Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
Add more filters










Publication year range
1.
Folia Histochem Cytobiol ; 51(1): 25-30, 2013.
Article in English | MEDLINE | ID: mdl-23690214

ABSTRACT

Caveolin-1, a major constituent of caveolae, has been implicated in endocytosis, signal transduction and cholesterol transport in a wide variety of cells. In the present study, the expression of caveolin-1 was examined by immunohistochemistry in rat brain with or without systemic injection of kainic acid (KA). Caveolin-1 immunoreactivity was observed in capillary walls in brains of control rats. From one to seven days after KA injection, caveolin-1 immunoreactivity appeared in activated microglia in the cerebral cortex, hippocampus and other brain regions. The strongest immunoreactivity of microglia was seen after 3 days after KA administration. The expression of caveolin-1 was confirmed by RT-PCR and Western blot analysis, respectively. The induction of caveolin-1 expression in microglia activated in response to kainic acid administration suggests its possible role in a modulation of inflammation.


Subject(s)
Brain/cytology , Caveolin 1/metabolism , Kainic Acid/pharmacology , Microglia/metabolism , Animals , Biomarkers/metabolism , Blotting, Western , Caveolin 1/genetics , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Male , Microglia/drug effects , Microscopy, Confocal , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction
2.
J Biol Chem ; 286(7): 5836-45, 2011 Feb 18.
Article in English | MEDLINE | ID: mdl-21163949

ABSTRACT

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter (VACHT) as candidate genes, which function together with ChAT in acetylcholine production. Using SH-SY5Y human neuroblastoma cells stably expressing wild-type human ChAT, we found that overexpressed ChAT enhanced transcription of the CHT1 gene but not the VACHT gene. In contrast, nuclear localization signal disrupted, and catalytically inactive mutant ChATs could not induce, CHT1 expression. Additionally, ChAT did not alter CHT1 expression in non-neuronal HEK293 cells. Our results suggest that ChAT activates the transcription of selected target genes in neuronal cells. Both enzymatic activity and nuclear translocation of ChAT are required for its transcriptional enhancement.


Subject(s)
Cell Nucleus/metabolism , Choline O-Acetyltransferase/metabolism , Gene Expression Regulation/physiology , Neurons/metabolism , Symporters/biosynthesis , Transcription, Genetic/physiology , Vesicular Acetylcholine Transport Proteins/biosynthesis , Acetylcholine/biosynthesis , Acetylcholine/genetics , Active Transport, Cell Nucleus/physiology , Cell Line , Cell Line, Tumor , Cell Nucleus/genetics , Choline O-Acetyltransferase/genetics , HEK293 Cells , Humans , Mutation , Organ Specificity/physiology , Symporters/genetics , Vesicular Acetylcholine Transport Proteins/genetics
3.
Mol Vis ; 15: 523-33, 2009.
Article in English | MEDLINE | ID: mdl-19279690

ABSTRACT

PURPOSE: MicroRNA-182 (miR-182) is expressed abundantly in the mammalian retina and is therefore thought to perform important roles for the retinal development and the function. To test this hypothesis, we generated miR-182 knockout mice. METHODS: northern blotting was performed to confirm the robust expression of miR-182 in the eye. The precursor sequence of miR-182 was replaced by the neomycin resistance gene under the control of the phosphoglycerate kinase 1 promoter in a targeting construct. The targeting vector was linearized and transfected into embryonic stem (ES) cells. Recombinant ES clones were selected and injected into blastocysts to generate male chimeras. Heterozygous and homozygous mice were obtained after five generations of backcrossing and were confirmed using genotyping and northern blotting. RESULTS: Heterozygous (+/-) and homozygous (-/-) knockout mice were morphologically normal, viable, and fertile. Immunohistochemical analysis of the miR-182-deficient retinas did not reveal any apparent structural abnormalities in the retinas. Consistently, global expression profiling using a repeated microarray did not identify significant fluctuations for potential target genes. CONCLUSIONS: We successfully generated miR-182 knockout mice and characterized the resulting miR-182-depleted retina. This is the first report describing the targeted deletion of a single miRNA that is highly expressed in the retina. The absence of significant transcriptional and phenotypic changes in miR-182-depleted retinas suggests that miR-182 is not a major determinant of retinal development or delamination. Further studies are required to elucidate any functional changes in the retina.


