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Immunohistochemical Localization of Ih Channel Subunits,HCN1-4,in the Rat Brain
https://ir.soken.ac.jp/records/1139
https://ir.soken.ac.jp/records/11398adf7b48-3266-4270-9c18-cebf60d369b4
名前 / ファイル | ライセンス | アクション |
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要旨・審査要旨 / Abstract, Screening Result (309.6 kB)
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本文 (31.0 MB)
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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公開日 | 2010-02-22 | |||||
タイトル | ||||||
タイトル | Immunohistochemical Localization of Ih Channel Subunits,HCN1-4,in the Rat Brain | |||||
タイトル | ||||||
タイトル | Immunohistochemical Localization of Ih Channel Subunits,HCN1-4,in the Rat Brain | |||||
言語 | en | |||||
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言語 | eng | |||||
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資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
資源タイプ | thesis | |||||
著者名 |
納冨, 拓也
× 納冨, 拓也 |
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フリガナ |
ノウトミ, タクヤ
× ノウトミ, タクヤ |
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著者 |
NOTOMI, Takuya
× NOTOMI, Takuya |
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学位授与機関 | ||||||
学位授与機関名 | 総合研究大学院大学 | |||||
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学位名 | 博士(理学) | |||||
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内容記述タイプ | Other | |||||
内容記述 | 総研大甲第728号 | |||||
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値 | 生命科学研究科 | |||||
専攻 | ||||||
値 | 20 生理科学専攻 | |||||
学位授与年月日 | ||||||
学位授与年月日 | 2003-09-30 | |||||
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値 | 2003 | |||||
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内容記述タイプ | Other | |||||
内容記述 | Hyperpolarization-activated cation currents, termed Ih, were discovered in the heart and brain. These currents contribute to various physiological properties and functions, including neuronal pacemaker activity, the setting of resting potential and dendritic integration. The Hyperpolarization-activated and Cyclic-Nucleotide-gated nonselective cation channels (HCNs: HCN1-4), which generate Ih, have been cloned recently. To understand the functional diversity of Ih in the brain, precise immunohistochemical localization of all four HCNs is needed. Here I present the distribution and subcellular localization of immunoreactivity for HCNs in the rat brain using newly raised guinea pig polyclonal antibodies against fusion proteins containing rat HCN sequences. Immunoblot analyses of the rat brain with these antibodies showed single bands, which disappeared after adsorption of the antibodies with the respective antigens, suggesting specificity of these antibodies to each HCN subunit.<br />Then, I performed immunohistochemical investigation of HCNs in the rat brain. Immunoreactivity for all HCN subunits was detected with various intensities in neuropil throughout the brain. For HCN3 and HCN4 but not for HCN1 and HCN2, immunoreactive neuronal cell bodies and processes were clearly visible in light microscopic level in many brain regions. HCN1: Intense HCN1-like immunoreactivity (LI) was observed in the main olfactory bulb, cerebral cortex, lacunosum moleculare (LM) of the hippocampal CA areas, superior colliculus, inferior olive, area postrema, hypoglossal nucleus and molecular layer of the cerebellum. HCN2: HCN2-LI was widely distributed throughout the brain being most intense in the cerebral cortex, thalamus, inferior colliculus, brain stem and granular layer of the cerebellum. The immunoreactivities for HCN1 and HCN2 were overlapped in layer I of the neocortex and the LM of CA areas. In electron microscopic level, immunogold particles for HCN1 and HCN2 were found dense along the plasma membrane of distal dendrites of pyramidal cells in the neocortex and CA1 area. The HCN2-immunopositve small glia-like cells were observed throughout the brain including the white matter. HCN3:HCN3-LI was mainly distributed in the main and accessory olfactory bulbs, piriform cortex, preoptic area, habenular nucleui, hypothalamus, interpeduncular nuclei and inferior olivary complex. In the flocculus of the cerebellum, intense HCN3-LI was observed in the cholinergic terminal and axon. HCN4: The distribution pattern of HCN4-LI was restricted to the thalamic nuclei and some other regions such as the external plexiform layer of the main olfactory bulb, nucleus of the olfactory tract, fasciculus retroflexous, lateral lemniscus, ventral cochlear nucleus, superior olivary complex and area postrema.<br />I found that all four HCN-LIs were localized to presynaptic elements as well as to postsynaptic elements. In presynaptic elements, immunoreactivity for HCNs was often found in preterminal and axonal parts rather than axon terminals. For example, in cerebellar basket cells, most of immunogold particles for HCN1 were found in the ponceau of the basket cell, but only rarely localized to their terminals with symmetrical synapses. I also found immunoreactivity for HCNs in plasma membrane of myelinated axons in various regions including the fasciculus retroflexus and hippocampus.<br />Next, to identify HCN2-immunopositive small cells, I tried double immunostaining using the guinea pig anti-HCN2 antibody and mouse or rabbit antibodies for various markers including NeuN for matured neurons, GFAP for astrocytes, NG2 for oligoprogenitors, IbaI for microglia-macrophages, and GST-π for oligodendrocytes. I found that all of the HCN2-positive cells were labeled only for GST-π throughout the brain except the amygdaloid areas and cingulated, prirhinal, and lateral entorhinal cortices, where I found only single labeled cells GST-π. In addition, some of the double-immunopositive cells were particularly close to neuronal cell bodies and processes in the hippocampal pyramidal cell layer and cerebellar granule cell layer. In electron microscopic level, the HCN2-immunopositive cells had some clumps of heterochromatin along nuclear membrane and those clumps were eccentrically located in the soma, having similar properties to those of previously described perineuronal oligodendrocytes. These results suggest that HCN2-immunopositive smallcells belong to a subpopulation of oligodendrocytes, including perineuronal oligodendrocytes.<br />In conclusion four HCNs have distinct distribution patterns in neuropil and neuronal cell bodies throughout the brain consistent with the reported patterns of distribution of mRNAs for HCNs. My results indicate that HCNs are localized not only in somato-dendritic compartments, but also in axonal compartments of neurons. The HCN1 and HCN2 are co-localized in the hippocampus and neocortex and had similar distribution patterns in electron microscopic level, suggesting that these subunits could form the heteromeric channels. Interestingly, HCN2 is extensively expressed in a subpopulation of oligodendrocytes including perineuronal oligodendrocytes. These results support previous electrophysiological findings and further suggest diversified roles of Ih channels in the brain, which are not yet fully identified. | |||||
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値 | 有 | |||||
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内容記述タイプ | Other | |||||
内容記述 | application/pdf |