Search PubMed⌕ Search

Biomedical subjects

C Aoki

Publications and source records attributed to C Aoki.

At least 19 recordsLinked to original sources
PubMed1998

Preservation of ultrastructure and antigenicity for EM immunocytochemistry following intracellular recording and labeling of single cortical neurons in brain slices.

Knowledge of the distribution of neurotransmitters, neuromodulators, and transmitter receptors operating at specific synaptic sites on cortical neurons is essential for understanding the precise mechanisms that underlie the dynamic properties of cortical microcircuitry. We report on a new combination of techniques for analyzing chemically-specified synaptic input to individual cortical neurons first electrophysiologically characterized in the in vitro brain slice preparation. We tested the feasibility of this approach by performing intracellular recordings and biocytin injections in guinea pig medial prefrontal cortex slices and then by performing dual preembedding immunocytochemistry in order to localize neuronal nitric oxide synthase relative to single biocytin-filled neurons. The recorded cell and nitric oxide synthase immunoreactivity were visualized by light and electron microscopy utilizing both peroxidase and silver intensified gold stains. Single neurons were also dually visualized with fluorescence for light microscopy and with silver intensified gold for electron microscopy. Our findings indicate that both antigenicity and ultrastructure can be well preserved in tissue first used for in vitro slice experiments. This combination of methods should be widely applicable for analyzing the subcellular distribution of neuronal molecules such as receptors, channels and enzymes on physiologically characterized mammalian neurons.

Animals↗
PubMed1998

A method of combining biocytin tract-tracing with avidin-biotin-peroxidase complex immunocytochemistry for pre-embedding electron microscopic labeling in neonatal tissue.

A new method that allows the combination of avidin-biotin-peroxidase visualization of antigens and silver-intensified gold labeling of biocytin, a rapid tract-tracer, is described. The method provides a practical tool for in vivo and in vitro studies of chemically specified afferent-target relationships and particularly in developing neural pathways where biocytin is invaluable as a rapidly transporting, sensitive tracer requiring little permeabilizing agents. Transported biocytin was first visualized with silver-intensified colloidal gold conjugated to anti-biotin IgG. This was followed by blocking of all unbound biotin groups of biocytin in the tissue with an Avidin-Biotin blocking kit. Finally, a second antigen, neuronal nitric oxide synthase NOS or GluR2/3 subunit of AMPA receptors, was visualized selectively with avidin-biotin-peroxidase/DAB. This protocol allowed visualization of two chromagens that could be distinguished by electron microscopy. The presence of biocytin was evident by silver particles, while accumulation of peroxidase reaction product marked only the antibody labeling: no cross-reaction between biocytin and the avidin-biotin-peroxidase was observed.

Animals↗
PubMed1998

Stimulatory effects of 4-methylcatechol, dopamine and levodopa on the expression of metallothionein-III (GIF) mRNA in immortalized mouse brain glial cells (VR-2g).

Metallothionein (MT)-III, originally discovered as a growth inhibitory factor (GIF), is a brain specific isomer of MTs and is markedly reduced in the brain of Alzheimer's disease patients (AD) and in several other neurodegenerative diseases. We analyzed the level and regulation of mRNA expression of MT-III in immortalized fetal mouse brain glial cells (VR-2g) by reverse transcriptase-polymerase chain reaction (RT-PCR). The basal expression level of MT-III mRNA is very low in VR-2g cells. 4-Methylcatechol, dopamine (DA) and levodopa (l-3, 4-dihydroxyphenylalanine), which stimulate the synthesis of nerve growth factor (NGF), further increased the expression of MT-III mRNA in VR-2g cells.

Animals↗
PubMed1998

The subcellular distribution of nitric oxide synthase relative to the NR1 subunit of NMDA receptors in the cerebral cortex.

