Piwi-interacting RNAs (piRNAs) are a unique class of small regulatory RNAs which interact specifically with the Piwi-like proteins. Through this interaction, piRNAs can modulate gene expression via RNA interference and epigenetic mechanisms. The Piwi pathway has been defined by its role in transposon control during spermatogenesis in mammals, and despite an increasing number of studies demonstrating its expression outside the testes, relatively little is known about its function in mammalian somatic tissues. We have discovered that the Piwi-like proteins Piwil1 and Piwil2 are expressed in neurons throughout the mouse brain, and Piwil2 is upregulated by neuronal activation. Simultaneous knockdown of Piwil1 and Piwil2 in the mouse hippocampus enhances fear learning and the memory of conditioned fear 24 hours later, without affecting generalised anxiety. However, knockdown of either Piwil1 or Piwil2 in isolation fails to recapitulate this effect. This suggests that the role of Piwil1 and Piwil2 in the neuronal Piwi pathway may be redundant. Preliminary results from small RNA sequencing indicate that some hippocampally-expressed piRNAs are responsive to the fear conditioning paradigm, suggesting that the function of Piwi proteins in neurons may occur through piRNA-mediated epigenetic control of transposons or protein-coding genes. Our laboratory is performing small RNA sequencing on cytoplasmic and nuclear fractions of mouse hippocampus to look for neuronally expressed piRNAs which are activity regulated or which shuttle into the nucleus in response to learning. We are also performing FRIP-seq (formaldehyde-crosslinked RNA immunoprecipitation and sequencing) to confirm the interaction between Piwi proteins and putative neuronal piRNAs. and investigate the regulatory targets of the Piwi pathway in the mammalian brain.