PROJECT SUMMARY GABAergic inhibitory synapses are critical for controlling neuronal excitability, fine-tuning neuronal output, and coordinating neuronal firing. Inhibitory synapses undergo bi-directional, activity-dependent changes that result in both changes to synaptic clustering and synaptic strength. In particular, one form of inhibitory long-term potentiation (iLTP) results in increased post-synaptic clustering of the GABAA receptors (GABAARs) and the inhibitory synaptic scaffolding protein, gephyrin. The clustering of both GABAARs and gephyrin at early stages of iLTP is through trafficking of these proteins to synapses, but later stages of iLTP require de novo protein synthesis in order to sustain this potentiation long-term. We have found that two microRNAs (miRNAs: miR376c and miR153) repress the translation of GABAARs and gephyrin under basal conditions. Following iLTP stimulation, miR376c and miR153 expression is reduced, resulting in increased GABAAR and gephyrin translation required for sustained increases in inhibitory synaptic strength. This decrease in miRNA expression is a result of transcriptional repression, mediated by the transcription factor, nuclear factor of activated T-cells (NFAT), which is regulated upstream by the phosphatase calcineurin (CaN). However, there is still a major gap in our knowledge of signaling mechanisms that control long-term changes in gene expression during inhibitory synaptic plasticity. In this proposal, I will address this gap in knowledge by identifying the upstream signaling cascades responsible for miR376c and miR153 transcriptional repression following iLTP, specifically focusing on the role of both NFAT and the scaffolding protein, A-kinase anchoring protein (AKAP), which mediates the anchoring of CaN in close proximity of calcium influx. I hypothesize NFAT transcription factors regulated by AKAP-anchored CaN signaling are required for long-term strengthening of inhibitory synapses during iLTP. I will utilize live-cell confocal microscopy and whole-cell electrophysiology to identify the requirement for NFAT in increases in inhibitory synapse size, number and strength following iLTP stimulation (Aim 1). Live-cell confocal microscopy, 3D-structured illumination microscopy (SIM), and slice electrophysiology will be performed to characterize the role of AKAP-anchored CaN in regulating NFAT translocation and inhibitory synaptic potentiation following iLTP stimulation (Aim 2). Together, this proposal will help identify the upstream signaling mechanisms that are required to regulate altered gene expression during long-term iLTP and will provide clarity on the mechanisms that regulate inhibitory synaptic plasticity, which are vital for regulating proper circuit function and learning, memory, and cognition.