Project Summary/Abstract Extensive effort has been committed to more fully characterize the human brain transcriptome within and across cell types to better understand changes in RNA expression associated with brain development and aging, developmental or psychiatric brain disorders, and local genetic variation. Large consortia, including psychENCODE, have primarily focused on the molecular profiling of RNA extracted from homogenate/bulk tissue from different brain regions across hundreds of individuals, though single nuclei expression approaches are increasingly being utilized in the second phase of projects. While these differences in signatures across brain regions relate to the unique cell types underlying each region, the specific cell types and their corresponding spatial landscapes are largely unknown. Neurons in different cortical and hippocampal layers show distinct expression patterns, morphology, physiology and patterns of connectivity. Converging evidence suggests that impairments in the formation or maintenance of synapses may be involved in schizophrenia, and studies in the postmortem brains of subjects have pointed to specific cell types and revealed differences in neuronal and synaptic structure that are localized to specific layers, suggesting that genetic risk for schizophrenia may manifest with laminar specificity. In this application, we propose to generate detailed spatial transcriptomics maps of the human DLPFC and hippocampus. These spatial expression maps will be combined with complementary single nuclei sequencing data from the same tissue blocks to develop spatial "registration" approaches that can add spatial information to existing single nuclei datasets in the psychENCODE project. We will combine these spatial and cell type-specific maps to implicate layer- and cell-specific populations in schizophrenia genetic risk and illness state that will be validated using complementary in situ hybridization techniques. These rich spatial transcriptome maps will add another dimension to existing and forthcoming single nuclei RNA-seq datasets in the frontal cortex and hippocampus to further refine the cell types in the human brain and their subsequent dysregulation in debilitating brain disorders.