Results from several electrophysiological studies predict that the neuronal NO-synthesizing enzyme, nNOS, resides within spines formed by pyramid-to-pyramid axo-spinous synaptic junctions of the cortex. On the other hand, light microscopic neuroanatomical detection of nNOS within pyramidal neurons has been difficult, suggesting that these neurons contain nNOS at levels below threshold for detection. Our results obtained by electron microscopic immunocytochemistry indicate that nNOS occurs within spiny neurons, such as those of pyramidal neurons, albeit discretely within their spines. Dual electron microscopic immunocytochemistry, whereby antigenic sites to the NR1 subunit of NMDA receptors are probed simultaneously with sites immunoreactive for nNOS, reveals that some, although not all, nNOS within spines co-exist with NR1 subunits. Additionally, immunoreactivity for the NR1 subunit is detectable within nNOS-axons, indicating that NO may be generated in response to axo-axonic interactions with glutamatergic axons in the vicinity and independently of action potential propagation. Immunoreactivity for NR1 subunits within axons (with or without nNOS-immunoreactivity) may additionally serve to confer receptivity of these axons to NO generated coincidentally with activity. Analysis of the visual cortex of monocular adult animals indicates that the level of nNOS within neurites is dependent on chronic activity levels of the surrounding neuropil and independent of somatic input level. Together, these findings point to plasticity of nNOS neurons within adult brain tissue, involving regulation of subcellular nNOS distribution.

Animals↗
PubMed1998

Bone and calcium metabolism in Werner's syndrome.

In order to compare the bone aging process in Werner's syndrome, a disease characterized by premature aging, and that in natural aging, we have assessed the bone status in a total of 19 cases (11 males and eight postmenopausal females) with Werner's syndrome. The spinal deformity index was determined for a total of 87 vertebral bodies from nine male patients and for a total of 61 vertebral bodies from six female patients. In the male patients, 15 vertebral deformities among 87 vertebrae in seven patients were observed, and the incidences of patients bearing deformity and deformed vertebrae were 77.8 (7/9) and 17.2% (15/87), respectively. In the female patients, the corresponding incidences were 50.0% (3/6) and 4.9% (3/61), respectively. The chi2-values for the incidences of patients bearing deformity and deformed vertebrae in the male and the female patients were 1.25 (P=0.26) and 5.24 (P<0.03), respectively. The metacarpal cortical thickness (MCI) was also measured on hand X-ray films, and the Z scores for MCI in the male (n=9) and female (n=3) patients were -0.8+/-0.3 and -1.6+/-0.3, respectively. This gender-based difference in the MCI was not statistically significant but, the Z scores for MCI in both the males and the females were significantly smaller than those of corresponding age-matched controls (P<0.05 and 0.01, respectively). The serum levels of calcium, intact PTH, 25-OH vitamin D and 1,25(OH)2 vitamin D in three male and three female Werner's syndrome patients were within the reference ranges with one exception. The serum intact osteocalcin levels in the male (2.2+/-0.6 ng/ml) and female (2.6+/-0.8 ng/ml) patients were lower than the age-matched control values (P<0.05 for males) and the postmenopausal control values (P<0.05 for females). In conclusion, the incidence of spinal bone deformity was higher in males with Werner's syndrome than that in female patients. The cortical bone thickness decreased in both the male and the female patients. A low serum intact osteocalcin level was a characteristic finding in the male and in post-menopausal Werner's syndrome, so that the bone changes such as vertebral fracture incidence and the serum intact osteocalcin level in Werner's syndrome may differ from those in normal aging in the general population.

Adult↗
PubMed1997

The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide.

