Akio Wanaka - Academia.edu (original) (raw)

Papers by Akio Wanaka

Voluntary Exercise Induces Astrocytic Structural Plasticity in the Globus Pallidus

Frontiers in cellular neuroscience, 2016

Changes in astrocyte morphology are primarily attributed to the fine processes where intimate con... more Changes in astrocyte morphology are primarily attributed to the fine processes where intimate connections with neurons form the tripartite synapse and participate in neurotransmission. Recent evidence has shown that neurotransmission induces dynamic synaptic remodeling, suggesting that astrocytic fine processes may adapt their morphologies to the activity in their environment. To illustrate such a neuron-glia relationship in morphological detail, we employed a double transgenic Olig2(CreER/WT); ROSA26-GAP43-EGFP mice, in which Olig2-lineage cells can be visualized and traced with membrane-targeted GFP. Although Olig2-lineage cells in the adult brain usually become mature oligodendrocytes or oligodendrocyte precursor cells with NG2-proteoglycan expression, we found a population of Olig2-lineage astrocytes with bushy morphology in several brain regions. The globus pallidus (GP) preferentially contains Olig2-lineage astrocytes. Since the GP exerts pivotal motor functions in the indirec...

A sequence-specific splicing activator, Tra2�, is up-regulated in response to nerve injury

Mol Brain Res, 1998

Tra2β is the first mammalian protein which is proved to activate mRNA splicing in sequence-specif... more Tra2β is the first mammalian protein which is proved to activate mRNA splicing in sequence-specific manner. Following hypoglossal nerve injury, the expression of Tra2β mRNA was elevated in injured motoneurons transiently. The up-regulation of Tra2β mRNA was observed from post-operative day 3 to 21. In addition to the nerve injury in PNS, a brain lesion in CNS also enhanced the expression of Tra2β mRNA. The present study could be the first observation showing that an expression of the sequence-specific splicing activator is enhanced in neuronal cells in response to nerve injury, and indicates that Tra2β may participate in the control of injury-specific splicing patterns in order to express molecules which are necessary for regeneration.

β-Adrenergic receptors in the vasopressin-containing neurons in the paraventricular and supraoptic nucleus of the rat

Brain Research, 1989

Immunohistochemical Demonstration of Beta-Arenoreceptor in the Rat Brain - with Reference to the Central Control of Blood Pressure

Development, Feb 1, 1991

We examined the expression of FGF-receptor (FGF-R) mRNA during rat development with in situ hybri... more We examined the expression of FGF-receptor (FGF-R) mRNA during rat development with in situ hybridization histochemistry. Embryonic tissues (E9, E12, E14, E17) and postnatal neural tissues (PI, P7, P14, adult) were examined. We detected significant levels of FGF-R mRNA in various tissues at different developmental stages. As postulated by previous studies using other methods, FGF-R gene expression was observed primarily in mesoderm-and neuroectoderm-derived tissues. In the nervous system, the pattern of gene expression was developmentally regulated; in embryos, FGF-R mRNA was mainly detected in the ependymal layer of the central nervous system (CNS). Postnatally, FGF-R transcripts were observed in specific neuronal populations, such as hippocampal neurons. FGF-R mRNA was also found in sensory systems such as trigeminal and dorsal root ganglia in late stage embryos; however, FGF-R mRNA decreased in the postnatal period. FGF-R mRNA expression was modulated in the developing retina: FGF-R messages were observed in the pigment epithelium and neuroblast layer at embryonic stages; in the postnatal period, they were found in the ganglion cell and inner granular layer. In non-neuronal embryonic tissues, a wide variety of organs expressed FGF-R message. Particularly, the prevertebral column, bone, kidney and skin showed high levels of expression. These observations reinforce the idea that FGF exerts effects on the development of various tissues.

Journal of Central Nervous System Disease, Feb 12, 2009

The traditional Chinese herbal medicine yi-gan san has been used to cure neuropsychological disor... more The traditional Chinese herbal medicine yi-gan san has been used to cure neuropsychological disorders. Schizophrenia can be one of the target diseases of yi-gan san. We aimed at evaluating the possible use of yi-gan san in improving the schizophrenic symptoms of an animal model. Yi-gan san or distilled water was administered to mice born from pregnant mice injected with polyinosinic-polycytidilic acid or phosphate buffered saline. The former is a model of schizophrenia based on the epidemiological data that maternal infection leads to psychotic disorders including schizophrenia in the offspring. Prepulse inhibition and sensitivity to methamphetamine in open fi eld tests were analyzed and the total glutathione content of whole brains was measured. Yi-gan san reversed the decrease in prepulse inhibition, hypersensitivity to methamphetamine and cognitive defi cits found in the model mice to the level of control mice. Total glutathione content in whole brains was reduced in the model mice but was restored to normal levels by yi-gan san treatment. These results suggest that yi-gan san may have ameliorating effects on the pathological symptoms of schizophrenia.

