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Kazuto Kobayashi

Publications and source records attributed to Kazuto Kobayashi.

At least 19 recordsLinked to original sources
PubMed2006

Selective loss of nigral dopamine neurons induced by overexpression of truncated human alpha-synuclein in mice.

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and presence of Lewy bodies, whose major component is alpha-synuclein. In the present study, we generated transgenic mice termed Syn130m that express truncated human alpha-synuclein (amino acid residue number: 1-130) in dopaminergic neurons. Notably, dopaminergic neurons were selectively diminished in the substantia nigra pars compacta of Syn130m, while transgenic mice that expressed comparable amount of full-length human alpha-synuclein did not develop such pathology. Therefore, the truncation of human alpha-synuclein seems to be primarily responsible for the loss of nigral dopaminergic neurons. The nigral pathology resulted in impairment of axon terminals in the striatum and concomitant decrease in striatal dopamine content. Behaviorally, spontaneous locomotor activities of Syn130m were reduced, but the abnormality was ameliorated by treatment with L-DOPA. The loss of nigral dopaminergic neurons was not progressive and seemed to occur during embryogenesis along with the onset of expression of the transgene. Our results indicate that truncated human alpha-synuclein is deleterious to the development and/or survival of nigral dopaminergic neurons.

Animals↗
PubMed2006

Spontaneous recanalization at subacute phase of stroke may be dramatic: A case report.

Spontaneous recanalization of the occluded cerebral arteries has occasionally occurred in an acute phase of stroke patients. We report a stroke patient with recanalization at 7 days of onset detected by using continuous transcranial Doppler (TCD) monitoring. After the presence of microembolic signals on TCD display, dynamic flow changes of the middle cerebral artery (MCA) occurred, which represented recanalization of the occluded MCA.

Adult↗
PubMed2006

Decline of striatal dopamine release in parkin-deficient mice shown by ex vivo autoradiography.

Parkin is the causal gene of autosomal recessive juvenile parkinsonism (AR-JP). Dopamine (DA) metabolism has been linked to Parkinson's disease (PD). To understand the pathogenesis of AR-JP, we generated parkin-deficient mice to assess the status of DA signaling pathway and examine DA release and DA receptor by ex vivo autoradiography. Ex vivo autoradiography using [11C]raclopride showed a clear decrease in endogenous DA release after methamphetamine challenge in parkin-deficient mice. Furthermore, parkin deficiency was associated with considerable upregulation of DA (D1 and D2) receptor binding in vivo in the striatum and increased DA levels in the midbrain. Our results suggest that dopaminergic neurons could behave abnormally before neuronal death.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2006

Ultrasonic speed microscopy for imaging of coronary artery.

We have been developing a scanning acoustic microscope (SAM) system for medicine and biology featuring quantitative measurement of ultrasonic speed and attenuation of soft tissues. In the present study, we will propose a new concept ultrasonic speed microscopy that can measure the thickness and ultrasonic speed using fast Fourier transform of a single pulsed wave instead of continuous waves used in conventional SAM systems. Six coronary arteries were frozen and sectioned approximately 10 microm in thickness. They were mounted on glass slides without cover slips. The scanning time of a frame with 300 x 300 pixels was 121 s and two-dimensional distribution of ultrasonic speed was obtained. The ultrasonic speed was 1720 m/s in the thickened intima with collagen fiber, 1520 m/s in lipid deposition underlying fibrous cap and 1830 m/s in calcified lesion in the intima. These basic measurements will help understanding echogenecity in intravascular ultrasound (IVUS) images. Imaging of coronary artery with the ultrasonic speed microscopy provides important information for study of IVUS coronary imaging.

Algorithms↗
PubMed2006

Separation of mu-opioid receptor desensitization and internalization: endogenous receptors in primary neuronal cultures.

