Evaluation of synaptic connectivity and dysfunction in aging mouse brains using an RNAscope multiomic spatial imaging assay (MSIA) that detects RNA, proteins, and protein interactions

Advancements in spatial imaging technologies (SIT) have broadened transcriptomic discovery capacity. Yet, development of orthogonal technologies for follow-up translational studies have trailed. We have developed MSIA that utilizes non-temporal, optical barcodes by combining the advantages of RNAscope™ double-Z probes and Hiplex v2 signal amplification technology.Fresh-frozen and FFPE brain coronal sections were collected from C57BL/6J mice of 3 age groups: 1 month, 6 months, and 12 months, respectively. MSIA was executed in 3 cycles, with 100 RNA targets in cycles 1&2 and then up to 4 protein targets in cycle 3 that collectively mark different neuronal and non-neuronal cells. Fluorescent images were collected using Zeiss Axioimager, digitally decoded at single-cell scale with a fast deep-learning algorithm and analyzed with a proprietary bioinformatics pipeline.All 100 genes in the panel were detected by MSIA at an average of 70 transcripts/cell with >100,000 cells analyzed in each whole brain section. UMAP revealed 22-24 distinct cell populations in the brain including astrocytes, microglia/macrophage, pericytes/vascular cells, oligodendrocytes, OPC, and various GABAergic/glutamatergic neuron subclasses. Twelve-month-old brain exhibited an enrichment of oligodendrocytes and reduction in astrocytes and microglia compared to the one-month-old. Moreover, synaptic connectivity and their dysfunction in striatum were also evaluated in normal and elder mouse brains using a panel of 53 genes followed by a new RNAscope-based protein/protein interaction assay to specifically interrogate synaptic interactions.With only 3 manual cycles and a Deep-learning algorithm, MSIA assay achieved efficient spatial transcriptomics profiling of brain cell states with high robustness and accuracy.