Molecular Brain Research, Dec 1, 1995

In the course of studies to identify new members of the eph family of receptor tyrosine kinases, ... more In the course of studies to identify new members of the eph family of receptor tyrosine kinases, MDK1 and one of its splicing variants lacking a kinase domain, MDK1-T1, were identified. To gain insight into the functions of these subtypes, expression patterns of their mRNAs in the developing mouse nervous system were examined by Northern blotting and in situ hybridization histochemistry. Colocalization of their mRNAs was observed, but the levels of expression of each mRNA were developmentally regulated. These findings suggest functional differences between full-length and truncated forms of MDKI receptor tyrosine kinase.

Bone, Nov 30, 2001

Previous studies have demonstrated that FGF-FGFR signal transduction plays a crucial role in skel... more Previous studies have demonstrated that FGF-FGFR signal transduction plays a crucial role in skeletal development (1). Recent reports have shown that exogenous bFGF enlarges fracture calluses and accerelates the healing of osteotomized long bones (2, 3). Thus, it is possible that FGF ligand-receptor signals play important roles not only in embryogenesis but also in skeletal repair. In the processes of fracture healing, however, detailed expression of FGF ligands and receptors have not yet been fully established. In the present study, we employed a standardized rat model of fracture healing and analyzed the spatial and temporal gene expression for bFGF and FGFR1. Based on the results, we discuss a possible role for FGF-FGFR1 signaling in fracture repair. Materials and Methods: Fracture model: Closed middiaphyseal fractures were created in the right femora of SD rats (4). The rats were euthanized at 1, 4, 7, 14, 21 and 28 days postoperatively, and fractured femora were fixed, decalcified, and embedded in paraffin. In situ hybridization (ISH): Sections were hybridized with DIG-labeled cRNA probes for bFGF and FGFR1. To evaluate matrix production and bone remodeling, sections were also hybridized with probes for collagen types I and V, and osteocalcin. Tartarate resistent acid phosphate (TRAP) staining: For the identification of osteoclasts, TRAP staining was performed. Immunohistochemistry: To evaluate cell growth activity, sections were reacted with an antibody for PCNA. RNAse protection assay (RPA): After the isolation of total RNA from fracture calluses, RPA was carried out with 32 P-labeled cRNA probes for bFGF, FGFR1 and GAPDH. Results: ISH: In normal femoral shafts, a weak FGFR1 signal was seen in cells in the periosteum, endosteum and medullary cavity. On day 1 after fracture, a moderate or relatively strong signal for FGFR1 was detected in osteoprogenitor cells under the periosteum , arrows) and in roundshaped mesenchymal cells among the muscle layers . Such FGFR1-positive cells at this stage were PCNA-positive, but negative for collagen types I and V. From day 4 to day 7, periosteal hard callus was formed. At this stage, a strong FGFR1 signal was detected in cuboidal osteoblasts in the deep layer of the hard callus, but the signal was weak in the superficial layer. Similarly, osteocalcin gene expession preferentially occurred in the deep layer. From day 14, cartilage was replaced by bony tissues and endochondral ossification rapidly progressed. At the ossification front, TRAPpositive multinuclear cells , presumably mature osteoclasts, expressed a moderate signal for FGFR1 . At the central portion of the hard callus, TRAP-positive cells not attaching to the trabeculae, presumably immature osteoclasts, expressed FGFR1 gene, but the signal was very weak. During bone remodeling of the hard callus, a strong FGFR1 signal was detected in mature osteoblasts lining the trabeculae even in the later stages of fracture healing. In spite of such widespread gene expression for FGFR1, we could not detect any bFGF signals throughout the healing stages. RPA: By quantification, a small amount of FGFR1 mRNA was detected in normal femora. On day 1, FGFR1 mRNA levels were approximately 3-fold higher than in normal femora ). They reached a maximum level on day 14, and remained at relatively high levels even on day 28 . In contrast to the enhanced expression for FGFR1 mRNA, bFGF mRNA expression was hardly detected . Discussion: The present results demonstrate that gene expression for FGFR1 is rapidly upregulated after fracture, and maintained at relatively high levels even in the later stages of healing. In spite of such enhanced expression of FGFR1, little expression for bFGF mRNA was detected by our ISH and RPA analyses. Previous studies showed that a considerable amount of bFGF peptide is stored in bone matrix (5). Thus we suggest that, during fracture healing, FGFR1 is produced locally but its ligand (bFGF) is mainly released from the bone matrix. From the expression patterns of FGFR1, we suggest that FGF-FGFR1 signaling may preferentially contribute to cell proliferation during initial callus formation, and to bone remodeling in the later stages. In this study, we also revealed that FGFR1 mRNA was expressed not only in

