Morphological properties of tectal neurons that project to the nucleus geniculatus lateralis, pars ventralis (GLv) and the surrounding ventral thalamus in chicks.

Layer 10 neurons of the chick tectum were morphologically investigated. The layer 10 neurons displayed heterogeneous immunoreactivities to calcium-binding proteins (CaBPs). Calbindin (CB)-immunoreactive (ir) neurons had pyramidal or round somata, primarily found in layers 5, 9, and 13. Parvalbumin (PV)-ir neurons were of various shapes with small to large somata (109.7±48.6µm(2)) that were located mainly in layers 4 and 10. Calretinin (CR)-ir neurons had small to middle-sized somata (79.3±9.7µm(2)) located primarily in layers 10 and 13, and most of them were similar to typical radial cells in size and shape. Two distinct types of neurons that projected to the nucleus geniculatus lateralis, pars ventralis (GLv) and ventral thalamus were demonstrated in layer 10. Type 1 cells had small to middle-sized somata (74.3±33µm(2)), and each cell had a single apical dendrite that ramified into bush-like branches in layer 7. These cells corresponded to CR-ir neurons and radial cells in size and shape. Type 2 cells had larger somata (124.7±52.6µm(2)), and their shapes were pyramidal, polygonal, or oval. They had multiple obliquely ascending dendrites that ramified into bush-like branches in layer 7. These cells often appeared similar to PV-ir neurons.

Retrograde tracing with fluorescent microspheres reveals bifurcating projections from central retina to tectum and thalamus in chicks.

The goal of this study is to demonstrate the dual-projection pattern of retinal ganglion cells (RGCs) projecting to the tectum and visual thalamus in chick using retrograde fluorescent tracers and also to define the morphological properties of these RGCs with dual projections by intracellular injection of Lucifer Yellow (LY) combined with immunohistochemistry. Thirty-two chicks received double injections of green and red fluorescent microspheres into their thalamus and tectum in the same side. In the central retina, most of the labelled RGCs were tec-RGCs (RGCs projecting to the tectum), a quarter was tha-RGCs (RGCs projecting to the thalamus), and almost all of the tha-RGCs were double-labelled RGCs. An intracellular injection of LY into the double-labelled RGCs showed all six groups of RGCs without specific populations in each group (J. Comp. Neurol., 2004, 469: 360). These dendritic patterns were mostly mono- and bistrata, which extended horizontally in the deeper part of the inner plexiform layer.

Statin suppresses apoptosis in osteoblastic cells: role of transforming growth factor-beta-Smad3 pathway.

Statins possess pleiotropic effects in several tissues. Among them, their bone anabolic actions have been recently noted. We have proposed that Smad3, a TGF-beta-signaling molecule, is a promoter of bone formation. However, whether statins would affect TGF-beta-Smad3 pathway in osteoblasts is still unknown. The present study was performed to examine the effects of statin on Smad3 expression and cell apoptosis by employing mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Statins (pitavastatin, mevastatin, and simvastatin) as well as alendronate increased the levels of Smad3 in MC3T3-E1 cells. The effects of pitavastatin on Smad3 levels were observed from 3 hours and later. Pitavastatin induced the expression of TGF-beta, and cycloheximide, a protein synthesis inhibitor, antagonized the increased levels of pitavastatin on Smad3. On the other hand, pitavastatin antagonized dexamethasone- or etoposide-induced apoptosis in a dose-dependent manner, and Smad3 inactivation by dominant negative Smad3 or an inhibition of endogenous TGF-beta action by SB431542 antagonized anti-apoptotic effects of pitavastatin, indicating that pitavastatin suppressed osteoblast apoptosis partly through TGF-beta-Smad3 pathway. In conclusion, the present study has demonstrated for the first time that statin suppressed cell apoptosis partly through TGF-beta-Smad3 pathway in osteoblastic cells.

Serum soluble factors induce the proliferation, alkaline phosphatase activity and transforming growth factor-beta signal in osteoblastic cells in the patient with hepatitis C-associated osteosclerosis.

