Takahiro Satoda

Immunochemical and immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine tooth germ

Abstract Our previous report identified 27- and 29-kDa calcium-binding proteins in porcine immature dental enamel. In this study we revealed that the N-terminal amino acid sequences of the two proteins were identical: LLANPXGXIPNLARGPAGRSRGPPG. The sequence matches a portion of the amino acid sequence of the porcine sheath protein, sheathlin. Porcine tooth germs were investigated immunochemically and immunohistochemically using specific antibodies raised against synthetic peptide that included residues 13–25 of this sequence. The affinity-purified antibodies reacted with several proteins extracted from newly formed immature enamel in immunochemical analyses, especially protein bands migrating at 62, 35–45, 29, and 27 kDa in SDS-polyacrylamide gels. The largest protein detected was a weak band near 70 kDa. In immunochemical analyses of proteins extracted from the inner (old) immature enamel, the antibody reacted faintly with the 27- and 29-kDa proteins. In immunohistochemical preparations, the Golgi apparatus and secretory granules of the secretory ameloblast, and the surface layer of immature enamel showed immunoreactivity. The immunoreactivity of immature enamel just beneath the secretory face of the Tomes’ process was intense. No immunoreactivity was found in the Golgi apparatus of the maturation ameloblast. These results suggest that the 70-kDa protein, whose degradation might be very fast, is the parent protein of the 27- and 29-kDa proteins.

Distribution of axons exhibiting both enkephalin- and serotonin-like immunoreactivities in the lumbar cord segments: an immunohistochemical study in the cat

Axons exhibiting both enkephalin- and serotonin-like immunoreactivities were observed by the double immunofluorescence method in the lumbar cord segments of the cat. Double-labeled axons were seen most frequently in laminae I, IIa and the lateral part of lamina V. They were also distributed in other parts of the dorsal horn and lamina X (especially in the dorsal part), but rarely found in laminae VII, VIII and IX. After cervical hemicordotomy the vast majority of double-labeled axons disappeared from the spinal gray ipsilateral to the lesion.

Amygdalospinal projections in the macaque monkey

Injection of horseradish peroxidase into the cervical cord in the macaque monkey led to the retrograde labeling of neurons in the caudal part of the central nucleus of the amygdala ipsilateral to the spinal half where the injection was made. Although the number of labeled neurons in the amygdala was small, they were constantly found in 7 macaque monkeys (3 Japanese monkeys, 3 crab-eating monkeys and 1 rhesus monkey) which were injected with the enzyme into the upper and middle cervical cord segments.

Amygdaloid pathway to the trigeminal motor nucleus via the pontine reticular formation in the rat

The connections of the amygdala with the trigeminal motor nucleus were studied by light and electron microscopy. Horseradish peroxidase (HRP) experiments showed that the pontine reticular formation, ventromedial to the spinal trigeminal nucleus at the level rostral to the genu of the facial nerve, receives fibers from the central nucleus of the amygdala ipsilaterally and sends fibers to the trigeminal motor nucleus contralaterally. Electron microscopic observations were carried out on the pontine reticular formation after electrolytic lesions in the central nucleus of the amygdala and HRP injections into the contralateral trigeminal motor nucleus were made on the same animal. These experiments using the combined degeneration and HRP technique clearly demonstrated that degenerating amygdaloid fibers made synaptic contacts with retrogradely labeled neurons.

Convergence of serotonin-, enkephalin- and substance P-like immunoreactive afferent fibers on single pudendal motoneurons in Onuf's nucleus of the cat: a light microscope study combining the triple immunocytochemical staining technique with the retrograde HRP-tracing method

Convergence of serotonin (5-HT)-, enkephalin (ENK)-, and substance P (SP)-like immunoreactive (LI) afferent fibers on single pudendal motoneurons within Onufs nucleus was demonstrated: pudendal motoneurons were retrogradely labeled with horseradish peroxidase applied to the pudendal nerve. Subsequently, 5-HT-LI fibers were stained by the immunoperoxidase method, and then ENK- and SP-LI fibers were stained by the double immunofluorescence method.

Representation of the main branches of the facial nerve within the facial nucleus of the Japanese monkey (Macaca fuscata).

The facial nucleus of the Japanese monkey was divided cytoarchitectonically into the ventral, medial, intermediate, dorsal and lateral divisions. When horseradish peroxidase (HRP) was applied to the inferior labial, cervical or posterior auricular branch of the facial nerve, HRP-labeled neurons were seen in the lateral, ventral or medial division of the facial nucleus, respectively. After applying HRP to the anterior auricular-zygomatico-orbital branch, labeled neurons were observed mainly in the intermediate and dorsal divisions. HRP applied to the superior labial branch labeled neurons within the dorsal and lateral divisions.

