PROJECT SUMMARY This revised R01 application is submitted in response to PAR-14-309 and seeks to understand the mechanisms by which a microRNA enriched in cortical parvalbumin-expressing (PV) GABAergic interneurons regulates the activity of these cells and behaviors under their control. In prefrontal cortex (PFC), microRNA-206 (miR-206) is highly enriched in PV interneurons and its expression levels in post-mortem PFC correlate with psychosis in schizophrenia and bipolar patients. In exciting preliminary data, we show that newly generated miR-206 knockout mice demonstrate cellular and cognitive deficits consistent with the hypothesis that this microRNA regulates PV interneuron function in PFC. The goals of this proposal are threefold: First, cellular and behavioral abnormalities in miR-206 KO mice, and in KO mice in which miR-206 expression has been “rescued” in cortical PV interneurons, will be assessed. To accomplish this Aim we have generated miR-206 knockout mice that express Cre recombinase in PV neurons. We will use a Cre-dependent expression vector to re-express the otherwise deleted miRNA in cortical PV interneurons. We will then assess the strength of synaptic connectivity between cortical PV interneurons and neighboring pyramidal neurons in PFC of miR-206 KO mice, KO mice with rescued miR-206 expression in cortical PV interneurons, and appropriate control groups. We will also characterize aspects of cognition and emotionality controlled by cortical PV interneurons in these mice. Second, the intracellular mechanisms by which miR-206 controls activity of PV interneurons, and hence mPFC function, will be investigated. We will use High-Throughput Sequencing of RNA isolated by CrossLinking ImmunoPrecipitation (HITS-CLIP) to identify genes targeted by miR-206 in cortical PV interneurons. We will verify that identified genes are direct targets for miR-206 using 3'UTR luciferase reporter assays, RNA expression analysis and protein immunoblotting. Third, the molecular mechanisms by which miR- 206 controls activity of PV interneurons, and hence mPFC function, will be investigated. Specifically, we will use in vivo CRISPR technology to delete the most promising genes targeted by miR-206 in PV interneurons and assess the behavioral consequences. The cutting-edge experiments proposed in this application will facilitate greater understanding of the molecular mechanisms by which cortical PV interneuron activity is regulated and may yield new insights into the pathophysiology of PV interneuron-related psychiatric disorders.