Hepatitis C-associated osteosclerosis (HCAO) is a rare syndrome characterized by severe, acquired, generalized osteosclerosis and hyperostosis in adults who are infected with the hepatitis C virus. However, the detail of the pathogenesis of HCAO is still unknown. We examined the effects of serum of the HCAO patient on the proliferation, alkaline phosphatase (ALP) activity and transforming growth factor (TGF)-beta-Smad signaling in mouse osteoblastic cells. The patient was compatible with HCAO, characterized by high bone mass, bone thickening and bone pain with normal lamelar bone. The serum from the HCAO patient increased the levels of TGF-beta and Smad3 expression in osteoblastic MC3T3-E1 cells, compared with the control subject. Moreover, the serum from the HCAO patient significantly augmented TGF-beta-induced transcriptional activity with luciferase assay using 3TP-Lux with a Smad3-specific responsive element. In addition, the serum from the HCAO patient significantly stimulated the MTT intensity, the level of proliferating cell nuclear antigen expression, a proliferation marker, and ALP activity in MC3T3-E1 cells, compared with that from the control subject. In conclusion, the present study indicated that the serum from the HCAO patient stimulated TGF-beta-Smad signaling, as well as the proliferation and ALP activity in osteoblastic cells. Some soluble factors other than parathyroid hormone might be related to the pathogenesis of HCAO.

Smad3 differently affects osteoblast differentiation depending upon its differentiation stage.

Smad3, a critical component of the TGF-beta signaling pathways, plays an important role in the regulation of bone formation. However, how Smad3 affects osteoblast at the different differentiation stage remains still unknown. In the present study, we examined the effects of Smad3 on osteoblast phenotype by employing mouse bone marrow ST-2 cells and mouse osteoblastic MC3T3-E1 cells at the different differentiation stage. Smad3 overexpression significantly inhibited bone morphogenetic protein-2 (BMP-2)-induced ALP activity in ST-2 cells, indicating that Smad3 suppresses the commitment of pluripotent mesenchymal cells into osteoblastic cells. Smad3 increased the levels of COLI and ALP mRNA at 7 day cultures in MC3T3-E1 cells, and its effects on COL1 were decreased as the culture periods progress, although its effects on ALP were sustained during 21 day cultures. Smad3 overexpression enhanced the level of Runx2 and OCN mRNA at 14 day and 21 day cultures. Smad3 increased the levels of MGP and NPP-1 mRNA, although the extent of increase in MGP and NPP-1 was reduced and enhanced during the progression of culture period, respectively. Smad3 did not affect the level of ANK mRNA. On the other hand, Smad3 enhanced the level of MEPE mRNA at 14 and 21 day cultures, although Smad3 decreased it at 7 day cultures. In conclusion, Smad3 inhibits the osteoblastic commitment of ST-2 cells, while promotes the early stage of differentiation and maturation of osteoblastic committed MC3T3-E1 cells. Also, Smad3 enhanced the expression of mineralization-related genes at the maturation phase of MC3T3-E1 cells.

Body composition and vertebral fracture risk in female patients treated with glucocorticoid.

INTRODUCTION: Glucocorticoid (GC) causes bone loss and an increase in bone fragility. However, fracture risk was found to be only partly explained by bone mineral density in GC-treated patients (GC patients). Although GC causes a change in the distribution of fat in the body, the relationship between body composition and fracture risk in GC patients remains unknown. METHODS: The present study examined the relationship between the presence or absence of vertebral fractures and various indices, including body composition, in 92 premenopausal GC patients, 122 postmenopausal GC patients and 122 postmenopausal age-matched control subjects. Dual-energy X-ray absorptiometry was employed to analyze body composition. RESULTS: Percentage lean body mass (LBM), % fat and % trunk fat were not significantly different between postmenopausal GC patients and the control women. When groups with and without vertebral fractures were compared, % LBM and % fat were significantly higher and lower in groups with vertebral fractures, respectively, in postmenopausal GC patients, but not in the postmenopausal control women, although % trunk fat was not significantly different between groups with and without vertebral fractures. Femoral neck BMD was negatively correlated with % LBM and positively correlated with % fat. In premenopausal GC patients, % trunk fat was significantly higher in the fracture group, although % LBM and % fat were not significantly different between groups with and without vertebral fractures. CONCLUSION: The present study revealed that body composition is related to vertebral fracture risk in GC-treated patients. Lower % fat can be included in the determination of vertebral fractures in postmenopausal GC-treated patients. The influence of body composition on vertebral fracture risk may be different between the pre- and postmenopausal state in GC patients.

