Short-Hairpin RNA Silencing of Endogenous Fibroblast Growth Factor 2 in Rat Hippocampus Increases Anxiety Behavior

Abstract

The fibroblast growth factor system has been implicated in the pathophysiology of mood disorders in humans and in affective behavior in animal models. However, the studies have been either correlative or involved exogenous administration of fibroblast growth factor 2 (FGF2). None of them have directly linked endogenous FGF2 to changes in emotional responses. Therefore, we began a series of studies to knockdown FGF2 by RNA interference to examine the role of brain FGF2 in emotional responsiveness. We assessed the efficacy of short-hairpin RNA (shRNA) sequences targeted to FGF2 in COS7 cells transfected with a plasmid vector containing the full-length FGF2 sequence. We then sought to assess the effects of knocking down FGF2 gene expression in vivo on behavior. We microinjected a lentiviral vector containing either a shRNA targeting FGF2 or a nonsilencing sequence bilaterally into the dentate gyrus of the rat. In a reporter assay system, three different shRNA sequences resulted in significant FGF2 knockdown in vitro. Five weeks following a single microinjection of one of those sequences in vivo, we observed a significant decrease in FGF2 gene expression by messenger RNA in situ hybridization in the hippocampus. The FGF2 knockdown increased the time spent in the closed arms of the elevated-plus maze, a test of anxiety behavior. The FGF2 knockdown in the hippocampus resulted in an anxiogenic effect. Together with our findings of an inverse correlation between anxiety and FGF2 expression levels, these results implicate FGF2 in the genesis and expression of anxiety disorders.