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

点击翻译按钮获取中文摘要

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.