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

用电生理学方法研究了神经网络的功能和性质，使其可视化。本项目建立了利用技术鉴定记录神经元中的神经递质或调节性，以及脑内功能分子mRNA的含量和分布的方法。研究表明，催产素（OXT）在雌性大鼠视上核中的神经传递对神经活动有抑制作用，并且在分娩或切除后，抑制反应逆转为兴奋反应。为了研究卵巢类固醇对OXT作用的影响，我们使用了切除卵巢的大鼠的脑切片，分别给予或不给予雌激素。利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。记录结束后，以正矩形脉冲注射神经生物素（N-（2-氨基乙基）生物酰胺）。切片用多聚甲醛固定，石蜡包埋，切成5 μm的切片。标记后的神经元用生物素复合物可视化，荧光显微镜下拍照。为鉴定OXT神经元，经乙醇褪色后，用抗OXT-神经physin单克隆抗体孵育切片，再用FITC偶联的山羊抗小鼠IgG孵育切片，在荧光显微镜下观察。比较脱水大鼠和配对喂养大鼠杏仁核中央核和室旁核CRH mRAN的含量和分布。在本实验中，建立了应用于脑的原位PCR/杂交技术。

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.