Toshihiko Inage

Gene Expression and Localization of Amelogenin in the Rat Incisor

Gene expression and localization of amelogenin were studied in the developing rat incisor by the methods of in situ hybridization and immunohistochemistry. ISH revealed the first expression of amelogenin mRNA in the inner enamel epithelium of the cervical loop. The signals were clearly observed in pre-ameloblasts in the region bordering on predentin formation and became more intense toward the cells on the initial enamel matrix secretion. The maximal signals were found in the cytoplasm of secretory ameloblasts. From the terminal secretion zone, the signals then became gradually weaker toward the incisal edge but were still evident in the cytoplasm of shortening, transitional ameloblasts and those at the early maturation stage. No signals were found in the cells of the stratum intermedium and stellate reticulum throughout amelogenesis. Immunohistochemistry by means of an antibody against amelogenin C-telopeptide consisting of 12 amino acids revealed immunoreaction in the secretory ameloblasts reacting to the ISH. When a polyclonal antibody against amelogenin was used, immunoreaction was found in the distal ends of ruffle-ended ameloblasts (RA) in the maturation zone. Those results indicated that amelogenin is synthesized by ameloblastic cells from the inner enamel epithelium to the early maturation stage and is then resorbed by the RA.

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.

Gene expression of TGF-β1 and elaboration of extracellular matrix using in situ hybridization and EM radioautography during dentinogenesis

The expressions of TGF‐β1 and Type I collagen mRNA were studied by in situ hybridization and immunohistochemistry then the secretory pathway of dentin phosphoprotein was investigated electron microscopic radioautography in rat incisors.

Morphological analysis of cat masseteric motoneurons after intracellular staining with horseradish peroxidase

Intracellular injection of horseradish peroxidase (HRP) into 58 masseteric motoneurons identified by antidromic activation was performed in cats under pentobarbital anesthesia. Monosynaptic EPSPs were evoked by masseteric nerve stimuli in 52 cells, and were absent in the remaining six cells. The antidromic nature of the evoked spikes was confirmed by IS-SD separation observed at high frequency (50 Hz) stimulation. Motoneurons with monosynaptic excitation from masseter afferents showed IPSPs following stimulation of lingual and inferior alveolar nerves. Motoneurons which did not show monosynaptic excitation from masseter afferents showed no IPSPs from the above nerves. There were no differences in cell size or the number of stem dendrites between motoneurons with and without monosynaptic EPSPs. No recurrent collaterals were observed in any motor axons. Motoneurons with monosynaptic EPSPs were located at all rostrocaudal levels throughout the trigeminal motor nucleus, whereas motoneurons without such EPSPs were encountered only at the middle level. Dendrites of motoneurons with monosynaptic EPSPs did not extend into the medial portion of the nucleus where motoneurons innervating the anterior belly of the digastric muscle were located. In contrast, motoneurons without monosynaptic EPSPs had dendrite branches extending well into the medial part. The results show that there are two subpopulations of masseteric motoneurons that differ in peripheral inputs as well as dendritic morphology.

The location of brainstem neurons with bilateral projections to the motor nuclei of jaw openers in the cat

Symmetrical motor output is the rule in the masticatory system. We examined morphologically how this functional symmetry might be reflected in the organization of premotor neurons that could mediate excitation of jaw-opener motoneurons. Premotor neurons projecting bilaterally to jaw-opener motoneurons by way of axon collaterals were identified by retrograde dual-labeling with cholera toxin B-conjugated fluorescein isothiocyanate (CTb-FITC) and tetramethylrhodamine (TMR). In each cat, CTb-FITC and TMR were injected into the digastric motoneuron pools, respectively, on the left and right sides. In three animals, 69-147 neurons were labeled with both tracers, comprising approximately 44% of all retrogradely labeled cells. Double-labeled cells were located bilaterally in the trigeminal oral nucleus (Vo) and the adjacent reticular formation (RF), the former containing a larger number of cells. Neurons labeled with only one tracer were also distributed bilaterally in the Vo and RF. The results indicated that the bilaterally projecting premoter neurons identified mainly in the Vo and RF represent neuronal substrates for the symmetry that characterizes most jaw movements.