Histology-guided spatial lipidomics and proteomics of the trisynaptic circuit in the human hippocampus

The human hippocampal trisynaptic circuit activity is essential for learning and memory. This canonical circuit has spatially distinct populations of neurons, but their unique contributions to neurodevelopment, as well as to dysfunction in neurodegenerative disorders, are missed when analyzing bulk tissue homogenates. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to guide laser capture microdissection (LCMD) of regions of interest for spatial multimodal analyses is a relatively new approach to study topographically distinct neuronal cell populations in heterogenous tissues. However, MALDI-MSI may not identify region-defining molecular mass-to-charge ions. Here, we apply a multimodal approach of MALDI-MSI-LCMD-lipidomic and proteomic analysis to the trisynaptic circuit. Our MALDI-MSI of the hippocampus revealed that the of mass-to-charge ions of the cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) did not segment from the surrounding tissue. Thus, we developed a novel histology-guided MALDI-MS imaging-LCMD-spatial lipidomic/proteomic pipeline with four steps which does not rely on segmentation analysis to determine and co-register regions of interest in tissue sections. Our pipeline allows MALDI imaging, LCMD, lipidomic and proteomic analysis from the same tissue section and does not require co-registration across serial sections. In addition, poly-l-lysine coating for improving tissue/cell adherence on indium-tin-oxide microscopy slides did not impact MALDI-MSI or spatial proteomics. We show that the human trisynaptic circuit proteomes of CA1 and CA3 pyramidal neurons are more similar to each other than those of the dentate gyrus (DG), which is consistent with previously reported transcriptomics studies. The spatial distributions of several phospholipids and proteins, however, were significantly different in cell bodies from the CA1, CA3 and DG regions, and these lipids correlated with some lipid metabolizing enzymes in those regions. As little is known about lipid metabolism in the hippocampus, our pipeline provides an initial step in studying the combined and differential spatial regulation of the lipids and proteins within the trisynaptic circuit that will provide insights into the development and disease-related molecular changes in these important hippocampal regions.