Profiling lamina specific pyramidal neurons using postmortem human formalin fixed paraffin embedded frontal cortex tissue in combination with digital spatial profiling
Digital spatial profiling (DSP) is an innovative approach to perform RNA sequencing (RNA-seq) and bioinformatic analysis combined with precise spatial information from individually identified regions or cell types, including neurons. This approach enables expression profiling linking RNA-seq data to spatially characterized samples utilizing tissue bound probes. We employ the GeoMx DSP system to allow spatial characterization of transcriptomic data from lamina specific pyramidal neurons as well as cortical laminar ribbons containing neurons and admixed cell types in human postmortem brain tissue. We optimized the protocol to establish a robust and reproduceable method in human postmortem formalin fixed paraffin embedded (FFPE) frontal cortex tissue using nondemented human control brains. Layer III (L3) and Layer V (L5) pyramidal neurons from Brodmann area 9 were identified with the neuronal marker Ca2+/calmodulin-dependent protein kinase II and selected for probe collection. The present protocol significantly reduced the amount of FFPE tissue needed for robust RNA-seq using the GeoMx DSP platform. We demonstrate ~20 identified L3 or L5 pyramidal neurons or  one lamina-specific cortical ribbon from a single 5 µm thick section is sufficient to generate RNA-seq results. Bioinformatic analysis of isolated neurons and ribbons showed notable similarities and differences, reflective of the single neuron and multiple admixed cell types within the former and latter, respectively. This revised DSP assay provided high resolution RNA-seq data demonstrating utility and versatility of GeoMx platform for individually characterized neurons as well as isolated cortical ribbons within postmortem FFPE human brain tissue for downstream analyses. Overall design: This study is focused on profiling of layer III (L3) and layer V (L5) pyramidal neurons within their respective cortical laminae in non pathologic human postmortem FFPE frontal cortex brain tissue sections for gene expression analysis and downstream bioinformatic inquiry.