Spatial Transcriptomic Dissection of the Cellular and Molecular Architecture of Fear Memory and its Association with...

Fear memory (FM) is a neurophysiological process regulated by diverse neural cell populations and closely linked to general memory function. However, the precise cellular and molecular mechanisms underlying FM remain insufficiently understood.

In this study, spatial transcriptomic data from four sagittal mouse brain sections obtained from 10xGenomics were systematically analyzed. Cell2location deconvolution was applied to characterize cellular composition and identify key cell populations associated with FM. CellChat and Monocle analyses were used to investigate intercellular communication and cellular activation trajectories during FM progression. Signaling pathways identified utilizing CellChat, together with pathways enriched from hypervariable genes identified by MFUZZ and DESeq2, were analyzed to clarify the molecular basis of FM. To further explore the mechanisms through which FM influences general memory function, key ligands, receptors, transcription factors (TFs), and downstream targets were identified using scMLnet.

Spatial transcriptomic analysis revealed extensive interactions among M2 macrophages, astrocytes, oligodendrocytes, and cholinergic neurons during FM, while M1 macrophages and dopaminergic neurons exhibited supportive roles in cellular co-occurrence networks. These interactions mediated autocrine and paracrine transmission through JAM, EPHB, NCAM, NRXN, and AMPK signaling pathways. Among the identified genes, Opalin, Thbs4, and Cyp2j12 emerged as potential biomarkers associated with dominant cellular interactions and were upregulated in FM-related regions. FM-associated molecular alterations may impair memory function through EPH/Ephrin-mediated ligand-receptor interactions that activate TFs, including CREB, E1A-binding proteins, and estrogen receptors, subsequently regulating Ras-related proteins and epidermal growth factor receptors. These findings suggest that estrogen signaling may represent a potential strategy for FM modulation.

Astrocytes, cholinergic neurons, M1/M2 macrophages, and oligodendrocytes appear to play central roles in FM regulation. Notably, M1 macrophages may promote the transcriptional transition toward M2 macrophages, thereby contributing to neuroinflammatory resolution. EPHB and NRXN signaling pathways demonstrated prominent regulatory associations with FM, whereas the functional significance of pathways such as JAM requires further investigation. In addition, competitive interactions between FM and general memory processes were closely linked to EPH/Ephrin signaling. Estrogen-mediated regulation may therefore provide a therapeutic avenue for suppressing maladaptive FM, although the underlying mechanisms remain incompletely defined.

This study provides a spatially resolved characterization of cellular composition, intercellular communication, and molecular regulation associated with FM. The findings generate new hypotheses regarding the relationship between emotional regulation and memory function, particularly the possibility that FM formation and consolidation compromise general memory processes. Collectively, these results offer new insights into the cellular and molecular neurobiology of FM.