Subject(s)
Mice, Knockout , MicroRNAs/physiology , Retina/embryology , Retina/metabolism , Animals , Female , Gene Expression , Gene Expression Profiling , Gene Expression Regulation, Developmental , Male , Mice , Mice, Inbred C57BL , MicroRNAs/antagonists & inhibitors , Retina/cytology
4.
J Comp Neurol ; 509(4): 436-48, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18521856

ABSTRACT

Rat trigeminal ganglion neurons have been shown to contain a splice variant of choline acetyltransferase (pChAT). Here we report the distribution pattern of pChAT-containing afferents from the trigeminal ganglion to the brainstem, compared with that of calcitonin gene-related peptide (CGRP) and substance P (SP), by use of the immunohistochemical techniques in the rat. Most of CGRP(+) SP(+) ganglion cells contain pChAT, whereas half of the pChAT(+) ganglion cells possess neither CGRP nor SP. In the brainstem, pChAT(+) nerve fibers are found exclusively in the trigeminal and solitary systems, although the distribution pattern differs from that of CGRP(+) or SP(+) fibers. First, the ventral portion of the principal sensory nucleus contains many pChAT(+) fibers, with few CGRP(+) or SP(+) fibers. Because this portion receives projections of nociceptive corneal afferents, a subpopulation of pChAT(+) CGRP(-) SP(-) primary afferents is most probably nonpeptidergic nociceptors innervating the cornea. Second, the superficial laminae of the medullary dorsal horn, the main target of nociceptive afferents, contain dense CGRP(+) and SP(+) fibers but sparse pChAT(+) fibers. Because pChAT occurs in most CGRP(+) SP(+) ganglion cells, such sparseness of pChAT(+) fibers implies poor transportation of pChAT to axon branchlets. Another important finding is that pChAT(+) axons are smooth and nonvaricose, whereas CGRP(+) or SP(+) fibers possess numerous varicosities. Our confocal microscopy suggests colocalization of these three markers in the same single axons in some brainstem regions. The difference in morphological appearance, nonvaricose or varicose, appears to reflect the difference in intraaxonal distribution between pChAT and CGRP or SP.


Subject(s)
Alternative Splicing , Brain Stem/enzymology , Calcitonin Gene-Related Peptide/genetics , Choline O-Acetyltransferase/genetics , Genetic Variation , Substance P/genetics , Trigeminal Ganglion/enzymology , Animals , Antibodies , Brain Stem/physiology , Goats/immunology , Immunohistochemistry , Male , Rats , Rats, Wistar , Trigeminal Ganglion/physiology
5.
Brain Res ; 1141: 92-8, 2007 Apr 13.
Article in English | MEDLINE | ID: mdl-17291466

ABSTRACT

We have previously demonstrated that a variant form of choline acetyltransferase (pChAT) is expressed in rat trigeminal neurons. To assess the significance of pChAT in sensory functions, we characterized immunohistochemically pChAT-positive trigeminal neurons in the rat. pChAT-immunoreactivity was observed in a rather uniform pattern in about half of all trigeminal neurons throughout the trigeminal ganglion. The majority of pChAT-positive neurons had small to medium-sized cell bodies. Double immunofluorescent study showed that more than 90% of substance P (SP)-positive trigeminal cells and about 80% of calcitonin gene-related peptide (CGRP)-positive cells exhibited pChAT-immunoreactivity. pChAT-positive cells formed a larger population of neurons than SP-positive or CGRP-positive cells, but they were a different population from calbindin-D(28k)-positive neurons. In addition, pChAT-immunoreactivity was present in a subset of neurons positive for neuronal nitric oxide synthase. The present results suggest that pChAT plays roles not only in nociception, but also in other sensory functions such as mechanoreception mediating tactile sensation.


