| Project/Area Number |
12680798
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
神経・脳内生理学
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| Research Institution | Dokkyo Medical University |
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Principal Investigator |
HORI Yuuichi Dokkyo University School of Medicine, Department of Physiology and Biological Information, Professor, 医学部, 教授 (60190229)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | GFP / Enkephalin / Preproenkephalin Promoter / Spinal Dorsal Horn Neurons / Patch-Clamp Recording / Organotypic Slice Culture / Biolistic Gene Gun / エンケファリンプロモーター / 脊髄スライスカルチャー / エンケファリン プロモーター |
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| Research Abstract |
Recently, the biolistic gene gun has been used to transfect neurons from various regions of the central nervous system in organotypic cultures. In the present study, we transfected cultures of mouse spinal cord slices with the enhanced green fluorescent protein (GFP) gene driven by the promoter for preproenkephalin, using the particle-mediated gene transfer system adapted for small neurons in the superficial dorsal horn.
A high density of GFP fluorescence was expressed in the superficial dorsal horn, reminiscent of the previously reported distribution of enkephalinergic neurons. In contrast, the slices transfected with CMV promoter GFP plasmid had a uniform distribution of GFP fluorescence throughout the spinal cord.
The number of GFP-expressing neurons increased in response to activation of adenylate cyclase, suggesting that GFP expression was controlled by the preproenkephalin promoter with its cAMP responsive elements.
The soma diameter of GFP-expressing neurons ranged from 5μm to 12μm
… More
. While GFP-expressing neurons varied in the morphology of cell body and neurite, most of them resembled "stalked cells" and "islet cells", which have been described in the spinal cord superficial dorsal horn by S. Gobel.
Single-cell RT-PCR analysis showed that the incidence of N-methyl-D-aspartate (NMDA) receptor NR2B subunit was significantly larger in enkephalin-expressing neurons compared with neurons not expressing enkephalin. It was apparent that expression of the NMDA receptor subunit is differentially regulated between enkephalinergic neurons and non-enkephalinergic neurons, although the functional significance of this differential regulation is unknown at present.
Finally, the organotypic slice culture of spinal cord transfected with preproenkephalin-promoter-GFP plasmid described in the present study provided an opportunity to investigate trans-synaptical regulation of preproenkepahlin gene expression and enabled rapid analysis of changes in preproenkephalin gene expression in highly restricted populations of neurons from specific areas of the CNS. Less
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