Afferent and efferent connections of the nucleus geniculatus lateralis ventralis demonstrated by WGA-HRP in the chick.

Fibre connections of the chick nucleus geniculatus lateralis ventralis (GLv) were investigated using the axonal tracing method with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After an injection of WGA-HRP into the GLv, many labelled neurons were observed in layer i of the stratum griseum et fibrosum superficiale (SGFS) in the ipsilateral tectum opticum (TO) and in the nucleus lentiformis mesencephali (LM). In the TO-GLv projection, cells of origin were located in the deeper part of layer i of the TO and were topographically distributed along the direction from the rostrodorsal part to the caudoventral part of the TO relating to a rostrocaudal axis of the GLv. In the LM-GLv connection, the dorsal and ventral parts of the LM connected reciprocally with the rostral and caudal halves of the GLv, respectively. In contrast, in the GLv efferent connection, labelled axon terminals spread widely in the ipsilateral area pretectalis without any clear topographical arrangement.

Quantitative analysis of cells in the ganglion cell layer of the chick retina: developmental changes in cell density and cell size.

Changes in cell density and size in the ganglion cell layer (GCL) of the retina were studied in chick embryos and post-hatching chicks. The total number of cells in the GCL increased from 3.64 million at embryonic day 8 (E8) to the maximal 7.85 million at E14. After E14, the number of cells decreased to 6.08 million at post-hatching day 1 (P1) and 4.87 million at P8. Cell density in the GCL decreased unevenly according to retinal regions; cell density in the presumptive central area (pCA) of P8-chicks decreased to approximately 45% of that in E8-embryos. Densities of the nasal peripheral retina (NP) and temporal peripheral retina (TP) of P8-chicks decreased to 23 and 18% of E8-embryos, respectively. Differentiation of the central (44,000 cells/mm(2) in pCA) - peripheral (28,000 cells/mm(2) in TP) gradient in cell density was formed by E8. The presumptive dorsal area (pDA) was shaped by E11, but became obscure with age. Although ganglion cell sizes were basically uniform at E8, differentiation occurred with the appearance of larger ganglion cells after E14. Mean size of retinal ganglion cells increased 2.8-fold in the pCA and 3.8-fold in the TP between E8 and P8, accompanying a similar scale of decreases in cell densities.

Afferent and efferent connections of the nucleus rotundus demonstrated by WGA-HRP in the chick.

Organization of the fibre connections in the chick nucleus rotundus (Rt) was investigated by an axonal tracing method using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After an injection of WGA-HRP into the Rt, labelled neurones were observed in the striatum griseum centrale (SGC) in both sides of the tectum (TO) and in the ipsilateral nucleus subpretectalis/nucleus interstito-pretecto-subpretectalis (SP/IPS). Labelled fibres and terminals were also found in the ipsilateral ectostriatum (Ect). These fibre connections were topographically organized rostrocaudally. In the TO-Rt projection, the rostral and the dorsocaudal parts of the Rt received afferents from the superficial part of the SGC, the middle part of the Rt received afferents from the intermediate part of the SGC, and the ventrocaudal part of the Rt received mainly fibres from the deep part of the SGC. These topographic projections were accompanied by a considerable number of diffuse projections to the thalamic regions surrounding the Rt. In addition, the rostral and middle caudal parts of the Rt received afferents from the lateral and medial parts of the SP/IPS, respectively, and respective parts of the Rt sent efferents to the lateral and medial parts of the Ect.

Transmural heterogeneity of myocardial integrated backscatter in diabetic patients without overt cardiac disease.

