Biomedical subjects

Kazuto Kobayashi

Publications and source records attributed to Kazuto Kobayashi.

26 records · Page 2Linked to original sources

PubMed2004

Survival of developing motor neurons mediated by Rho GTPase signaling pathway through Rho-kinase.

A variety of neurons generated during embryonic development survive or undergo programmed cell death (PCD) at later developmental stages. Survival or death of developing neurons is generally considered to depend on trophic support from various target tissues. The small GTPase Rho regulates diverse cellular processes such as cell morphology, cell adhesion, cell motility, and apoptosis. Rho-dependent serine-threonine protein kinase (Rho-kinase-ROK-ROCK), one of the effector proteins, transmits signals for some Rho-mediated processes. Here, we report the in vivo role of the Rho signaling pathway through Rho-kinase during development of motor neurons (MNs) in the spinal cord. We performed conditional expression of a dominant-negative form for RhoA (RhoA DN) or for Rho-kinase (Rho-K DN) in transgenic mice by using the Cre-loxP system to suppress the activity of these signaling molecules in developing MNs. Expression of RhoA DN reduced the number of MNs in the spinal cord because of increased apoptosis while preserving the gross patterning of motor axons. Expression of Rho-K DN produced developmental defects similar to those observed in RhoA DN expression. In addition, analysis of transgenic mice expressing Rho-K DN showed that the increased apoptosis of MNs was induced at the early embryonic stages before the initiation of PCD, and that MN death at the late embryonic stages corresponding to the period of PCD was moderately enhanced in the transgenic mice. These findings indicate that the Rho signaling pathway, primarily through Rho-kinase, plays a crucial role in survival of spinal MNs during embryogenesis, particularly at the early developmental stages.

Animals↗

PubMed2004

Development of an ultra-portable echo device connected to USB port.

In practical cardiology, a stethoscope based auscultation has been used to reveal the patient's clinical status. Recently, several hand-held echo devices are going on market and they are expected to play a role as "visible" auscultation instead of stethoscope. We have developed a portable and inexpensive echo device which can be used for screening of cardiac function. Two single element transducers were attached 180 degrees apart to a rotor with 14-mm diameter. The mechanical scanner, integrated circuits for transmitting and receiving ultrasonic signals and an A/D converter were encapsulated in a 150 x 40 mm probe weighing 200 g. The scan was started and the image was displayed on a Windows based personal computer (PC) as soon as the probe was connected to USB 2.0 port of the PC. The central frequency was available between 2.5 and 7.5 MHz, the image depth was 15 cm and the frame rate was 30/s. The estimated price of this ultra-portable ultrasound is about 3000 US dollars with software. For 69 cardiac patients with informed consent, image quality was compared with those obtained with basic range diagnostic echo machines. Left ventricular ejection fraction (EF) derived from normal M-mode image of standard machines (EFm) were compared with visual EF of the ultra-portable ultrasound device (EFv). The image quality was comparable to the basic range diagnostic echo machines although short axis view of aortic root was not clearly visualized because the probe was too large for intercostal approach. EFv agreed well with EFm. The ultra-portable ultrasound may provide useful information on screening and health care.

Echocardiography↗

PubMed2004

Identification of GABAA receptor subunit variants in midbrain dopaminergic neurons.

Modulation of the activity of dopamine (DA)-producing neurons by GABA plays an important role in the control of DA-mediated brain functions. Ionotropic GABA(A) receptors exist as heteropentametric structures assembling different subunits composed of various subtypes. However, the expression pattern of these subunits in DA neurons in the ventral midbrain has not been fully defined. In the present study, we investigated the subunit composition of GABA(A) receptors in DA neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). We isolated DA neurons from the ventral midbrain of transgenic mice that express green fluorescent protein under the control of the tyrosine hydroxylase (TH) gene promoter and analyzed expression of various GABA(A) receptor subunits in single cells by using the reverse transcription-polymerase chain reaction. This analysis showed the presence of the transcripts encoding alpha2, alpha3, alpha4, beta1, beta3 and gamma2 subunits in the isolated DA neurons. Double fluorescence in situ hybridization with probes for TH and GABA(A) receptor subunit mRNAs revealed the expression of these six subunits in the majority of DA neurons in the SNc and the VTA.