A close relationship between desensitization and internalization of mu-opioid receptors (MORs) has been proposed based on differential actions of series of agonists. The role that these two processes have in the development of tolerance and dependence to opioids has been a controversial subject that has been studied in a variety of model systems. Here, we examine desensitization and internalization of endogenous MORs simultaneously in primary cultures of locus ceruleus neurons using fluorescently tagged peptide agonists. With the use of two fluorescent opioid peptides, dermorphin-Bodipy Texas Red and dermorphin-Alexa594 (Derm-A594), desensitization was measured electrophysiologically and trafficking was followed by the accumulation of intracellular fluorescent puncta. Blocking endocytosis with concanavalin A eliminated the accumulation of fluorescent puncta but desensitization induced by Derm-A594 was unaffected. Likewise, after treatment with concanavalin A, there was no change in either desensitization or recovery from desensitization induced by [Met]5enkephalin. The results demonstrate that desensitization and the recovery from desensitization are not dependent on receptor internalization and suggest that the activity of endogenous MORs in primary neurons can be modulated at the level of the plasma membrane.

Animals↗
PubMed2006

Granulocyte-colony stimulating factor is neuroprotective in a model of Parkinson's disease.

We have recently shown that the hematopoietic Granulocyte-Colony Stimulating Factor (G-CSF) is neuroprotective in rodent stroke models, and that this action appears to be mediated via a neuronal G-CSF receptor. Here, we report that the G-CSF receptor is expressed in rodent dopaminergic substantia nigra neurons, suggesting that G-CSF might be neuroprotective for dopaminergic neurons and a candidate molecule for the treatment of Parkinson's disease. Thus, we investigated protective effects of G-CSF in 1-methyl-4-phenylpyridinium (MPP+)-challenged PC12 cells and primary neuronal midbrain cultures, as well as in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Substantial protection was found against MPP+-induced dopaminergic cell death in vitro. Moreover, subcutaneous application of G-CSF at a dose of 40 microg/Kg body weight daily over 13 days rescued dopaminergic substantia nigra neurons from MPTP-induced death in aged mice, as shown by quantification of tyrosine hydroxylase-positive substantia nigra cells. Using HPLC, a corresponding reduction in striatal dopamine depletion after MPTP application was observed in G-CSF-treated mice. Thus our data suggest that G-CSF is a novel therapeutic opportunity for the treatment of Parkinson's disease, because it is well-tolerated and already approved for the treatment of neutropenic conditions in humans.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2006

Dynamic temporal and cell type-specific expression of Wnt signaling components in the developing midbrain.

Wnt1 and -5a have been shown to modulate the proliferation and differentiation of midbrain dopaminergic (DA) neurons. However, it is not known whether other Wnts or which Frizzled (Fz) receptors are expressed in the developing midbrain. We found that 13 out of 19 Wnts, all 10 Fzs, and several intracellular Wnt signaling modulators, including Axin, FRAT, Naked, Par-1, and Ltap are developmentally regulated between embryonic days (E) 10.5 and 15.5. Next, we studied whether Fzs are differentially expressed in different cell types and examined neuronal-progenitor- or glial-enriched cultures and DA neurons isolated from TH-GFP reporter mice. We found that Fz8 is expressed at high levels in DA neurons at E11.5 and E13.5. Fz6 and -7 are the predominant transcripts in glial precursors, and Fz9, which is absent in DA neurons at E11.5, is the main receptor expressed in neuronal precursors. We therefore examined the function of Fz9 in DA cells and found that overexpression of Fz9 reduced Wnt5a- but not Wnt3a-induced hyperphosphorylation of Dishevelled. Thus, our results show that Fzs are developmentally regulated and differentially expressed in VM precursors, DA neurons, and glia. These findings suggest that Fz expression contributes to provide specificity to Wnt-mediated effects.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Altered gene expression in the subdivisions of the amygdala of Fyn-deficient mice as revealed by laser capture microdissection and mKIAA cDNA array analysis.