It is important to detect early changes in diabetic myocardium, because some diabetic patients suffer from diabetic cardiomyopathy, especially those with poorer glycemic control or hypertension (HT). To clarify whether ultrasonic tissue characterization can noninvasively detect ultrastructural changes in diabetic myocardium, we analyzed the transmural heterogeneity in myocardial integrated backscatter (THIB) in 20 diabetic patients and 16 normal subjects. THIB was defined as the absolute value of difference of integrated backscatter between the endocardial and epicardial half of the myocardium. THIB in diabetic patients was significantly greater than that in normal subjects. In diabetic patients, there was a significant correlation between glycosylated hemoglobin and THIB, and the greater THIB was shown in patients with HT compared with those without HT. Early changes in the myocardium, related to increased interstitial collagen deposition or other occult cardiomyopathic changes, may be detected on the basis of quantitative analysis of THIB in diabetic patients.

Morphological analogy of the rostral midline area in the chicken midbrain to the anteromedian nucleus in the mammalian midbrain.

Substance P-immunoreactive neurons projecting from the midbrain to the spinal cord of the chicken were examined by the use of the retrograde tract-tracing method combined with immunohistochemical techniques. Many small neurons were densely clustered in the rostral midline area of the midbrain (RMA), and showed substance P-immunoreactivity. These substance P-immunoreactive neurons sent axons to the intermediomedial cell column (avian autonomic preganglion) and its vicinity in the lumbar spinal segments. On the basis of the strong neuroanatomical analogy in the cytoarchitectural features, immunoreactivity, and fiber connections, the RMA was assumed to be the avian homologue of the anteromedian nucleus in the mammalian midbrain.

The developmental study on lamination of the optic tectum in relation to the retinotectal projection in chicks and chick embryos.

The tectal lamination was investigated in the central part of the chick embryonic tectum. Two and 5 layers were observed above the neuroepithelium (NE) on embryonic day 6 (E6) and E8, respectively. Optic fibers extended on the surface of the tectum by E8. On E10-11, the outer tectum was composed of 2 layers, that is, a fibrous layer forming the optic fiber layer on the tectal surface and a cellular layer showing the gradient of cell density. In the inner tectum, the lamination was almost completed. On E12-13, the outer tectal layers, which showed the gradient of cell density, was divided into dark and light cellular layers. The dark cellular layer was divided into 2 layers on E14-15 and further into 4 layers (layer C-F in chick) on E18. On the other hand, the light cellular layer did not change until E18, but finally, it was divided into 2 layers (layer A and B in chick) by E20. Optic fibers reached the bottom of the outer tectum by E14 showing different densities of terminals. Stratification by optic fibers was going to step into the final stage on E18. On E20, laminations according to cytoarchitectural features and the optic fiber terminals were substantially completed. In the tectum affected by destruction of the contralateral embryonic eye (E4), some cellular layers were incompletely discriminated by differences of cell density.

Substance P-immunoreactive neurons in the rostromedian area of the midbrain send axons to the lower spinal cord in the chicken.

Substance P-immunoreactivity in neurons projecting to the spinal cord was examined using retrograde tract-tracing method combined with immunohistochemical techniques in chickens. Many small substance P-immunoreactive neurons were densely clustered in the midline area in the rostral midbrain, the rostromedian area (80% of the neurons in the rostromedian area). Some of these substance P-immunoreactive neurons in the rostromedian area (about 20% of substance P-immunoreactive neurons) were retrogradely labeled by small injections of wheat germ agglutinin conjugated horseradish peroxidase into the central part of the lumber segments including the intermediomedial nucleus, suggesting the projections from the rostromedian area to the lower spinal preganglionic regions. From the present data and mammalian previous studies, it was suggested that the midline area in the midbrain has fiber connections with the regions related autonomic functions, and all of which exhibit substance P-immunoreactivity.

Biosynthesis of L-alanine, a major amino acid of fibroin in Samia cynthia ricini.