Localization of chondroitin sulfate proteoglycan versican in the adult brain with special reference to large progection neurons

Neurosci Res, 2009

ABSTRACT Versican is a chondroitin sulfate proteoglycan belonging to the lectican family. Versica... more ABSTRACT Versican is a chondroitin sulfate proteoglycan belonging to the lectican family. Versican has two glycosaminoglycan attachment regions, named the GAG alpha and GAG beta domains, which are both regulated by alternative splicing and yield four protein isoforms. We have investigated the expression and localization of versican in the developing and adult brain by using anti-versican GAG alpha and GAG beta antibodies. Western analysis revealed that GAG alpha-reactive isoform was dominant in the adult brain. Immunohistochemical study demonstrated that GAG alpha immunoreactivity was detectable from neonatal periods to adulthood, whereas GAG beta immunoreactivity completely disappeared within 3 weeks of birth. In the adult brain, GAG alpha immunoreactivity was seen in the white matter regions and was also localized in the gray matter including somata and dendrites of cortical and hippocampal pyramidal neurons and cerebellar Purkinje cells. In contrast, GAG alpha immunoreactivity was not localized on parvalbumin-positive interneurons and cerebellar stellate cells. Furthermore, GAG alpha immunoreactivity was not co-localized with perineuronal net markers such as Wisteria floribunda agglutinin lectin and phosphacan. Thus, versican was localized on large projection neurons rather than small interneurons. To confirm the binding mechanism of versican to neurons, hyaluronan and chondroitin sulfates were enzymatically removed from brain sections before the immunolabeling of versican. These treatments had no effect on the labeling pattern of versican, suggesting that other versican-interactive molecules are involved in the binding of versican to neurons.

Journal of Neurochemistry, Jul 1, 2009

These authors contributed equally to this work.

Neuroscience Letters, Jun 6, 1997

After inoculation of a highly neuro-invasive strain of herpes simplex virus (HSV) type 2 (186) in... more After inoculation of a highly neuro-invasive strain of herpes simplex virus (HSV) type 2 (186) into the mouse hind-paw planter skin, many virus-positive neurons and glial cells were detected in the dorsal root ganglia (DRGs) and lumbar spinal cord by immunohistochemistry for HSV-2 antigen. A number of apoptotic cells were also observed in the spinal cord and DRGs by the terminal dUTP nick-endlabeling (TUNEL) method. Double labeling with the immunohistochemistry for HSV-2 and the TUNEL method revealed further that some glial and neuronal cells in the spinal cord, either infected or non-infected by HSV-2, showed apoptotic signs. In DRGs, however, apoptosis was detected in no neuronal cells, although some of HSV-2 -infected glial cells were apoptotic.

Environmental stimulation changes tissue-type plasminogen activator activity in the adult mouse hippocampus

Neurochemistry International, Jan 31, 2011

Tissue-type plasminogen activator (tPA) plays an important role in synaptic plasticity and contri... more Tissue-type plasminogen activator (tPA) plays an important role in synaptic plasticity and contributes to several brain functions such as memory, learning, and behaviours. Although a number of studies have demonstrated that various kinds of stimuli such as electrophysiological stimulation, excitotoxic injury, and stress change tPA activity in the brain, no studies have ever examined whether environmental stimulation affect tPA activity in the brain. The aim of this study is to clarify the effect of environmental enrichment on tPA activity in the hippocampus and cerebral cortex of adult mouse brain. Zymographic analysis revealed that hippocampal tPA activity was higher in enriched mice than in standard-caged mice, whereas no significant changes in the cerebral cortex were detected between enriched and control mice. Enrichment-enhanced tPA activity reverted to the pre-enrichment level after mice were returned to a standard housing condition, suggesting that tPA upregulation is stimulation-dependent and reversible. These results suggest that tPA activity is regulated reversibly in an experience-dependent manner in the adult hippocampus.