The major contribution of this paper is the finding of a glycolytic source of ATP in the isolated postsynaptic density (PSD). The enzymes involved in the generation of ATP are glyceraldehyde-3-phosphate dehydrogenase (G3PD) and phosphoglycerate kinase (PGK). Lactate dehydrogenase (LDH) is available for the regeneration of NAD+, as well as aldolase for the regeneration of glyceraldehyde-3-phosphate (G3P). The ATP was shown to be used by the PSD Ca2+/calmodulin-dependent protein kinase and can probably be used by two other PSD kinases, protein kinase A and protein kinase C. We confirmed by immunocytochemistry the presence of G3PD in the PSD and its binding to actin. Also present in the PSD is NO synthase, the source of NO. NO increases the binding of NAD, a G3PD cofactor, to G3PD and inhibits its activity as also found by others. The increased NAD binding resulted in an increase in G3PD binding to actin. We confirmed the autophosphorylation of G3PD by ATP, and further found that this procedure also increased the binding of G3PD to actin. ATP and NO are connected in that the formation of NO from NOS at the PSD resulted, in the presence of NAD, in a decrease of ATP formation in the PSD. In the discussion, we raise the possible roles of G3PD and of ATP in protein synthesis at the PSD, the regulation by NO, as well as the overall regulatory role of the PSD complex in synaptic transmission.

Actins↗
PubMed1997

NMDA-R1 subunit of the cerebral cortex co-localizes with neuronal nitric oxide synthase at pre- and postsynaptic sites and in spines.

The majority of nitric oxide's (NO) physiologic and pathologic actions in the brain has been linked to NMDA receptor activation. In order to determine how the NO-synthesizing enzyme within brain, neuronal NO synthase (nNOS), and NMDA receptors are functionally linked, previous studies have used in situ hybridization techniques in combination with light microscopic immunocytochemistry to show that the two are expressed within single neurons. However, this light microscopic finding does not guarantee that NMDA receptors are distributed sufficiently close to nNOS within single neurons to allow direct interaction of the two. Thus, in this study, dual immuno-electron microscopy was performed to determine whether nNOS and NMDA receptors co-exist within fine neuronal processes. We show that nNOS and the obligatory subunit of functional NMDA receptors, i.e. the NMDA-R1, co-exist within dendritic shafts, spines and terminals of the adult rat visual cortex. Axon terminals form asymmetric synaptic junctions with the dually labeled dendrites, suggesting that the presynaptic terminals release glutamate. Axons and dendrites expressing one without the other also are detected. These results indicate that it is possible for the generation of NO to be temporally coordinated with glutamatergic synaptic transmission at axo-dendritic and axo-axonic junctions and that NO may be generated independently of glutamatergic synaptic transmission. Together, our observations point to a greater complexity than previously recognized for glutamatergic neurotransmission, based on the joint versus independent actions of NO relative to NMDA receptors at pre- versus postsynaptic sites.

Animals↗
PubMed1997

Postnatal changes in the laminar and subcellular distribution of NMDA-R1 subunits in the cat visual cortex as revealed by immuno-electron microscopy.

Although it is recognized that nearly all synapses in the cerebral cortex form postnatally, little is known about the emergence of molecules necessary to render these synapses functional. This study visualized the emergence of synaptically localized NMDA receptors by immuno-electron microscopic labeling of the receptor's obligatory subunit, NMDA-R1, in the developing cat visual cortex. Prior to eye-opening (postnatal day 2-10), NMDA-R1 immunoreactivity is already present within dendritic and growth cones, even though these profiles are devoid of synaptic specializations. This indicates that synthesis and incorporation of NMDA-R1 into plasma membranes are independent of form vision. During the next 2-3 weeks, i.e., preceding the onset of the critical period for ocular dominance plasticity (around the fourth week), NMDA-R1 immunoreactivity changes from a diffuse distribution within dendrites to a more discrete aggregation over postsynaptic densities of axo-spinous junctions. Such clustering of NMDA-R1 at synapses may be a prerequisite for stabilization and strengthening of synapses activated by visual stimulation during the critical period. Furthermore, only during the first several weeks, intensely NMDA-R1-immunoreactive neurons are present in the infragranular layers and the white matter. Enrichment of NMDA-R1 in the deep-layer neurons may reflect the neurons' supportive role in the development of cortical circuitry, serving as transient synaptic targets for geniculate and cortico-cortical afferents while these afferents 'wait' in the infragranular for their ultimate, life-long target neurons to become receptive in the upper layers.

Animals↗
PubMed1997

Insulin-like growth factors I and II are autocrine factors in stimulating proteoglycan synthesis, a marker of differentiated chondrocytes, acting through their respective receptors on a clonal human chondrosarcoma-derived chondrocyte cell line, HCS-2/8.