Subject(s)
Choline O-Acetyltransferase/metabolism , Neurons, Afferent/metabolism , Neuropeptides/metabolism , Nitric Oxide Synthase/metabolism , S100 Calcium Binding Protein G/metabolism , Trigeminal Ganglion/cytology , Animals , Calbindins , Calcitonin Gene-Related Peptide/metabolism , Immunohistochemistry , Male , Rats
6.
J Histochem Cytochem ; 55(3): 287-99, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17142806

ABSTRACT

Cholinergic innervation of the heart has been analyzed using cholinergic markers including acetylcholinesterase, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). In the present study we demonstrate putative cholinergic nerves in the rat heart using an antibody to ChAT of a peripheral type (pChAT), which is the product of a splice variant of ChAT mRNA and preferentially localized to peripheral cholinergic nerves. Expression of mRNAs for pChAT and the conventional form of ChAT (cChAT) were verified in the rat atrium by RT-PCR. Localization of both protein products in the atrium was confirmed by Western blotting. Virtually all neurons and small intensely fluorescent cells in the intrinsic cardiac ganglia were stained immunohistochemically for pChAT. The density of pChAT-positive fibers was very high in the conducting system, high in both atria, the right atrium in particular, and low in the ventricular walls. pChAT and VAChT immunoreactivities were closely associated in some fibers and fiber bundles in the ventricular walls. These results indicate that intrinsic cardiac neurons homogeneously express both pChAT and cChAT. Furthermore, innervation of the ventricular walls by pChAT- and VAChT-positive fibers provides morphological evidence for a significant role of cholinergic mechanisms in ventricular functions.


Subject(s)
Choline O-Acetyltransferase/biosynthesis , Myocardium/enzymology , Peripheral Nervous System/enzymology , Animals , Biomarkers/analysis , Blotting, Western , Choline O-Acetyltransferase/genetics , Heart/innervation , Immunohistochemistry , Myocardium/cytology , Nerve Fibers/enzymology , Neurons/enzymology , RNA, Messenger/biosynthesis , Rats , Reverse Transcriptase Polymerase Chain Reaction
7.
Neurosci Lett ; 409(1): 35-40, 2006 Nov 27.
Article in English | MEDLINE | ID: mdl-17014960

ABSTRACT

To evaluate the roles of complement in kainic acid (KA)-induced neuronal damages, the immunohistochemical localization of the complement protein C3 was examined in rat hippocampus after systemic KA injection. The immunoreactivity for C3 was found in glial cells in control rats, and such glial cells were increased in number after KA injection. Our confocal study showed that C3-positive glial cells were microglia. Three to seven days after KA, C3 immunoreactivity appeared in CA1 and CA3 pyramidal neurons. Double staining for C3 combined with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling showed that occurrence of C3 immunoreactivity in neurons coincided well with that of DNA fragmentation. Western blot analysis and RT-PCR experiments suggested local synthesis of C3 by brain cells. Our results suggest that C3 contributes greatly to neuronal death after systemic KA administration, and that microglia and neurons are the local source of C3 in KA-induced brain injury.


Subject(s)
Complement C3/metabolism , Excitatory Amino Acid Agonists/toxicity , Hippocampus/metabolism , Kainic Acid/toxicity , Pyramidal Cells/metabolism , Animals , Blotting, Western , CD11a Antigen/metabolism , Cell Death/physiology , Clusterin/metabolism , DNA Fragmentation/drug effects , Glial Fibrillary Acidic Protein/metabolism , Hippocampus/cytology , Immunohistochemistry , In Situ Nick-End Labeling , Male , Microscopy, Confocal , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction
8.
J Comp Neurol ; 492(2): 234-49, 2005 Nov 14.
Article in English | MEDLINE | ID: mdl-16196032