Expression of Heparin-Binding Growth Factors and Their Receptors in Neural Development

解剖學雜誌, Dec 1, 1995

Identification and expression of DP5, a programmed neuronal death-related gene

Neurosci Res, 1997

Identification of differentially expressed mRNAs during neuronal differentiation of P19 embryonal carcinoma cells

Neuroscience Research the Official Journal of the Japan Neuroscience Society, Aug 31, 1995

Hedgehog Signaling Components Are Expressed in Choroidal Neovascularization in Laser-induced Retinal Lesion

Acta Histochemica et Cytochemica, 2016

Choroidal neovascularization is one of the major pathological changes in age-related macular dege... more Choroidal neovascularization is one of the major pathological changes in age-related macular degeneration, which causes devastating blindness in the elderly population. The molecular mechanism of choroidal neovascularization has been under extensive investigation, but is still an open question. We focused on sonic hedgehog signaling, which is implicated in angiogenesis in various organs. Laser-induced injuries to the mouse retina were made to cause choroidal neovascularization. We examined gene expression of sonic hedgehog, its receptors (patched1, smoothened, cell adhesion molecule down-regulated by oncogenes (Cdon) and biregional Cdon-binding protein (Boc)) and downstream transcription factors (Gli1-3) using real-time RT-PCR. At seven days after injury, mRNAs for Patched1 and Gli1 were upregulated in response to injury, but displayed no upregulation in control retinas. Immunohistochemistry revealed that Patched1 and Gli1 proteins were localized to CD31-positive endothelial cells that cluster between the wounded retina and the pigment epithelium layer. Treatment with the hedgehog signaling inhibitor cyclopamine did not significantly decrease the size of the neovascularization areas, but the hedgehog agonist purmorphamine made the areas significantly larger than those in untreated retina. These results suggest that the hedgehog-signaling cascade may be a therapeutic target for age-related macular degeneration.

Differential display analysis of gene expression in the long-term cultured astrocyte

Neurosci Res, 1997

Hedgehog Signaling Modulates the Release of Gliotransmitters from Cultured Cerebellar Astrocytes

Neurochemical research, 2016

Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family, plays essential roles in the developm... more Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family, plays essential roles in the development of the central nervous system. Recent studies suggest that the Hh signaling pathway also functions in mature astrocytes under physiological conditions. We first examined the expression of genes encoding Hh signaling molecules in the adult mouse cerebellum by in situ hybridization histochemistry. mRNA for Patched homolog 1 (Ptch1), a receptor for Hh family members, was expressed in S100β-positive astrocytes and Shh mRNA was expressed in HuC/D-positive neurons, implying that the Hh signaling pathway contributes to neuro-glial interactions. To test this hypothesis, we next examined the effects of recombinant SHH N-terminal protein (rSHH-N) on the functions of cultured cerebellar astrocytes. rSHH-N up-regulated Hh signal target genes such as Ptch1 and Gli-1, a key transcription factor of the Hh signaling pathway. Although activation of Hh signaling by rSHH-N or purmorphamine influenced n...

Molecular cloning of mouse Doc2a and distribution of its mRNA in adult mouse brain

Mol Brain Res, 1997

We have previously isolated from a human brain cDNA library, a new protein having two C2-like dom... more We have previously isolated from a human brain cDNA library, a new protein having two C2-like domains which interact with Ca2+ and phospholipid, and named Doc2α. Doc2α is abundantly expressed in brain, where it is highly concentrated on the synaptic vesicle fraction, and is implicated in Ca2+-dependent exocytosis. We have isolated here a mouse Doc2α cDNA and determined the localization of its mRNA in adult mouse brain. The amino acid sequence of the mouse Doc2α cDNA is 92% identical with that of the human counterpart. Northern blot analysis and in situ hybridization on adult mouse brain sections have revealed that Doc2α is predominantly expressed in mouse brain, where it is expressed in neuronal cells, but not in non-neuronal cells. Doc2α is highly expressed in the olfactory bulb, cerebral cortex, hippocampus, amygdaloid complex, and ventromedial hypothalamus nucleus, but not in the cerebellum, caudate-putamen, or ventral thalamus. These results indicate that Doc2α is expressed heterogeneously in mouse brain, where it is predominantly expressed in neuronal cells, and suggest that Doc2α plays a specific role in the area where it is expressed.

