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.