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.