Voluntary Exercise Induces Astrocytic Structural Plasticity in the Globus Pallidus

Frontiers in cellular neuroscience, 2016

Changes in astrocyte morphology are primarily attributed to the fine processes where intimate con... more Changes in astrocyte morphology are primarily attributed to the fine processes where intimate connections with neurons form the tripartite synapse and participate in neurotransmission. Recent evidence has shown that neurotransmission induces dynamic synaptic remodeling, suggesting that astrocytic fine processes may adapt their morphologies to the activity in their environment. To illustrate such a neuron-glia relationship in morphological detail, we employed a double transgenic Olig2(CreER/WT); ROSA26-GAP43-EGFP mice, in which Olig2-lineage cells can be visualized and traced with membrane-targeted GFP. Although Olig2-lineage cells in the adult brain usually become mature oligodendrocytes or oligodendrocyte precursor cells with NG2-proteoglycan expression, we found a population of Olig2-lineage astrocytes with bushy morphology in several brain regions. The globus pallidus (GP) preferentially contains Olig2-lineage astrocytes. Since the GP exerts pivotal motor functions in the indirec...

A sequence-specific splicing activator, Tra2�, is up-regulated in response to nerve injury

Mol Brain Res, 1998

Tra2β is the first mammalian protein which is proved to activate mRNA splicing in sequence-specif... more Tra2β is the first mammalian protein which is proved to activate mRNA splicing in sequence-specific manner. Following hypoglossal nerve injury, the expression of Tra2β mRNA was elevated in injured motoneurons transiently. The up-regulation of Tra2β mRNA was observed from post-operative day 3 to 21. In addition to the nerve injury in PNS, a brain lesion in CNS also enhanced the expression of Tra2β mRNA. The present study could be the first observation showing that an expression of the sequence-specific splicing activator is enhanced in neuronal cells in response to nerve injury, and indicates that Tra2β may participate in the control of injury-specific splicing patterns in order to express molecules which are necessary for regeneration.

β-Adrenergic receptors in the vasopressin-containing neurons in the paraventricular and supraoptic nucleus of the rat

Brain Research, 1989

Immunohistochemical Demonstration of Beta-Arenoreceptor in the Rat Brain - with Reference to the Central Control of Blood Pressure

Development, Feb 1, 1991

We examined the expression of FGF-receptor (FGF-R) mRNA during rat development with in situ hybri... more We examined the expression of FGF-receptor (FGF-R) mRNA during rat development with in situ hybridization histochemistry. Embryonic tissues (E9, E12, E14, E17) and postnatal neural tissues (PI, P7, P14, adult) were examined. We detected significant levels of FGF-R mRNA in various tissues at different developmental stages. As postulated by previous studies using other methods, FGF-R gene expression was observed primarily in mesoderm-and neuroectoderm-derived tissues. In the nervous system, the pattern of gene expression was developmentally regulated; in embryos, FGF-R mRNA was mainly detected in the ependymal layer of the central nervous system (CNS). Postnatally, FGF-R transcripts were observed in specific neuronal populations, such as hippocampal neurons. FGF-R mRNA was also found in sensory systems such as trigeminal and dorsal root ganglia in late stage embryos; however, FGF-R mRNA decreased in the postnatal period. FGF-R mRNA expression was modulated in the developing retina: FGF-R messages were observed in the pigment epithelium and neuroblast layer at embryonic stages; in the postnatal period, they were found in the ganglion cell and inner granular layer. In non-neuronal embryonic tissues, a wide variety of organs expressed FGF-R message. Particularly, the prevertebral column, bone, kidney and skin showed high levels of expression. These observations reinforce the idea that FGF exerts effects on the development of various tissues.

Journal of Central Nervous System Disease, Feb 12, 2009

The traditional Chinese herbal medicine yi-gan san has been used to cure neuropsychological disor... more The traditional Chinese herbal medicine yi-gan san has been used to cure neuropsychological disorders. Schizophrenia can be one of the target diseases of yi-gan san. We aimed at evaluating the possible use of yi-gan san in improving the schizophrenic symptoms of an animal model. Yi-gan san or distilled water was administered to mice born from pregnant mice injected with polyinosinic-polycytidilic acid or phosphate buffered saline. The former is a model of schizophrenia based on the epidemiological data that maternal infection leads to psychotic disorders including schizophrenia in the offspring. Prepulse inhibition and sensitivity to methamphetamine in open fi eld tests were analyzed and the total glutathione content of whole brains was measured. Yi-gan san reversed the decrease in prepulse inhibition, hypersensitivity to methamphetamine and cognitive defi cits found in the model mice to the level of control mice. Total glutathione content in whole brains was reduced in the model mice but was restored to normal levels by yi-gan san treatment. These results suggest that yi-gan san may have ameliorating effects on the pathological symptoms of schizophrenia.

