OPTOGENETIC ACTIVATION OF ASTROCYTIC CALCIUM SIGNALING REGULATES OBSERVATIONAL FEAR MEMORY IN MICE

Observational fear learning enables animals to acquire threat-related information by observing conspecifics and critically depends on anterior cingulate cortex (ACC) circuits. While accumulating evidence indicates that astrocytic calcium (Ca²⁺) signaling is actively involved in neural circuit dynamics, its causal role in higher-order brain functions, including observational fear memory, remains poorly understood. Here, we employed monSTIM1, an ultra-light-sensitive optogenetic tool that selectively enhances astrocytic Ca²⁺ signaling, to investigate astrocytic contributions to observational fear learning. Optogenetic activation of monSTIM1-expressing astrocytes in the right ACC during observational fear conditioning significantly impaired memory expression 24 hours later, while leaving fear acquisition intact. By contrast, elevating astrocytic Ca²⁺ levels in hippocampal CA1 during contextual fear conditioning disrupted both acquisition and retrieval, revealing region- and process-specific roles of astrocytic Ca²⁺ signaling in memory processing. In vivo fiber photometry confirmed that blue light stimulation robustly increased Ca²⁺ activity in monSTIM1-expressing astrocytes and revealed the temporal dynamics of ACC astrocytic Ca²⁺ signals during observational fear learning. Mechanistically, bulk RNA sequencing of cultured astrocytes showed that monSTIM1-mediated Ca²⁺ elevation induced a MAPK/ERK-driven, activity-dependent transcriptional program enriched for growth factor signaling and extracellular matrix remodeling, implicating astrocyte-mediated synaptic modulation pathways. Furthermore, whole-cell patch-clamp recordings revealed that activation of monSTIM1-expressing astrocytes reduced the excitability of neighboring neurons, providing a functional link between astrocytic Ca²⁺ signaling and circuit-level inhibition. Together, our findings establish astrocytic Ca²⁺ signaling as a key regulator of observational fear memory through region-specific modulation of neuronal excitability and memory stabilization.