Silencing Hypothalamic Galanin with RNA Interference

描述（由申请人提供）：该试点和可行性拨款申请（NIDDK R21 PA-02-008 公告）寻求资金来进行一系列探索性实验，以表征使用 RNA 干扰 (RNAi) 作为抑制哺乳动物大脑中特定神经肽表达的方法。 RNAi 是最近发现的一种过程，它利用双链 RNA (dsRNA) 的高度保守监视机制，引起同源 mRNA 的选择性转录后基因沉默。因此，将细胞暴露于与特定 mRNA 一致的 dsRNA 中应该会沉默该 mRNA 的表达，并随后沉默肽的表达。 RNAi 应用于大脑中的神经元，可能被证明是一种选择性消除任何神经化学特异性神经元组的方法，这些神经元组涉及神经元信号传导机制中的蛋白质（例如神经肽递质、生物合成酶）。例如，该方法可应用于抑制下丘脑室旁核（PVN）中特定的食欲调节肽神经递质，以检查它们在食物摄入调节中的作用。这种适应性强的方法对神经科学的普遍效用可能会非常显着。虽然初步数据揭示了该程序的可行性，但必须对其实用性和可靠性进行评估和表征。我们建议通过使用针对神经肽甘丙肽 (GAL) 和甘丙肽受体 1 (GAL-R1) 的 dsRNA 来评估和表征 RNAi 的局部应用。我们将确定在 PVN 中选择性使用 dsRNA 对抗 GAL 和 GAL-R1 的关键参数（剂量、体积、丢失和恢复的时间过程）。然后，我们将使用这些最佳参数来评估 PVN GAL 和 GAL-R1 的功能效果。饮食行为将成为检查的功能输出，因为 GAL 对食物摄入的作用已得到很好的表征。研究结果完成后，将提供可靠地使用 RNAi 作为一种高度选择性、神经化学特异性“损伤”方法的详细程序，并证明其在研究大脑中特定神经元亚群的生理功能中的实用性。

DESCRIPTION (provided by applicant): This pilot and feasibility grant application (NIDDK R21 PA-02-008 announcement) seeks funding to conduct a series of exploratory experiments to characterize the use of RNA interference (RNAi) as a method to suppress the expression of specific neuropeptides in the mammalian brain. RNAi is a recently discovered process that exploits a highly conserved surveillance mechanism for double stranded RNA (dsRNA), to cause a selective, post-transcription gene silencing of homologous mRNA. Thus, exposure of cells to dsRNA coinciding to a specific mRNA should silence the expression of that mRNA and subsequently the peptide. Applied to neurons in brain, RNAi could prove to be a method for the selective elimination of any neurochemically-specific set of neurons that involve a protein in the neuron's signaling mechanism (e.g. neuropeptide transmitters, biosynthetic enzymes). For example, the method could be applied to the suppression of specific appetite modifying peptide neurotransmitters in the paraventricular nucleus of the hypothalamus (PVN) to examine their roles in food intake regulation. The general utility of such an adaptable method to neuroscience would potentially be remarkable. While preliminary data reveal the feasibility of the procedure, it must be evaluated and characterized for its utility and reliability. We propose to evaluate and characterize local application of RNAi by using dsRNA against the neuropeptide galanin (GAL) and the galanin receptor-1 (GAL-R1). We will determine the key parameters (dose, volume, time course of loss and recovery) of using dsRNA against GAL and GAL-R1 selectively in the PVN. We will then use these optimal parameters to evaluate the functional effect of PVN GAL and GAL-R1. Eating behavior will be the functional output examined, because GAL's actions on food intake are well characterized. The results, when completed, will offer a detailed procedure for using RNAi reliably as a highly selective, neurochemically-specific "lesioning" method and evidence of its utility in studying physiological functions of particular neuronal subpopulations in the brain.