Molecular Brain Research, Dec 1, 1995

In the course of studies to identify new members of the eph family of receptor tyrosine kinases, ... more In the course of studies to identify new members of the eph family of receptor tyrosine kinases, MDK1 and one of its splicing variants lacking a kinase domain, MDK1-T1, were identified. To gain insight into the functions of these subtypes, expression patterns of their mRNAs in the developing mouse nervous system were examined by Northern blotting and in situ hybridization histochemistry. Colocalization of their mRNAs was observed, but the levels of expression of each mRNA were developmentally regulated. These findings suggest functional differences between full-length and truncated forms of MDKI receptor tyrosine kinase.

Bone, Nov 30, 2001

Previous studies have demonstrated that FGF-FGFR signal transduction plays a crucial role in skel... more Previous studies have demonstrated that FGF-FGFR signal transduction plays a crucial role in skeletal development (1). Recent reports have shown that exogenous bFGF enlarges fracture calluses and accerelates the healing of osteotomized long bones (2, 3). Thus, it is possible that FGF ligand-receptor signals play important roles not only in embryogenesis but also in skeletal repair. In the processes of fracture healing, however, detailed expression of FGF ligands and receptors have not yet been fully established. In the present study, we employed a standardized rat model of fracture healing and analyzed the spatial and temporal gene expression for bFGF and FGFR1. Based on the results, we discuss a possible role for FGF-FGFR1 signaling in fracture repair. Materials and Methods: Fracture model: Closed middiaphyseal fractures were created in the right femora of SD rats (4). The rats were euthanized at 1, 4, 7, 14, 21 and 28 days postoperatively, and fractured femora were fixed, decalcified, and embedded in paraffin. In situ hybridization (ISH): Sections were hybridized with DIG-labeled cRNA probes for bFGF and FGFR1. To evaluate matrix production and bone remodeling, sections were also hybridized with probes for collagen types I and V, and osteocalcin. Tartarate resistent acid phosphate (TRAP) staining: For the identification of osteoclasts, TRAP staining was performed. Immunohistochemistry: To evaluate cell growth activity, sections were reacted with an antibody for PCNA. RNAse protection assay (RPA): After the isolation of total RNA from fracture calluses, RPA was carried out with 32 P-labeled cRNA probes for bFGF, FGFR1 and GAPDH. Results: ISH: In normal femoral shafts, a weak FGFR1 signal was seen in cells in the periosteum, endosteum and medullary cavity. On day 1 after fracture, a moderate or relatively strong signal for FGFR1 was detected in osteoprogenitor cells under the periosteum , arrows) and in roundshaped mesenchymal cells among the muscle layers . Such FGFR1-positive cells at this stage were PCNA-positive, but negative for collagen types I and V. From day 4 to day 7, periosteal hard callus was formed. At this stage, a strong FGFR1 signal was detected in cuboidal osteoblasts in the deep layer of the hard callus, but the signal was weak in the superficial layer. Similarly, osteocalcin gene expession preferentially occurred in the deep layer. From day 14, cartilage was replaced by bony tissues and endochondral ossification rapidly progressed. At the ossification front, TRAPpositive multinuclear cells , presumably mature osteoclasts, expressed a moderate signal for FGFR1 . At the central portion of the hard callus, TRAP-positive cells not attaching to the trabeculae, presumably immature osteoclasts, expressed FGFR1 gene, but the signal was very weak. During bone remodeling of the hard callus, a strong FGFR1 signal was detected in mature osteoblasts lining the trabeculae even in the later stages of fracture healing. In spite of such widespread gene expression for FGFR1, we could not detect any bFGF signals throughout the healing stages. RPA: By quantification, a small amount of FGFR1 mRNA was detected in normal femora. On day 1, FGFR1 mRNA levels were approximately 3-fold higher than in normal femora ). They reached a maximum level on day 14, and remained at relatively high levels even on day 28 . In contrast to the enhanced expression for FGFR1 mRNA, bFGF mRNA expression was hardly detected . Discussion: The present results demonstrate that gene expression for FGFR1 is rapidly upregulated after fracture, and maintained at relatively high levels even in the later stages of healing. In spite of such enhanced expression of FGFR1, little expression for bFGF mRNA was detected by our ISH and RPA analyses. Previous studies showed that a considerable amount of bFGF peptide is stored in bone matrix (5). Thus we suggest that, during fracture healing, FGFR1 is produced locally but its ligand (bFGF) is mainly released from the bone matrix. From the expression patterns of FGFR1, we suggest that FGF-FGFR1 signaling may preferentially contribute to cell proliferation during initial callus formation, and to bone remodeling in the later stages. In this study, we also revealed that FGFR1 mRNA was expressed not only in

