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An approach to visualize the network in the central nervous system using In Situ PCR

使用原位 PCR 可视化中枢神经系统网络的方法

基本信息

批准号：
12557006
负责人：
NAKASHIMA Toshihiro
金额：
$ 5.38万
依托单位：
Kyoto Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557006/
关键词：
In Situ PCR Intracellular dve injection Brain slice Patch clamp ルシファーイエロー

项目摘要

To visualize the neural network which was studied the functions and properties by electro-physiology., this project established the methods identify the neurotransmitter or modulator in recorded neuron, and contents and distribution of mRNA of functioning molecule in the brain by using technique. It has been demonstrated that oxytocin (OXT) neurotransmission in the supraoptic nucleus has an inhibitory effect on neural activity in female virgin rat, and the inhibitory response reversed to excitatory one after parturition or overiectomy. To examine the effects of ovarian steroid on the OXT action, we used brain slice prepared from ovariectomized rats treated with or without estrogen. The effects of OXT on excitatory postsynaptic currents (EPSCs) were recorded by using perforated whole-cell patch-clamp technique. After recording session, neurobiotin (N-(2-aminoethyl) biotinamide) was injected ny positive rectangular pulses. The slice was fixed in paraformaldehide, embedded in paraffin and cut into 5 μm section. Labeled neuron was visualized by avidine-biotin complex and photographed with fluorescence microscope. To identify OXT neuron, after fading out with ethanol, the section was incubated with anti-OXT-neurophysin monoclonal antibory and then with goat anti-mouse IgG conjugated with FITC, and observed with fluorescent microscope. Next, the CRH mRAN contents and distribution was compared between dehydrated and pair-fed rats in the central nucleus of amygdale and paraventricular nucleus. During this experiment, the technique for apply In Situ PCR/hybridization to the brain was established.

通过电生理学研究神经网络的功能和特性，本课题建立了利用技术鉴定记录神经元中神经递质或调质的方法，以及脑内功能分子mRNA的含量和分布。雌性未孕大鼠视上核内催产素（oxytocin，OXT）的神经传递对神经元活动具有抑制作用，分娩或包皮切除后，这种抑制性反应逆转为兴奋性反应。为了研究卵巢类固醇对OXT作用的影响，我们使用了从切除卵巢的大鼠中制备的脑切片，这些大鼠接受了或没有接受雌激素治疗。采用穿孔全细胞膜片钳技术记录OXT对兴奋性突触后电流（EPSCs）的影响。记录期后，通过正矩形脉冲注射神经生物素（N-（2-氨基乙基）生物素酰胺）。切片用多聚甲醛固定，石蜡包埋，切成5 μm切片。标记的神经元用亲和素-生物素复合物显色，并在荧光显微镜下拍照。为鉴定OXT神经元，将切片经乙醇褪色后，分别与抗OXT-neurophysin单克隆抗体和FITC标记的羊抗鼠IgG孵育，荧光显微镜下观察。接下来，比较脱水和成对喂养大鼠杏仁中央核和室旁核中CRH mRAN的含量和分布。本实验建立了脑组织原位PCR/杂交技术。