The objective of this application is to develop RNA interference (RNAi) as a novel tool to completely silence gene expression in the central nervous system. Specifically, we will determine the feasibility of using RNAi as a method of choice to eliminate gene expression in a spatially- and temporally-controlled manner, and determine downstream effects on gene function and animal behavior. This approach will offer distinct advantage over conventional methods (knockouts, lesions, antagonists and antisense) by providing increased specificity and potency. Ever since the serendipitous discovery that double stranded RNA (dsRNA) functions as an extremely specific and potent inhibitor of gene expression in a manner phenotypically akin to a genetic knockout, there has been a surge in studies using RNAi to elucidate gene function in model systems such as the C. elegans, drosophila, and cultured cells. However, its application in vivo in mammals has been very limited. Thus, the aims of this study are to (1) establish and optimize the key parameters such as the dose and time course of RNAi action in rat brain. Towards this end, we have selected corticotropin-releasing factor (CRF) as the model neuropeptide and the hypothalamic paraventricular nucleus as the model locus. We will introduce dsRNA into the PVN using stereotaxic surgery. Effect of specifically silencing CRF in the PVN on neuroanatomy and gene expression will be determined by in situ and immunohistochemical studies. Downstream effect on hypothalamic-pituitary adrenal (HPA) axis function will be assessed by determining changes in basal and stress activated parameter directly influenced by CRF such as adrenocorticotropin hormone (ACTH) and corticosterone (B) using radioimmunoassays. (2) We will assess whether these optimized parameter can be applied more globally to inhibit CRF expression in another brain site (amygdala) that expresses CRF. Amygdala is the principal brain locus involved in fear/anxiety responses. We will determine role of amygdala CRF on acute restraint stress responses by measuring changes in basal and stress activated changes in ACTH and B. Furthermore, we will determine changes in expression levels of both RNA and protein of several genes and neuropeptides such as the CRF, vasopressin, galanin, urocortin III and c-fos. While application of RNAi in basic research is of vast importance, once established, it holds tremendous potential for its applicability in dissecting out neural circuitry and may be applied for silencing expression of genes involved in neurodegenerative diseases