Localization of chondroitin sulfate proteoglycan versican in the adult brain with special reference to large progection neurons

Neurosci Res, 2009

ABSTRACT Versican is a chondroitin sulfate proteoglycan belonging to the lectican family. Versica... more ABSTRACT Versican is a chondroitin sulfate proteoglycan belonging to the lectican family. Versican has two glycosaminoglycan attachment regions, named the GAG alpha and GAG beta domains, which are both regulated by alternative splicing and yield four protein isoforms. We have investigated the expression and localization of versican in the developing and adult brain by using anti-versican GAG alpha and GAG beta antibodies. Western analysis revealed that GAG alpha-reactive isoform was dominant in the adult brain. Immunohistochemical study demonstrated that GAG alpha immunoreactivity was detectable from neonatal periods to adulthood, whereas GAG beta immunoreactivity completely disappeared within 3 weeks of birth. In the adult brain, GAG alpha immunoreactivity was seen in the white matter regions and was also localized in the gray matter including somata and dendrites of cortical and hippocampal pyramidal neurons and cerebellar Purkinje cells. In contrast, GAG alpha immunoreactivity was not localized on parvalbumin-positive interneurons and cerebellar stellate cells. Furthermore, GAG alpha immunoreactivity was not co-localized with perineuronal net markers such as Wisteria floribunda agglutinin lectin and phosphacan. Thus, versican was localized on large projection neurons rather than small interneurons. To confirm the binding mechanism of versican to neurons, hyaluronan and chondroitin sulfates were enzymatically removed from brain sections before the immunolabeling of versican. These treatments had no effect on the labeling pattern of versican, suggesting that other versican-interactive molecules are involved in the binding of versican to neurons.

Journal of Neurochemistry, Jul 1, 2009

These authors contributed equally to this work.

Neuroscience Letters, Jun 6, 1997

After inoculation of a highly neuro-invasive strain of herpes simplex virus (HSV) type 2 (186) in... more After inoculation of a highly neuro-invasive strain of herpes simplex virus (HSV) type 2 (186) into the mouse hind-paw planter skin, many virus-positive neurons and glial cells were detected in the dorsal root ganglia (DRGs) and lumbar spinal cord by immunohistochemistry for HSV-2 antigen. A number of apoptotic cells were also observed in the spinal cord and DRGs by the terminal dUTP nick-endlabeling (TUNEL) method. Double labeling with the immunohistochemistry for HSV-2 and the TUNEL method revealed further that some glial and neuronal cells in the spinal cord, either infected or non-infected by HSV-2, showed apoptotic signs. In DRGs, however, apoptosis was detected in no neuronal cells, although some of HSV-2 -infected glial cells were apoptotic.

Environmental stimulation changes tissue-type plasminogen activator activity in the adult mouse hippocampus

Neurochemistry International, Jan 31, 2011

Tissue-type plasminogen activator (tPA) plays an important role in synaptic plasticity and contri... more Tissue-type plasminogen activator (tPA) plays an important role in synaptic plasticity and contributes to several brain functions such as memory, learning, and behaviours. Although a number of studies have demonstrated that various kinds of stimuli such as electrophysiological stimulation, excitotoxic injury, and stress change tPA activity in the brain, no studies have ever examined whether environmental stimulation affect tPA activity in the brain. The aim of this study is to clarify the effect of environmental enrichment on tPA activity in the hippocampus and cerebral cortex of adult mouse brain. Zymographic analysis revealed that hippocampal tPA activity was higher in enriched mice than in standard-caged mice, whereas no significant changes in the cerebral cortex were detected between enriched and control mice. Enrichment-enhanced tPA activity reverted to the pre-enrichment level after mice were returned to a standard housing condition, suggesting that tPA upregulation is stimulation-dependent and reversible. These results suggest that tPA activity is regulated reversibly in an experience-dependent manner in the adult hippocampus.