Animals↗

PubMed2003

Dopamine D4 receptor-induced postsynaptic inhibition of GABAergic currents in mouse globus pallidus neurons.

Dopamine D4 receptors (D4R) are localized in the globus pallidus (GP), but their function remains unknown. In contrast, dopamine D2 receptor activation hyperpolarizes medium spiny neurons projecting from the striatum to the GP and inhibits GABA release. However, using slice preparations from D2R-deficient [D2 knock-out (D2KO)] mice, we found that dopamine inhibited GABA(A)-receptor-mediated currents in GP neurons. The paired-pulse ratio was statistically unchanged after dopamine application but was significantly elevated in D2KO wild-type littermates (WT). Furthermore, in D2KO mice, outward currents elicited by iontophoretically applied GABA were suppressed by dopamine. Dopamine (30 microm) decreased the amplitude of miniature IPSCs in both WT and D2KO mice, but the decrease in the frequency was observed only in the former but not significantly in the latter. Dopamine-induced suppression of IPSCs was blocked by selective D4R antagonists (clozapine or 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo[2,3-b]pyridine trihydrochloride), and a D4R-selective agonist N-[[4-(2-cyanophenyl)-1-piperazinyl]methyl]-3-methyl-benzamide reversibly and dose-dependently suppressed IPSCs, whereas agonists [SKF38,393 ((+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride) or (+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol] or antagonists [SCH23,390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) or sulpiride] of other receptor subtypes had little effect. In GP neurons from D4R-deficient mice, dopamine-induced inhibition of GABAergic outward currents was undetectable. D4R activation suppressed the activity of protein kinase A in GP neurons, resulting in a decrease in the amplitude of GABAergic IPSCs. These findings showed that postsynaptic activation of D4R on the GP neurons reduces GABAergic currents through the suppression of PKA activity.

Animals↗

PubMed2003

Conditional ablation of striatal neuronal types containing dopamine D2 receptor disturbs coordination of basal ganglia function.

Dopamine (DA) exerts synaptic organization of basal ganglia circuitry through a variety of neuronal populations in the striatum. We performed conditional ablation of striatal neuronal types containing DA D2 receptor (D2R) by using immunotoxin-mediated cell targeting. Mutant mice were generated that express the human interleukin-2 receptor alpha-subunit under the control of the D2R gene. Intrastriatal immunotoxin treatment of the mutants eliminated the majority of the striatopallidal medium spiny neurons and cholinergic interneurons. The elimination of these neurons caused hyperactivity of spontaneous movement and reduced motor activation in response to DA stimulation. The elimination also induced upregulation of GAD gene expression in the globus pallidus (GP) and downregulation of cytochrome oxidase activity in the subthalamic nucleus (STN), whereas it attenuated DA-induced expression of the immediate-early genes (IEGs) in the striatonigral neurons. In addition, chemical lesion of cholinergic interneurons did not alter spontaneous movement but caused a moderate enhancement in DA-induced motor activation. This enhancement of the behavior was accompanied by an increase in the IEG expression in the striatonigral neurons. These data suggest that ablation of the striatopallidal neurons causes spontaneous hyperactivity through modulation of the GP and STN activity and that the ablation leads to the reduction in DA-induced behavior at least partly through attenuation of the striatonigral activity as opposed to the influence of cholinergic cell lesion. We propose a possible model in which the striatopallidal neurons dually regulate motor behavior dependent on the state of DA transmission through coordination of the basal ganglia circuitry.

Animals↗

PubMed2003

Cortical and striatal expression of tyrosine hydroxylase mRNA in neonatal and adult mice.