Fyn-tyrosine-kinase-deficient mice exhibit increased fearfulness and display enhanced excitability in the amygdala. To gain insight into the molecular changes associated with the increased excitability of the amygdala, we used a newly developed cDNA array system comprising mouse KIAA cDNA clones to identify novel genes differentially expressed in the amygdala of fyn(-/-) and fyn(+/-) mice following administration of N-methyl-D-aspartate (NMDA). Laser capture microdissection in combination with PCR-based cDNA amplification allowed us to analyze gene expression in each amygdalar subdivision. The statistical significance of the differential expressions was tested by one-way analysis of variance (ANOVA) by the false discovery rate controlling approach. Among the 805 mKIAA cDNA clones tested, only the expression level of mKIAA1577 (Zinc finger SWIM domain containing protein 6; gene name, Zswim6) showed statistically significant change in regard to the genotype and amygdalar subdivision. Namely, only the lowered expression of mKIAA1577 in the central nucleus of fyn(-/-) mice 1 h after NMDA administration (2.1-fold lower relative to fyn(+/-) mice) was statistically significant. In situ hybridization analysis confirmed the downregulation of the mRNA in the central nucleus of the fyn(-/-) mice 1 h after NMDA administration (3.2-fold lower relative to fyn(+/-) mice). The NMDA-induced change in gene expression was partially blocked by the NMDA antagonist D-AP-5. These results suggest that Fyn deficiency was responsible for the NMDA-induced downregulation of a specific gene in the amygdalar central nucleus.

2-Amino-5-phosphonovalerate↗
PubMed2005

Subthalamic neurons coordinate basal ganglia function through differential neural pathways.

The subthalamic nucleus (STN) is a key component of basal ganglia circuitry that mediates a variety of motor functions. The STN neurons send glutamatergic projections to the output structures of basal ganglia, including the substantia nigra pars reticulata (SNr) and the entopeduncular nucleus, and also innervate the globus pallidus (GP). However, the mechanism by which the STN regulates motor functions in the neural circuitry is not fully understood. Here we performed conditional ablation of the STN neurons by using immunotoxin-mediated cell targeting. We then analyzed dopamine (DA)-mediated motor behavior and firing activity of the SNr and GP neurons. Ablation of the STN neurons increased spontaneous movement and reduced hyperactivity in response to DA stimulation. Ablation of these neurons modulated the pattern and rate of spontaneous firing of the SNr neurons, although it did not substantially affect spontaneous firing of the GP neurons. The ablation attenuated DA-induced suppression of the firing rate of the SNr neurons and inhibited DA-induced elevation of the rate of the GP neurons. In addition, pharmacological blockade of GP activation in response to DA stimulation inhibited the suppression of SNr activity and the resultant motor activation. These results suggest that the STN neurons suppress spontaneous behavior through their direct projection to the output neurons and that, in response to DA, they contribute to expression of behavior by acting on the output neurons mainly through the GP-mediated pathways. We conclude that the STN coordinates motor behavior through differential neural pathways depending on the state of DA transmission.

Action Potentials↗
PubMed2005

Molecular genetics of tyrosine 3-monooxygenase and inherited diseases.

Tyrosine 3-monooxygenase (tyrosine hydroxylase, TH) catalyzes the initial and rate-limiting step in the catecholamine biosynthesis. Alteration in TH activity is involved in the pathogenesis of certain disorders derived from catecholaminergic dysfunction. In the present review, we focus on recent advances in molecular genetic study of TH function and inherited diseases. Knockout mice lacking TH gene show severe catecholamine depletion and perinatal lethality. Mice heterozygous for the TH mutation exhibit defects in some neuropsychological functions. Dopamine-deficient mice impair motor control and operant learning during postnatal development. In addition, some point mutations in the human TH gene underlie the inherited diseases, including the recessive form of L-DOPA-responsive dystonia, parkinsonism in infancy, or progressive encephalopathy. These mutations indeed appear to reduce TH activity or influence expression of TH protein. Advances in molecular genetic studies provide a deeper understanding of the relationship between the alteration in TH activity and the pathology of catecholaminergic systems.

Animals↗
PubMed2005

Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.