Expression of Heparin-Binding Growth Factors and Their Receptors in Neural Development

解剖學雜誌, Dec 1, 1995

Identification and expression of DP5, a programmed neuronal death-related gene

Neurosci Res, 1997

Identification of differentially expressed mRNAs during neuronal differentiation of P19 embryonal carcinoma cells

Neuroscience Research the Official Journal of the Japan Neuroscience Society, Aug 31, 1995

Hedgehog Signaling Components Are Expressed in Choroidal Neovascularization in Laser-induced Retinal Lesion

Acta Histochemica et Cytochemica, 2016

Choroidal neovascularization is one of the major pathological changes in age-related macular dege... more Choroidal neovascularization is one of the major pathological changes in age-related macular degeneration, which causes devastating blindness in the elderly population. The molecular mechanism of choroidal neovascularization has been under extensive investigation, but is still an open question. We focused on sonic hedgehog signaling, which is implicated in angiogenesis in various organs. Laser-induced injuries to the mouse retina were made to cause choroidal neovascularization. We examined gene expression of sonic hedgehog, its receptors (patched1, smoothened, cell adhesion molecule down-regulated by oncogenes (Cdon) and biregional Cdon-binding protein (Boc)) and downstream transcription factors (Gli1-3) using real-time RT-PCR. At seven days after injury, mRNAs for Patched1 and Gli1 were upregulated in response to injury, but displayed no upregulation in control retinas. Immunohistochemistry revealed that Patched1 and Gli1 proteins were localized to CD31-positive endothelial cells that cluster between the wounded retina and the pigment epithelium layer. Treatment with the hedgehog signaling inhibitor cyclopamine did not significantly decrease the size of the neovascularization areas, but the hedgehog agonist purmorphamine made the areas significantly larger than those in untreated retina. These results suggest that the hedgehog-signaling cascade may be a therapeutic target for age-related macular degeneration.

Differential display analysis of gene expression in the long-term cultured astrocyte

Neurosci Res, 1997

Hedgehog Signaling Modulates the Release of Gliotransmitters from Cultured Cerebellar Astrocytes

Neurochemical research, 2016

Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family, plays essential roles in the developm... more Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family, plays essential roles in the development of the central nervous system. Recent studies suggest that the Hh signaling pathway also functions in mature astrocytes under physiological conditions. We first examined the expression of genes encoding Hh signaling molecules in the adult mouse cerebellum by in situ hybridization histochemistry. mRNA for Patched homolog 1 (Ptch1), a receptor for Hh family members, was expressed in S100β-positive astrocytes and Shh mRNA was expressed in HuC/D-positive neurons, implying that the Hh signaling pathway contributes to neuro-glial interactions. To test this hypothesis, we next examined the effects of recombinant SHH N-terminal protein (rSHH-N) on the functions of cultured cerebellar astrocytes. rSHH-N up-regulated Hh signal target genes such as Ptch1 and Gli-1, a key transcription factor of the Hh signaling pathway. Although activation of Hh signaling by rSHH-N or purmorphamine influenced n...

Molecular cloning of mouse Doc2a and distribution of its mRNA in adult mouse brain

Mol Brain Res, 1997

We have previously isolated from a human brain cDNA library, a new protein having two C2-like dom... more We have previously isolated from a human brain cDNA library, a new protein having two C2-like domains which interact with Ca2+ and phospholipid, and named Doc2α. Doc2α is abundantly expressed in brain, where it is highly concentrated on the synaptic vesicle fraction, and is implicated in Ca2+-dependent exocytosis. We have isolated here a mouse Doc2α cDNA and determined the localization of its mRNA in adult mouse brain. The amino acid sequence of the mouse Doc2α cDNA is 92% identical with that of the human counterpart. Northern blot analysis and in situ hybridization on adult mouse brain sections have revealed that Doc2α is predominantly expressed in mouse brain, where it is expressed in neuronal cells, but not in non-neuronal cells. Doc2α is highly expressed in the olfactory bulb, cerebral cortex, hippocampus, amygdaloid complex, and ventromedial hypothalamus nucleus, but not in the cerebellum, caudate-putamen, or ventral thalamus. These results indicate that Doc2α is expressed heterogeneously in mouse brain, where it is predominantly expressed in neuronal cells, and suggest that Doc2α plays a specific role in the area where it is expressed.