ABSTRACT

Unlike the brains of most mammals, the mouse brain appears unique in the massive appearance of cells showing IgG-like immunoreactivity, which has repeatedly been shown via immunohistochemistry. In the present study, we first examined possible species differences in IgG-like immunohistochemical staining in the brains of various rodents, including mice. In four of six mouse strains examined (ICR, Balb/c, C57BL/6, and AKR/J), antibodies against mouse IgG revealed positive staining in many brain microglia. However, no such positive staining was detected in brains of the rat, hamster, guinea pig, or two other mouse strains (CBA/N and CBA/J). We purified IgG-like-immunoreactive molecule(s) biochemically from brain of the ICR mouse as a representative mouse strain. Our amino-acid-sequence analysis proved that the purified protein was identical to serum IgG. The possibility of IgG synthesis by brain microglia in the ICR mouse was denied by our RT-PCR experiments and in situ hybridization histochemistry. In addition, Fcgamma-receptor-deficient double-knockout mice of the C57BL/6 genetic background contained no IgG-immunoreactive microglia in the brain. These results clearly indicate that microglial IgG staining is due to the uptake of serum IgG through Fcgamma receptors. However, the strain-specific mechanisms resulting in microglial IgG uptake remain to be elucidated, in that Fcgamma receptors are omnipresent in microglia of all rodents examined here.


Subject(s)
Brain/immunology , Immunoglobulin G/metabolism , Microglia/immunology , Animals , Brain/anatomy & histology , Cricetinae , Guinea Pigs , Immunohistochemistry , In Situ Hybridization , Male , Mesocricetus , Mice , Mice, Inbred Strains , Rats , Rats, Wistar , Spleen/cytology , Spleen/immunology
9.
Neurochem Int ; 46(5): 423-33, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15737440

ABSTRACT

Choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type (pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type (cChAT) using green fluorescent protein (GFP) in living human embryonic kidney cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.


Subject(s)
Acetylcholine/biosynthesis , Alternative Splicing/physiology , Central Nervous System/enzymology , Choline O-Acetyltransferase/metabolism , Peripheral Nervous System/enzymology , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Animals , Cell Nucleus/enzymology , Cells, Cultured , Choline O-Acetyltransferase/genetics , Corpus Striatum/enzymology , Cytoplasm/enzymology , Enzyme Inhibitors/pharmacology , Fatty Acids, Unsaturated/pharmacology , Ganglia, Parasympathetic/enzymology , Green Fluorescent Proteins , Humans , Male , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Transport/drug effects , Protein Transport/physiology , Rats
10.
J Comp Neurol ; 472(2): 232-45, 2004 Apr 26.
Article in English | MEDLINE | ID: mdl-15048690

ABSTRACT

We have recently discovered a splice variant of choline acetyltransferase (ChAT) mRNA and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, the presence of pChAT in rat iris was examined by immunohistochemistry and Western blot using a pChAT antiserum, in combination with RT-PCR analysis and ChAT enzyme assay. For comparison, the conventional ChAT (cChAT) was studied in parallel. By pChAT immunohistochemistry, intense labeling was found to occur in nerve fibers of the iris and in neurons of the ciliary and trigeminal ganglia. Denervation studies, analyzed by semiquantitative morphometry, indicated that these iridial pChAT fibers originated about half from the ciliary ganglion and the other half from the trigeminal ganglion. The presence of pChAT protein in the iris and trigeminal ganglion was confirmed by Western blot. The expression of pChAT mRNA in the ciliary and trigeminal ganglia was proved by RT-PCR. Although cChAT protein and mRNA were detected in the ciliary ganglion, neither was detectable in the trigeminal ganglion. The contributions of the ciliary and trigeminal ganglia to the iridial ChAT enzyme activity were verified by the present ChAT assay. Here, we provide evidence that iridial pChAT nerves are composed of postganglionic parasympathetic efferents from the ciliary ganglion and, more interestingly, somatic sensory afferents of the trigeminal ophthalmic nerve.