Both insulin-like growth factor (IGF)-I and IGF-II increased the synthesis of cartilage-type, large proteoglycan in a human chondrosarcoma-derived chondrocyte cell line, HCS-2/8. In contrast to the stimulatory effects of IGFs on costal chondrocytes of the young rabbit, the stimulatory effect of IGF-II on proteoglycan synthesis in HCS-2/8 cells was more potent than that of IGF-I. IGF-II, but not IGF-I, increased calcium influx into HCS-2/8 cells, and there was a close relation between the stimulation of proteoglycan synthesis and the calcium influx. [125I]IGF-I bound to HCS-2/8 cells, and this binding was competitively inhibited by low concentrations of unlabeled IGF-I, higher concentrations of IGF-II, and much higher concentrations of insulin. [125I]IGF-II also bound to the cells, and its binding was competitively inhibited by IGF-II and slightly inhibited by higher concentrations of IGF-I and much higher concentrations of insulin. When radioligand-receptor complexes were separated by SDS-PAGE and subjected to autoradiography, two major bands at 260 and 130 kDa were observed, which correspond to the IGF type II receptor (IGF-IIR) and the alpha subunit of the IGF type I receptor (IGF-IR), indicating the presence of both receptors. When confluent cultures of HCS-2/8 cells were maintained in serum-free medium, proteoglycan synthesis did not decrease unless the medium was repeatedly replaced. Conditioned medium of HCS-2/8 cells stimulated the HCS-2/8 cells to synthesize proteoglycans. RIA revealed that the cells produced both IGF-II and IGF-I. Transcripts of messenger RNAs of both IGF-I and IGF-II and both IGF-IR and IGF-IIR also were detectable by Northern analysis. Both anti-IGF-IR antibody and anti-IGF-II antibody inhibited proteoglycan synthesis. Mannose-6-phosphate, which is known to bind to IGF-IIR, stimulated proteoglycan synthesis, potentiated IGF-II-stimulated proteoglycan synthesis, and enhanced the binding affinity for IGF-II but not for IGF-I. Even in the presence of anti-IGF-IR antibody, IGF-II and mannose-6-phosphate stimulated proteoglycan synthesis in the cells. [Leu27]IGF-II, an IGF-II analogue with high affinity only for IGF-IIR, strongly stimulated proteoglycan synthesis in HCS-2/8 cells but [Arg54, Arg55]IGF-II, which binds to only IGF-IR, also stimulated proteoglycan synthesis in the cells. These findings indicate that IGF-I and IGF-II act as autocrine differentiation factors for this chondrocytic permanent cell line, HCS-2/8, mainly via respective receptors.

Analysis of Variance↗
PubMed1997

Association of bone mineral density with apolipoprotein E phenotype.

The phenotypes of apolipoprotein E (Apo E) and their relationship with the bone mineral density (BMD) were examined in 284 unrelated postmenopausal Japanese women aged 47-82 years (64.0 +/- 1.0 years, mean +/- SE). The Apo E phenotype was analyzed by the isoelectric focusing method, followed by immunoblotting. The relationship between the Apo E phenotype and the vitamin D receptor (VDR) gene or estrogen receptor (ER) gene genotypes was also studied in the same population. The Apo E phenotypic frequencies in our population were 9.9% for E3/2, 66.5% for E3/3, 1.8% for E4/2, 19.7% for E4/3, and 2.1% for E4/4. We classified these phenotypes into three categories: Apo E4-/- (E3/2 and E3/3, n = 217, Apo E4 +/- (E4/3 and E4/2, n = 61), and Apo E4+/+ (E4/4, n = 6). The age, body weight, body height, and years since menopause were not significantly different among these three categories. The lumbar BMD values in these three groups were significantly different in the order of E4-/- (0.91 +/- 0.01 g/cm2), E4 +/- (0.85 +/- 0.02 g/cm2), and E4+/+ (0.83 +/- 0.06 g/cm2) (p = 0.031). The same trend was also observed for the Z score of the total BMD (p = 0.022). The serum level of intact osteocalcin in E4+/+ (15.2 +/- 5.7 ng/ml) was higher than in E4-/- (7.7 +/- 0.3 ng/ml) or E4 +/- (7.7 +/- 0.7 ng/ml) (p = 0.004 by analysis of variance). However, there were no other significant differences in the serum or urinary levels of bone turnover markers. Serum cholesterol in the E4+/+ group tended to be higher than in the other two groups (p = 0.05). There were no significant associations of the VDR and ER genotypes with the Apo E4 phenotype. A multivariate linear regression analysis revealed Apo E4 to be a significant, independent predictor of the Z score of the lumbar BMD. The effect of the Apo E4 allele on the Z score of the lumbar BMD (-0.493 +/- 0.152) was not significantly different from that in the AAB of VDR (-0.616 +/- 0.225) or PPxx of ER (-0.785 +/- 0.314). In conclusion, the Apo E4 allele is associated with a low bone mass in postmenopausal Japanese.