The derivation of alanine in fibroin was investigated using NMR and selective isotopic labelling. 2H2O infused orally into 5th instar larvae was incorporated into the proton of the methyl group of alanine in fibroin. Proton exchange among alanine, glycine and serine was also found. Incorporation of 13C from [2-(13)C]acetate into alanine C2 and C3 and glycine C2 in fibroin, and also C4 of free glutamine plus glutamate was observed in vivo. Hemolymph contained a peak for C4 of glutamate plus glutamine, and an alanine C3 peak appeared transiently. Thus, it is suggested that the C-skeleton of alanine formed was derived from L-malate via the TCA-cycle, and that this alanine is utilized in part for fibroin synthesis. Spectra of the hemolymph extract of larvae infused orally with [15N2]urea showed no 15N-compounds, whereas those of larvae injected subcutaneously showed only one peak of urea, whose intensity decreased with time, as shown in the in vivo spectra of a living larva infused with [15N2]urea. The solution NMR spectrum of fibroin showed no 15N-labelled compounds. Temporal changes in the peak intensities of six compounds in the spectra of a living larva infused with [15N]ammonium demonstrated a process in which 15N was incorporated into fibroin containing 15N-alanine through the amide group of glutamine and the amino group of glutamate. Thus, alanine biosynthesis from the TCA-cycle originates mainly from water, L-malate and ammonium. The fact that no 15N-urea was detected in the hemolymph extract of larvae infused with [15N]ammonium suggests that 15N-urea found in the above in vivo spectra may be that accumulated in the hindgut. Thus, excess ammonium in the body causes the production of urea by the urea-cycle. In Samia larvae, urea was not reutilized but excreted. The metabolic relationships between the assimilation of ammonium and the function of the urea-cycle are discussed.

Regional specialization of ganglion cell layer of the chick retina.

Specialization of the ganglion cell layer (GCL) was studied by Nissl-staining and axonal tract-tracing methods in chicks and chick embryos. The changes in the retinal area and the cell number in the GCL produced a disparity in the cell density that occurred through the two different processes, cell generation (before embryonic days 10-14, E10-14) and cell loss (after E10-14). One high-density area was found in the retinal fundus on E8 (presumptive central area, pCA) and its density decreased toward the peripheral retina. Another high-density area was found in the dorsal retina on E11 (presumptive dorsal area, pDA). Cell densities of the pCA and the pDA on E11 decreased gradually to 25-30% by P1, and after that they further decreased to 40-60% by P30. The pCA was still identified on P30, but the pDA became very obscure by this age. In contrast, ganglion cell sizes increased 5-7 times in the pCA and pDA from E8 to P30, and increased 12 times in the temporal periphery. The present study suggests that the center-peripheral gradient of cell density results from lager scale of cell genesis in the pCA, but not from lager scale of cell loss in the peripheral retina. However, obscuration of the pDA results from equalization of cell density in cellular degeneration processes.

Immunocytochemical localization of serotonin, monoamine oxidase and assessment of monoamine oxidase activity in human dental pulp.

In the present study serotonin (5-hydroxytryptamine, 5-HT) and monoamine oxidase (MAO) were both found localized in the blood vessel walls of human dental pulp. Our discovery of MAO activity in human dental pulp suggests a functional relationship between serotonin and MAO in this region.

Changes in the retinotopical fiber order along the horizontal and dorsoventral axes of the nasal retina in the monkey optic chiasm.

The fiber arrangement of the retinogeniculate pathways was investigated in the Japanese monkey chiasm by iontophoretical injections into the lateral geniculate nucleus of wheat germ agglutinin conjugated to horseradish peroxidase. The present data demonstrated that the horizontal and dorsoventral axes of the nasal retina were rearranged in gross dorsoventral and posteroanterior orders in the chiasm. Thus, foveal/parafoveal fibers passed across the dorsal chiasm in a cluster and midperipheral nasal fibers passed across the central and ventral chiasms. Far-peripheral nasal fibers progressed in the ventral chiasm. Chiasmal fibers from the dorsal and ventral nasal retinas took pathways exactly similar to those from the midperipheral and the far-peripheral nasal retinas, respectively. In the anteroposterior direction, foveal/parafoveal fibers crossed the chiasmal midline extensively. However, midperipheral nasal fibers and dorsal nasal fibers crossed the posterior chiasm, and far-peripheral nasal fibers and ventral nasal fibers crossed the anterior chiasm. The correspondence of the retinotopical order in the chiasm with the chronological order is discussed.

