturation (M) phases of ameloblast activity at the start of fluoride dosing was determined from a tetracycline marker. The pattern of mineralization of the outer 150 μm of the cut labial enamel was assessed using microhardness testing. The SEM appearance of the acid-etched outer enamel was compared in S and M zones in 5 teeth. The enamel of the C tooth was translucent. Diffuse opacities, similar in appearance to human fluorosis, were present in all fluoride-treated teeth. Hardness values in the outer 70 μm of the enamel decreased as the fluoride dose increased and, in the HF teeth, were lower in the S zone than in the M zone. Fluoride given during the M phase induced a surface hypomineralization which increased in degree and depth when fluoride was also given during the S phase. The SEM appearance of M and S enamel was similar in 2 LF and 1 HF teeth but, in the other HF tooth, S enamel but not M enamel had a disordered prism structure and loosely-packed crystals in an abnormal organic matrix. Histological examination revealed that ameloblasts remained in only 4 of the 7 teeth and that their regression and the formation of the cementum adjacent only to the labial enamel were progressing abnormally.

The effect of streptozotocin on the secretory activity of ameloblasts in rat incisor as revealed by radioautography after 3H-proline administration.

The effect of a diabetogenic dose of streptozotocin on the secretory activity of ameloblasts was investigated in the rat incisor by radioautography. One group of male Sprague-Dawley rats was injected intravenously with streptozotocin in citrate buffer (pH 4.5). One hour later, this group was again injected intravenously with 3H-proline (2 mCi/kg). A control group of animals was injected with 3H-proline only. All the animals were sacrificed in groups of three at 5 min, 1 h, 2 h, 4 h and 8 h after 3H-proline injection by perfusion with 3% phosphate-buffered formaldehyde followed by an additional perfusion with 2.5% phosphate-buffered glutaraldehyde. The incisors were extracted with the jaws, demineralized, and prepared for radioautographic observations and analysis. The principal effects of streptozotocin were as follows: There was an inhibition of 3H-proline incorporation into the secretory ameloblasts at 5 min after injection. This was followed by a larger uptake and a slower passage of the label out of the cells into the enamel matrix than that seen in the control sample. Finally, there was a slower secretion of labeled proteins out of Tomes' processes between 1 and 4 h after injection. Therefore, streptozotocin had a temporary inhibitory effect on the incorporation and secretion of 3H-proline by the secretory ameloblasts of the rat incisor. This effect was present for about 4 h and was completely reversed 9 h after streptozotocin injection.

The effect of colcemid on the structure and secretory activity of ameloblasts in the rat incisor as shown by radioautography after injection of 3H-proline.

Enamel secretion by ameloblasts was investigated in the incisors of 100 gm normal and colcemid-injected male rats. Morphological studies were done on rats given a single intraperitoneal injection of 0.1 mg (1.25 mM) of colcemid and sacrified 1 to 4 hours after injection. Protein synthesis and secretion were investigated with radioautography in normal and colcemid-treated rats injected with 3H-proline and sacrificed at intervals between 0.5 and 3.5 hours after injection. Colcemid was injected 0.5 hours prior to 3H-proline in each experimental rat. Electron microscopic examination revealed several morphological alterations between 1 and 4 hours after injection of colcemid. These changes included fragmentation of the normally elongated rough endoplasmic reticulum into shorter profiles; a disorganization of the normally tubular configuration of the Golgi apparatus into a number of seples and profiles of smooth endoplasmic reticulum from Tomes' processes; and the accumulation of secretion granules at the mature face of the Golgi stacks, as well as in the infranuclear cytoplasm where thye are normally not found. Radioautography revealed that protein synthesis by the rough endoplasmic reticulum had continued in colcemid-altered ameloblasts. Labeled secretion granules were found at the mature surface of the Golgi stacks and in the infranuclear cytoplasm, however they did not migrate into Tomes' processes. Consequently, labeled enamel matrix did not appear extracellularly at the same time as in normal controls. Quantitative radioautography in the light microscope revealed that the effect of colcemid, although reversed within 4 hours, had temporarily inhibited normal migration, and exocytosis of secretion granules.