This study aimed to generate a mouse model of acquired glomerular sclerosis. A model system that allows induction of podocyte injury in a manner in which onset and severity can be controlled was designed. A transgenic mouse strain (NEP25) that expresses human CD25 selectively in podocytes was first generated. Injection of anti-Tac (Fv)-PE38 (LMB2), an immunotoxin with specific binding to human CD25, induced progressive nonselective proteinuria, ascites, and edema in NEP25 mice. Podocytes showed foot process effacement, vacuolar degeneration, detachment and downregulation of synaptopodin, WT-1, nephrin, and podocalyxin. Mesangial cells showed matrix expansion, increased collagen, mesangiolysis, and, later, sclerosis. Parietal epithelial cells showed vacuolar degeneration and proliferation, whereas endothelial cells were swollen. The severity of the glomerular injury was LMB2 dose dependent. With 1.25 ng/g body wt or more, NEP25 mice developed progressive glomerular damage and died within 2 wk. With 0.625 ng/g body wt of LMB2, NEP25 mice survived >4 wk and developed focal segmental glomerular sclerosis. Thus, the study has established a mouse model of acquired progressive glomerular sclerosis in which onset and severity can be preprogrammed by experimental maneuvers.

Animals↗
PubMed2005

Functional properties of dopaminergic neurones in the mouse olfactory bulb.

The olfactory bulb of mammals contains a large population of dopaminergic interneurones within the glomerular layer. Dopamine has been shown both in vivo and in vitro to modulate several aspects of olfactory information processing, but the functional properties of dopaminergic neurones have never been described due to the inability to recognize these cells in living preparations. To overcome this difficulty, we used a transgenic mouse strain harbouring an eGFP (enhanced green fluorescent protein) reporter construct under the promoter of tyrosine hydroxylase, the rate-limiting enzyme for cathecolamine synthesis. As a result, we were able to identify dopaminergic neurones (TH-GFP cells) in living preparations and, for the first time, we could study the functional properties of such neurones in the olfactory bulb, in both slices and dissociated cells. The most prominent feature of these cells was the autorhythmicity. In these cells we identified five main voltage-dependent conductances: the two having largest amplitude were a fast transient Na(+) current and a delayed rectifier K(+) current. In addition, we observed three smaller inward currents, sustained by Na(+) ions (persistent type) and by Ca(2)(+) ions (LVA and HVA). Using pharmacological tools and ion substitution methods we showed that the pacemaking process is supported by the interplay of the persistent Na(+) current and of a T-type Ca(2)(+) current. We carried out a complete kinetical analysis of the five conductances present in these cells, and developed a Hodgkin-Huxley model of TH-GFP cells, capable of reproducing accurately the properties of living cells, including autorhytmicity, and allowing a precise understanding of the process.

Animals↗
PubMed2005

Sound speed scanning acoustic microscopy for biomedical applications.

Since 1985, we have been developing a scanning acoustic microscope (SAM) system for biomedical use and have been investigating the acoustic properties of various organs and disease states by using this SAM system. In biomedicine, SAM is useful for intraoperative pathological examination, study of low-frequency ultrasonic images, and assessment of biomechanics at a microscopic level. Recently, we have proposed a new concept -- acoustic microscopy -- using a single pulsed wave instead of continuous waves used in conventional SAM systems. In the present study, we compared two systems by measuring the same biological material. The sound speed image obtained by sound speed microscopy corresponded well to that obtained using a conventional SAM system. Lesions with hyaline degeneration showed a lower sound speed when compared with that of normal myocardium. Frequency domain analysis of amplitude and phase by both methods also showed similar characteristics. Although the data acquisition time of one frame was greater than that in conventional SAM, the total time required for calculation was significantly shorter. The SAM system can be applied to intraoperative pathological examination.

Animals↗
PubMed2005

Ultrasonic tissue characterization of photodamaged skin by scanning acoustic microscopy.

The aim of this study was to ultrasonically characterize photodamaged skin of the elderly at the microscopic level using scanning acoustic microscopy which showed two-dimentional distribution of sound speed in the skin section. We confirmed that the expression level of the elastin gene was increased in the preauricular skin (photodamaged area), compared with postauricular skin (photo-protected area). The expression level of the procollagen gene was also increased in the preauricular skin compared with postauricular skin. The preauricular skin showed higher sound speed in the papillary dermis (Grenz zone). The site of progressive solar elastosis showed a somewhat sound speed velocity than that of the Grenz zone. Immunohistochemical staining showed conserved deposition of collagen in the Grenz zone even in the more photodamaged preauricular skin. These results suggest that fibrosis in the Grenz zone compensates tissue strength with the progress of solar elastosis. The sound speed analysis of skin will provide important information on heterogeneous mechanical changes in the skin during the process of photoaging.

