MECP2-duplication syndrome is caused by duplication of the MECP2 gene leading to progressive intellectual disability, autism, motor dysfunction, spasticity, and epilepsy in males with 100% penetrance. Synaptic plasticity mechanisms are causally implicated in multiple autism spectrum disorders, including Methyl-CpG-binding-protein-2 (MECP2) duplication syndrome. We recently described an abnormal synaptic plasticity phenotype in the Tg1 mouse model of MECP2-duplication syndrome, implicating the Ras/MAPK pathway. However, cell-specific investigation of plasticity pathways involved has yet to be undertaken. Characterizing common plasticity pathway themes of dysfunction in autism spectrum disorders (ASD) is potentially of great value for identifying new targets for intervention. Until recently it has not been possible to measure the transcriptome of individual cells in situ and relate it to their functional properties. Here, we adapt a recently developed high-throughput in-situ mRNA hybridization method (MERFISH), capable of resolving hundreds to thousands of distinct mRNA transcripts per imaging session, to investigate cortical plasticity mechanisms during learning in the Tg1 mouse model of the MECP2-duplication syndrome. We use a robust visual training paradigm introduced by M Bear, consisting of multiple presentations of a high contrast flickering grating at a specific orientation. This is known to form a true memory trace in area V1+ that requires sleep for consolidation, has a behavioral correlate and shares core molecular features with LTP. Aim-1: Compare how visual cortex circuits malfunction with learning in MECP2-duplication mice vs in littermate controls. Visual response properties, intra- and inter-layer functional connectivity profiles will be measured within area V1 and compared across genotypes before and after training. Hypothesis: The capacity of cortical circuits for learning differs in mutant vs control mice in a laminar specific way. Inter-layer functional connectivity profiles will be abnormal in MECP2-duplication syndrome resulting in abnormal signal transmission and processing across cortical laminae. This will be exacerbated post-training leading to rigid, less flexible, neural responses. Aim-2: Compare single-cell resolved transcriptomic profiles of plasticity and neurotransmitter pathways in the visual cortex of MECP2-duplication vs control animals undergoing the training paradigm introduced in aim-1. Relate neuronal responses obtained in aim-1 to cell-specific transcriptomic profiles obtained from the same neurons. Hypothesis: We will acquire a more complete, cell-specific, picture of how mechanisms of plasticity fail in the neocortex of the mouse model of MECP2-duplication syndrome. Data obtained will generate hypotheses for follow up experiments, helping to identify targets for future pharmacologic interventions. IMPACT: Relate for the first time, cell-specific plasticity & neurotransmitter/ion-channel transcriptomic profil...