Subject(s)
Alternative Splicing , Choline O-Acetyltransferase/biosynthesis , Ciliary Body/enzymology , Ciliary Body/innervation , Iris/enzymology , Iris/innervation , Trigeminal Nerve/enzymology , Animals , Choline O-Acetyltransferase/genetics , Gene Expression Regulation, Enzymologic/physiology , Male , Neurons/enzymology , Rats , Rats, Wistar
12.
J Neurosci ; 23(7): 2872-81, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12684474

ABSTRACT

Acetylcholine acts as a neurotransmitter in the retina. Although previous physiological studies have indicated that some retinal ganglion cells may be cholinergic, several immunohistochemical studies using antibodies to choline acetyltransferase (ChAT) have stained only amacrine cells but not ganglion cells. Recently, we identified a splice variant of ChAT mRNA, lacking exons 6-9, in rat peripheral nervous system. The encoded protein was designated as ChAT of a peripheral type (pChAT), against which an antiserum was raised. In the present study, we examined expression of pChAT in rat retina, both at the protein level by immunohistochemistry using the antiserum and at the mRNA level by RT-PCR. Immunohistochemistry revealed that although no positive neurons were found in untreated intact retinas, many neurons became immunoreactive for pChAT after intravitreal injection of colchicine. Damage of the optic nerve was also effective in disclosing positive cells. Such positive neurons were shown to be ganglion cells by double labeling with a retrograde tracer that had been injected into the contralateral superior colliculus. Western blot analysis and RT-PCR revealed a corresponding band to the pChAT protein and to the amplified pChAT gene fragment, respectively, in retinal samples. In addition, ChAT activity was definitely detected in retinofugal fibers of the optic nerve. These results indicate the presence of cholinergic ganglion cells in rat retina.


Subject(s)
Alternative Splicing , Choline O-Acetyltransferase/genetics , Retinal Ganglion Cells/enzymology , Adaptation, Ocular , Animals , Blotting, Western , Choline O-Acetyltransferase/analysis , Choline O-Acetyltransferase/biosynthesis , Cholinergic Fibers/enzymology , Dark Adaptation , Immunohistochemistry , Male , Optic Nerve/enzymology , Protein Isoforms , RNA, Messenger/metabolism , Rats , Rats, Wistar , Retina/cytology , Retina/enzymology , Retinal Ganglion Cells/cytology , Visual Pathways
13.
Epilepsia ; 43 Suppl 9: 39-43, 2002.
Article in English | MEDLINE | ID: mdl-12383279

ABSTRACT

PURPOSE: To clarify the relationship of neuronal death to cellular responses, we studied neuronal death as well as reactions of glia and progenitor cells in the hippocampus of two rat models of epilepsy. METHODS: Seizures were induced by either kainic acid (KA) administration or electrical kindling. Neuronal degeneration was assessed by in situ DNA fragmentation analysis. Reactions of glial cells were studied by immunohistochemistry. Progenitor cell division was evaluated using the bromodeoxyuridine (BrdU) labeling method. RESULTS: DNA fragmentation and reactive microglia were observed in the CA1, CA3, and hilus region for 24 h to 4 weeks after KA injection, but not detected in the kindling model. Reactive astrocytes and enhancement of progenitor cell division were seen in both animal models. The number of BrdU-positive cells began to increase on day 3 after KA injection, peaked on day 5, and returned to baseline on day 10. After kindling, the number of BrdU-positive cells began to increase after five consecutive experience of stage I seizures. CONCLUSIONS: These observations show that neuronal degeneration is not necessary for triggering the upregulation. Microglial activation is closely related to the neuronal death process induced by KA.


Subject(s)
DNA Fragmentation , Epilepsy/pathology , Hippocampus/pathology , Neurons/pathology , Stem Cells/cytology , Animals , Bromodeoxyuridine/administration & dosage , Cell Death , Cell Division , Disease Models, Animal , Excitatory Amino Acid Agonists/administration & dosage , Excitatory Amino Acid Agonists/pharmacology , Hippocampus/drug effects , Hippocampus/metabolism , Immunohistochemistry , In Situ Nick-End Labeling , In Vitro Techniques , Injections, Intraperitoneal , Kainic Acid/administration & dosage , Kainic Acid/pharmacology , Kindling, Neurologic , Male , Microglia/pathology , Neuroglia/pathology , Rats , Rats, Sprague-Dawley , Stem Cells/drug effects , Time Factors
SELECTION OF CITATIONS
SEARCH DETAIL
...