Aged↗
PubMed2004

Differentiation of the dopaminergic phenotype in the olfactory system of neonatal and adult mice.

Olfactory bulb (OB) interneurons are derived primarily postnatally from progenitors in the anterior subventricular zone (SVZa) and migrate to the OB in the rostral migratory stream (RMS). Progenitors differentiate into phenotypically diverse granule and periglomerular cells by as yet undefined mechanisms. To visualize spatiotemporal aspects of periglomerular dopamine (DA) neuron differentiation, two independently derived transgenic mouse lines were analyzed with a 9-kb tyrosine hydroxylase (TH) promoter to drive either a LacZ or an enhanced green fluorescent protein (EGFP) reporter gene. Both reporters showed similar neonatal expression that varied from low levels in RMS, to moderate in the superficial granule cell layer, to strong in relatively large cells, possibly external tufted cells, in the periglomerular region. TH mRNA and protein were not detected in the RMS but were colocalized with the transgenes in neonatal superficial granule and periglomerular cells. By comparison, TH protein in adults was further limited to periglomerular cells. To demonstrate that transcriptional regulation was the same for EGFP and TH, expression was shown to decline similarly in the OB ipsilateral to odor deprivation produced by adult unilateral naris closure. Of two genes previously hypothesized to regulate OB DA expression, only regulated expression of the orphan receptor Nurr1, but not the homeobox-containing genes Dlx-1 and -2, was consistent with a role in regulation of the DA phenotype. These data demonstrate for the first time that DA phenotypic differentiation in neonates begins with low-level transcription in migrating progenitors in the RMS and culminates with activity-dependent protein expression in periglomerular cells innervated by olfactory receptor cells.

Animals↗
PubMed2004

Fate of transient catecholaminergic cell types revealed by site-specific recombination in transgenic mice.

Catecholamine-producing cell types are generated from specified neuronal lineages during vertebrate development. The catecholaminergic phenotype is also expressed transiently in some cell types in non-catecholaminergic tissues, including the sensory ganglia, enteric ganglia, and ventral portions of the neural tube during embryonic development. The fate of the transient catecholaminergic cell types at later developmental stages, however, has not been elucidated. We developed a Cre-loxP-mediated recombination system under the control of the dopamine beta-hydroxylase (DBH) promoter, which drives gene expression in typical noradrenergic and adrenergic cell groups as well as in transient catecholaminergic cell types. Expression of Cre recombinase in transgenic mice resulted in an efficient recombination in noradrenergic and adrenergic cell groups at the adult stage. The recombination was also induced in the cranial nerve/spinal cord motor neurons and sensory/enteric ganglion neurons. Analysis of recombination patterns in transgenic mouse embryos showed the occurrence of recombination during prenatal development in both cell types exhibiting the typical and transient catecholaminergic phenotypes. Because the DBH gene promoter is expressed transiently in the ventral neural tube and sensory ganglion during embryonic development, our results provide evidence that the cell types showing a transient catecholaminergic phenotype in these tissues are destined to become mature motor neurons or sensory ganglion neurons during subsequent differentiation.

Animals↗
PubMed2004

Isolation and transplantation of dopaminergic neurons generated from mouse embryonic stem cells.

Embryonic stem (ES) cells differentiate into dopamine (DA)-producing neurons when co-cultured with PA6 stromal cells, but the resulting cultures contain a variety of unidentified cells. In order to label live DA neurons in mixed populations, we introduced a GFP reporter under the control of the tyrosine hydroxylase (TH) gene promoter into ES cells. GFP expression was observed in TH-immunoreactive cells that differentiated from the ES cells that carried the TH-GFP reporter gene. DA neurons expressing GFP were sorted from the mixed cell population by fluorescence-activated cell sorting of cells exhibiting GFP fluorescence, and the sorted GFP(+) cells obtained were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a partial recovery from parkinsonian behavioral defects. This strategy of isolation and transplantation of ES-cell-derived DA neurons should be useful for cellular and molecular studies of DA neurons and for clinical application in the treatment of Parkinson's disease.

Animals↗