Aged↗
PubMed1996

Reduced sympathetic innervation after alteration of target cell neurotransmitter phenotype in transgenic mice.

Neurotransmitters play a variety of important roles during nervous system development. In the present study, we hypothesized that neurotransmitter phenotype of both projecting and target cells is an important factor for the final synaptic linkage and its specificity. To test this hypothesis, we used transgenic techniques to convert serotonin/melatonin-producing cells of the pineal gland into cells that also produce dopamine and investigated the innervation of the phenotypically altered target cells. This phenotypic alteration markedly reduced the noradrenergic innervation originating from the superior cervical ganglia. Although the mechanism by which the reduction occurs is presently unknown, quantitative enzyme-linked immunoassay showed the presence of the equivalent amounts of nerve growth factor (NGF) in the control and transgenic pineal glands, suggesting that it occurred in a NGF-independent manner. The results suggest that target neurotransmitter phenotype influences the formation of afferent connections during development.

Animals↗
PubMed1996

Cellular and subcellular distribution of alpha 2A-adrenergic receptors in the visual cortex of neonatal and adult rats.

Activation of alpha 2-adrenergic receptors (alpha 2AR) in the cerebral cortex has been shown to modulate visually guided delayed response tasks as well as anxiety and depression. We used an antiserum directed specifically against the A subtype of alpha 2AR (alpha 2AAR) to determine the cell types and subcellular sites for noradrenergic reception mediated by this receptor in the adult and the developing rat visual cortices. Light microscopic examination of adult tissue revealed numerous labeled perikarya in layers II-VI, many of which appeared distinctly pyramidal. A few perikarya in layer I also were immunoreactive. In all layers, alpha 2AAR immunoreactivity (alpha 2AAR-ir) was present within proximal dendrites and fine processes. In neonatal tissue, there was an intense, distinct band of immunoreactivity spanning the layer composed of tightly packed immature cell bodies, i.e., the cortical plate. The band dissipated as this tier differentiated postnatally into the supragranular layers. Electron microscopy showed that the supragranular layers, which contain the highest density of noradrenergic fibers, also contain the highest areal density of labeled postsynaptic junctions beyond 2 weeks of age. Throughout the ages, the majority of immunoreactivity occurred at sites which, in single ultrathin sections, appeared to be nonjunctional sites of axons, dendrites, and in glial processes. Our observations indicate that (1) both pyramidal and nonpyramidal neurons are receptive to norepinephrine via alpha 2AAR, (2) alpha 2AAR synthesis is robust prior to synaptogenesis, and (3) alpha 2AAR operates both pre- and postsynaptically.

Animals↗
PubMed1996

Second case of zoonotic Onchocerca infection in a resident of Oita in Japan.