Ommochrome deficiency in an albino strain of a terrestrial isopod, Armadillidium vulgare.

In order to clarify the cause of ommochrome deficiency in an albino strain of the terrestrial isopod, Armadillidium vulgare, levels of xanthommatin, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and tryptophan in whole body extracts of the albino and the wild type individuals were determined together with enzyme activities of kynurenine-3-hydroxylase, kynureninase and tryptophan-2,3-dioxygenase. Xanthommatin could not be detected in the albinos. The levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid were determined by high-performance liquid chromatography (HPLC) with electrochemical detection and were markedly low in the albinos compared with the wild type individuals. In contrast to those, the tryptophan levels determined by HPLC with fluorescence detection did not differ significantly between the two phenotypes. In the albino A. vulgare, kynurenine-3-hydroxylase activity was lower and kynureninase activity was higher than in the wild type, although the differences were not statistically significant. Tryptophan-2,3-dioxygenase activity in the albinos was less than 10% that in the wild type. Thus, ommochrome deficiency in the albino A. vulgare is considered to be caused by the extremely low activity of tryptophan-2,3-dioxygenase.

Morphological classification of ganglion cells in the central retina of chicks.

Classification of retinal ganglion cells (RGCs) in the chick central retina was studied by retrograde labeling of carbocyanine dye (DiI) and intracellular filling with Lucifer Yellow. Ganglion cells were divided into 4 groups, Group Ic/Is, Group IIc/IIs, Group IIIs, Group IVc, according to sizes of somal area and dendritic field and dendritic branching pattern. Group I cells had small somal area and small dendritic field. They were further divided into 2 subgroups by complexity (subgroup Ic) and simplicity (subgroup Is) of the dendritic arborization. Group II cells had medium-sized soma and dendritic field. They were also divided into subgroup IIc and IIs by the same definitions as those of subgroup Ic and Is. Group IIIs had medium-sized soma, large and simple dendritic arborization. Group IVc in which all cells had large soma, showed large and complex dendritic arborization. Cell populations of each group were 51.8% (subgroup Ic), 21.1% (subgroup Is), 6.2% (subgroup IIc), 14.6% (subgroup IIs), 4.2% (Group IIIs), and 2.1% (Group IVc). Subgroup Ic cells, which were very similar to beta-cells in the mammalian central area, represented about a half of the ganglion cell population. Cells in subgroup Is and IIs, which were not reported in the mammalian retina, were found in the chick central retina in relatively high population (35.7%). Morphological features of chick RGCs in the central retina were considered in comparison with those of other vertebrates.

Localization of sympathetic, parasympathetic and sensory neurons innervating the heart of the Beijing duck by means of the retrograde transport of horseradish peroxidase.

Sympathetic, parasympathetic and sensory neurons were labeled by injections of horseradish peroxidase into various regions of the heart in 33 Beijing ducks. Sympathetic postganglionic neurons innervating the heart were located in the paravertebral ganglia C15 (C16 is the last cervical segment in the duck) to T3, especially in the ganglion T1. The coronary sulcus and ventricle were more abundantly innervated by sympathetic neurons than the atrium. The left side of the heart was preferentially innervated by sympathetic postganglionic neurons in the left side of paravertebral ganglia but the right side of the heart were equally supplied from the right and left ganglia. Within the medulla oblongata, the number of labeled vagal preganglionic neurons in the nucleus ambiguus was much greater than that in the dorsal motor nucleus of the vagus nerve. Labeled neurons of the nucleus ambiguus were found in many ducks injected into the coronary sulcus. Cardiac sensory neurons were observed in the dorsal root ganglia C15 to T2 (highest in the ganglion T1) and in the nodose and jugular ganglia of the vagus nerve. These labeled neurons probably form the afferent and efferent limbs of cardiac reflexes and control circulation in the Beijing duck.