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シナプス後膜の形態と神経伝達物質受容体局在の高解像度解析

突触后膜形态和神经递质受体定位的高分辨率分析

基本信息

批准号：
17024060
负责人：
深澤有吾
金额：
$ 1.6万
依托单位：
National Institute for Physiological Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2005
资助国家：
日本
起止时间：
2005 至无数据
项目状态：
已结题

项目摘要

神経細胞膜上の分子局在の解析には電子顕微鏡レベルの観察が必要だが、従来法のpre-embedding法や、post-embedding法では、細胞膜上の分子と細胞質中の分子を見分けることは出来ず感度も低く定量性に欠けるため、シナプス機能を分子レベルで定量的に理解する事は不可能であった。そこで、細胞膜上の分子局在を電子顕微鏡レベルで2次元的に定量的に解析できるSDS処理凍結割断レプリカ免疫標識法(SDS-FRL法)を脳組織に適用し、電子顕微鏡レベルで細胞膜上のAMPA型グルタミン酸受容体(AMPA受容体)の局在を定量的に解析できるよう最適化し、海馬CA1錐体細胞。歯状回顆粒細胞樹状突起上の局在解析を行った。1)はじめに各AMPA受容体サブユニット欠損動物を入手し、レプリカ膜上での抗GluR1,抗GluR2,抗GluR3及び抗GluR2/3抗体の標識特異性を確認した。2)また、CA1野錐体細胞や歯状回顆粒細胞の樹状突起上には膜内粒子の凝縮を伴う受容体クラスタが高密度に分布し、レプリカ膜上のシナプスの同定に従来用いられてきた膜内粒子の凝集のみでは不十分と考えられたので、NMDA受容体標識を同時に行うことを検討し、この標識がレプリカ膜上のシナプスの同定に有効であることを明らかにした。LTP誘導後のAMPA受容体局在解析の結果、誘導後45分の時点ではシナプス内AMPA受容体の密度増加が観察され、LTP誘導実験によりAMPA受容体密度の低いシナプスがLTP誘導により増強されることを示唆する結果を得たが、GluR1サブユニットのシナプスへの輸送はまだ起こっていないことを示唆する結果を得た。3)レプリカ膜面の細胞種の同定に用いるレンチウィルス遺伝子導入技術も確立し、現在はレプリカ膜上で標識可能なタグの選定と凍結割断時の分子分配様式の解析を行っている。

The molecular bureau on the cell membrane is in the process of analyzing the necessary drugs, pre-embedding method, post-embedding method, molecular weight analysis on the cell membrane to detect the sensitivity of low molecular weight, low sensitivity, low molecular weight, low sensitivity, low sensitivity, low molecular weight, low molecular weight, low sensitivity, low molecular weight, low molecular weight, low sensitivity, low molecular weight, low molecular weight, low sensitivity, low molecular weight, low molecular weight, low sensitivity, low sensitivity, low molecular weight, low molecular weight, low sensitivity, low molecular weight, low molecular weight, low sensitivity, low molecular weight, low molecular weight, low sensitivity, low sensitivity, low molecular weight, low sensitivity, low sensitivity, The molecular bureau on the cell membrane was used in the quantitative analysis of the second dimension of the electron microarray, the SDS assay cut off the immunolabeling method (SDS-FRL method), the AMPA-type receptor (AMPA) was detected on the cell membrane of the cell membrane, and the AMPA receptor (AMPA) on the cell membrane of the cell membrane was quantitatively analyzed. The upper part of the granule cell-like process of the gyrus is analyzing the "line". 1) the specific identification of anti-GluR1, anti-GluR2, anti-GluR3 and anti-GluR2/3 antibodies was detected in each AMPA recipient. 2) the confluence of the particles in the membrane with the high density distribution of the acceptor on the cell-like processes of the cells of the CA1 and the CA1, the concentration of the particles in the membrane is the same as that of the particles in the membrane, the concentration of the particles in the membrane is different from that of the particles in the membrane, and the NMDA acceptor identity is the same as that of the receptor. On the film, there is a sign that there is a sign that there is a message on the film. After the LTP test, the AMPA recipient Bureau analyzed the results of the test, the density of the AMPA receptor in the test station was monitored at 45 minutes after the test, and the LTP guide was responsible for the detection of the bulk density of the AMPA capacitor. The LTP guide showed that the results of the test were satisfactory. GluR1, please tell me that the results of the test are not satisfactory. 3) in the same way that the system is used to determine the accuracy of the technology, it is possible to select a formula for molecular distribution when the device is cut off.