A non-gravid female Onchocerca was found in histopathological sections of a biopsy specimen taken from a painful nodule in the wrist of a 57-year old woman in Oita, in southern Japan. Six species of Onchocerca have been found in animals in Japan: two in wild bovids, one in equids, and three in domestic bovids of which one, Onchocerca sp., is only known by the microfilaria and infective stage. Distinctive morphological features of the worm, including a three-layered thick cuticle with prominent annular ridges at wide intervals, high somatic muscles and narrow lateral chords, resembled those of O. gutturosa, one of the three bovine Onchocerca species transmitted in the Oita region. However Onchocerca sp., which is also transmitted in this region, cannot be excluded. An ELISA test of the patient serum suggests that infections by Onchocerca spp. might be distinguished from those by Dirofilaria immitis, of which the number of human cases is increasing in Japan.

Animals↗
PubMed1995

Differential localization of NMDA and AMPA receptor subunits in the lateral and basal nuclei of the amygdala: a light and electron microscopic study.

Anatomical and physiological studies indicate that the amino acid L-glutamate is the excitatory transmitter in sensory afferent pathways to the amygdala and in intraamygdala circuits involving the lateral and basal nuclei. The regional, cellular, and subcellular immunocytochemical localizations of N-methyl-D-aspartate (NMDA) and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), two major classes of glutamate receptors, were examined in these areas of the amygdala. A monoclonal antibody and a polyclonal antiserum directed against the R1 subunit of the NMDA receptor were used. Each immunoreagent produced distinct distributions of perikaryal and neuropilar staining. Dendritic immunoreactivity was localized primarily to asymmetric (excitatory) synaptic junctions, mostly on spines, consistent with the conventional view of the organization and function of NMDA receptors. Whereas the anti-NMDAR1 antiserum produced sparse presynaptic axon terminal labeling and extensive glial labeling, the anti-NMDAR1 antibody labeled considerably fewer glia and many more presynaptic axon terminals. Labeled presynaptic terminals formed asymmetric and symmetric synapses, suggesting presynaptic regulation of both excitatory and inhibitory transmission. Immunoreactivity for different subunits of the AMPA receptor (GluR1, GluR2/3, and GluR4) was uniquely distributed across neuronal populations, and some receptor subunits were specific to certain cell types. Immunoreactivity for GluR1 and Glu2/3 was predominantely localized to dendritic shafts and was more extensive than that of GluR4 due to heavy labeling of proximal portions of dendrites. The distribution of GluR4 immunoreactivity was similar to NMDAR1: GluR4 was seen in presynaptic terminals, glia, and dendrites and was primarily localized to spines. The presynaptic localization of GluR4 in the absence of GluR2 suggests glutamate-mediated modulation of presynaptic Ca++ concentrations. These data add to our understanding of the morphological basis of pre- and postsynaptic transmission mechanisms and synaptic plasticity in the amygdala.

Amygdala↗
PubMed1995

The earliest events in vesicular stomatitis virus infection of the murine olfactory neuroepithelium and entry of the central nervous system.

After intranasal instillation of mice with vesicular stomatitis virus (VSV), olfactory receptor neurons are infected. By 12 to 24 hr postinfection, VSV antigens are observed in adjoining supporting and basal cells and in other structures of the olfactory epithelium and lamina propria. Peripheral deafferentation of the olfactory epithelium with Triton X-100 or bilateral surgical bulbectomy does not prevent spread of VSV to the central nervous system (CNS); the route of spread differs considerably from the route taken when the olfactory nerve is intact. In contrast to rabies virus and HSV-1, VSV does not use the trigeminal nerve for entry into the brain, as the trigeminal ganglion remains virus-free following intranasal infection. These results indicate that VSV has a strong tropism for olfactory receptor cells, using them for entry into the CNS. Both retrograde and anterograde transneuronal and nonneuronal (ependymal cells and cerebrospinal fluid) pathways are utilized by VSV within the CNS.

Animals↗
PubMed1995

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors.

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with 125I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with Kd values of 1 x 10(-10) M and 5 x 10(-9) M. The numbers of high- and low-affinity receptors were 1 x 10(4) and 2 x 10(5) per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-beta 1, IL-1 beta, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.

Animals↗