Distribution of axons showing calcitonin gene-related peptide- and/or substance P-like immunoreactivity in the sensory trigeminal nuclei of the cat.

Distribution of axons with calcitonin gene-related peptide (CGRP)-like and/or substance P (SP)-like immunoreactivity (LI) within the sensory trigeminal nuclei was examined in the cat before and after trigeminal rhizotomy. Axons with CGRP-LI or SP-LI were seen throughout the principal sensory trigeminal nucleus (Vp) and spinal trigeminal nuclei, including the medullary dorsal horn (MDH). They were densely distributed particularly in the dorsolateral part of the dorsal subnucleus of the Vp, ventromedial marginal zone of the ventral subnucleus of the Vp, dorsomedial and ventromedial parts of the oral spinal trigeminal nucleus, ventromedial and lateral marginal zones of the interpolar spinal trigeminal nucleus, and lamina I, outer part of lamina II and lamina V of the MDH. Most of the CGRP-LI axons exhibited SP-LI, while many SP-LI axons did not show CGRP-LI. After trigeminal rhizotomy, almost all CGRP-LI axons disappeared from the ipsilateral sensory trigeminal nuclei, while a considerable number of SP-LI axons remained intact throughout the nuclei; these SP-LI axons did not show CGRP-LI. The results indicate that CGRP-LI axons within the sensory trigeminal nuclei exhibit SP-LI and are of peripheral origin, and that SP-LI axons without CGRP-LI are of central origin.

Amygdaloid projections to commissural interneurons for masticatory motoneurons

Injections of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the trigeminal motor nucleus resulted in retrograde labeling of neurons, called commissural interneurons for masticatory motoneurons, in the contralateral supratrigeminal region. Further HRP studies, in which WGA-HRP injections were made into the amygdala and supratrigeminal region, indicated that the supratrigeminal region receives fibers from the central nucleus of the amygdala ipsilaterally. These findings raised the possibility of direct connections between the amygdala and commissural interneurons. In order to confirm the connections, electrolytic lesions in the central nucleus of the amygdala and WGA-HRP injections into the contralateral trigeminal motor nucleus were made on the same animal and electron microscopic observation was carried out on the supratrigeminal region. Of particular interest was that degenerating amygdalotegmental fibers synapsed upon HRP-labeled neurons.

Administration of MK-801 decreases c-Fos expression in the trigeminal sensory nuclear complex but increases it in the midbrain during experimental movement of rat molars.

Various studies reported c-Fos expression in the neurons in the trigeminal sensory nuclear complex (TSNC) following experimental tooth movement, which implies pain transmission to the central nervous system. Meanwhile, MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors, was shown to markedly reduce the expression of c-Fos in the trigeminal subnucleus caudalis (Vc) following noxious stimulation but to enhance c-Fos expression markedly in other brain regions, i.e., the neocortex, dorsal raphe and thalamic nuclei. In the present study, we examined the nature of c-Fos expression in the brainstem including the TSNC and midbrain following administration of MK-801 and/or experimental movement of the rat molars. Twelve hours after the beginning of experimental tooth movement, c-Fos was expressed bilaterally in the superficial laminae of Vc (Vc I/II), dorsomedial areas of the trigeminal subnucleus oralis (Vodm) and rostro-dorsomedial areas of the trigeminal subnucleus oralis (Vor) with the ipsilaterally dominant distribution, but hardly in the periaqueductal gray (PAG), dorsal raphe nucleus (DR) and Edinger-Westphal nucleus (EW). Intraperitoneal administration of MK-801 (0.03, 0.3 and 3.0 mg/kg) prior to the onset of experimental tooth movement reduced c-Fos in the TSNC (Vc I/II, Vodm and Vor) but increased it in the nucleus raphe magnus (NRM), ventrolateral PAG (vl PAG), DR and EW. These results highly emphasize that during experimental tooth movement, a blockade of NMDA receptors induces neuronal suppression in the TSNC but increases neuronal activity in the descending antinociceptive system including the NRM, vl PAG, DR and EW.

Immunohistochemical demonstration of coexistence of enkephalin- and substance P-like immunoreactivities in axonal components in the lumbar segments of cat spinal cord

Coexistence of enkephalin-like and substance P-like immunoreactivities (ENK-LI and SP-LI) was shown in axonal components in the lumbar segments of cat spinal cord by using the double immunofluorescence method. Although axonal components labeled with both ENK-LI and SP-LI were distributed throughout the spinal gray, they were most dense in the ventral horn, and most sparse in the superficial part of the dorsal horn.