HIGH-RESOLUTION SPATIOMOLECULAR MAPPING OF CA1 ENGRAM CELL SYNAPSES

Experience-driven changes in memory-encoding engram cells are fundamental to memory storage. These modifications occur particularly at the level of dendritic spines, where molecular adaptations play a crucial role in regulating synaptic strength, structure, and plasticity. The resulting augmented engram synaptic connectivity, shaped by alterations in synapse density, clustering and spine morphology, supports both memory storage and its retrieval to natural cues. However, it remains unclear how specific molecular changes at engram cell synapses relate to spine morphometry and how these features contribute to the spatial organization of spines along the dendritic arbor.
Here, we propose a comprehensive microscopy-based pipeline for the spatial and intensity-based analysis of proteins enriched in hippocampal CA1 engram cell synapses following contextual fear conditioning (CFC). By combining viral input-specific, activity-dependent labeling, global immunohistochemistry, high-resolution confocal microscopy, Imaris 3D dendritic tracing and an in-house developed R pipeline, we spatially resolve the dendritic distribution of specific glutamate receptors upregulated after CFC in CA1 engram cells. With this approach, we investigate how learning-induced molecular changes map onto input-defined synapse subtypes and their related structural adaptations within defined memory-encoding circuits. By correlating molecular and morphological features at single-spine resolution while assessing spatial patterning, our pipeline offers valuable insights into the spatiomolecular organization of memory-encoding engram cell synapses. Understanding this organization is critical for uncovering how stable patterns of synaptic connectivity emerge within engram cell networks to support memory storage.