1. Elucidating the mechanisms underlying regulation of the dopamine (DA) phenotype during development and in adult animals was a major focus of many of the students and postdoctoral fellows in the Laboratory of Dr Donald Reis. In one series of studies, expression of tyrosine hydroxylase (TH), the first enzyme in the DA biosynthetic pathway, was induced in primary cultures prepared from the cortical anlage of embryonic day 13 (E13)-E17 rat embryos. On the basis of these data, the current studies investigated whether under appropriate conditions TH expression might occur in forebrain regions that do not normally contain DA neurons. 2. A transgenic mouse strain harboring a 9-kb TH promoter/EGFP (enhanced green fluorescent protein) reporter construct was analyzed as adults for coexpression of the fluorescent reporter and the endogenous gene, the latter using a sensitive nonradioactive in situ hybridization procedure. The latter procedure was also used to determine the development of neonatal cortical endogenous TH expression. 3. Cortical and striatal cells containing TH mRNA were observed at postnatal day 5 (P5), but not P2, increased in number at P7 and were found in adults. Many cells in the cortex and striatum coexpressed TH mRNA and EGFP, but TH protein was not detected in these brain regions indicating independent transcriptional and translational regulation of TH expression. Overlapping expression of the two transcriptional indicators and TH protein in olfactory bulb occurred only in those DA neurons that receive afferent stimulation from receptor cells. 4. These findings suggest that partial DAergic differentiation may occur in some cortical and striatal cells, but that full expression of the phenotype requires synaptic activation or activity-dependent release of an as-yet unidentified factor(s).

Animals↗

PubMed2003

[Regulation of psychomotor functions by dopamine: integration of various approaches].

(1)The basal ganglia circuitry mediates a wide rage of brain functions such as motor control, behavioral planning, and reward prediction. Dopamine (DA) transmission plays an essential role in the regulation of these brain functions. DA action not only regulates the firing activity of target neurons but also is involved in the pattern formation of their firing. The striatopallidal neurons containing dopamine D(2) receptor plays a dual role in motor coordination dependent on DA transmission. (2)Activation of presynaptic D(2)-like receptors on GABAergic terminals onto striatal cholinergic interneurons selectively blocks N-type Ca(2+) channels, thereby inhibiting GABA release. In addition, contribution of N-type channels and D(2)-like receptor-mediated presynaptic inhibition decreases in parallel with development, implying some relationship between basal ganglia-related function or dysfunction and age. (3)As an approach to determine dopamine neuronal activity, we monitored neuronal activities by measuring cytosolic Ca(2+) concentration in VTA dopamine neurons. The present study indicates that VTA dopamine neurons are the direct targets of orexin-A and psychostimulants, and the [Ca(2+)](i) signaling is thought to play a significant role in the regulation of dopamine neuronal activity. (4)The excitability of neostriatal neurons is regulated by a balance of glutamatergic and dopaminergic inputs. Glutamate has been shown to modulate dopaminergic signaling. Studies on the regulation of DARPP-32 phosphorylation by glutamate provide a molecular basis for both the synergistic and antagonistic effects of glutamate on dopaminergic signaling. (5) Impairment of function of stem/progenitor cells may be implicated in the pathogenesis of schizophrenia. To test this hypothesis, several experiments are currently ongoing in our laboratory, and the preliminary results obtained are described here.

Animals↗

PubMed2002

Dynamics of tyrosine hydroxylase promoter activity during midbrain dopaminergic neuron development.

Dopamine (DA)-producing neurons in the ventral midbrain are generated from a specified neuronal lineage and form selective axonal pathways that mediate multiple CNS functions. Expression of the gene encoding tyrosine hydroxylase (TH), which is a key enzyme of catecholamine biosynthesis, is regulated during the development of midbrain DA neurons. In the present study, we report the developmental regulation and cell type specificity of TH gene promoter in the ventral midbrain by using a green fluorescent protein (GFP) reporter system. Transgenic mice were generated that express GFP in the majority of midbrain DA neurons under the control of the 9-kb upstream region of the rat TH gene. At an early embryonic stage, GFP expression was induced in the developing DA neurons, and the expression was then markedly down-regulated at later embryonic stages. However, the expression was reactivated and approached the adult levels during early post-natal development. These developmental changes in GFP expression patterns suggest the presence of multistep regulatory mechanisms for TH gene expression during DA neuron development. The TH promoter appears to possess transcriptional elements at least necessary for the induction of TH expression at the early embryonic stage and its reactivation during the post-natal development.

Animals↗