A study reveals that the DNA-sensing TLR9 pathway plays a critical role in the formation of memory assemblies in hippocampal neurons. Following learning, some excitatory CA1 neurons exhibit DNA damage, nuclear envelope ruptures, and the release of histone and DNA fragments. These events trigger TLR9 signaling, leading to the accumulation of DNA repair complexes and the recruitment of neurons into memory circuits. TLR9 is essential for centrosome function, ciliogenesis, and the formation of perineuronal nets. Neuron-specific Tlr9 knockdown impairs memory formation and increases genomic instability, which is linked to cognitive impairments. The study also shows that TLR9 signaling is involved in the regulation of gene expression in memory-related neuronal clusters. TLR9-mediated DNA sensing is crucial for maintaining genomic integrity and preventing cognitive deficits. The findings suggest that TLR9 signaling is a key mechanism for integrating DNA damage repair with memory formation, and that maintaining TLR9 function is essential for preserving neurocognitive health. The study highlights the importance of TLR9 in the formation and maintenance of memory circuits, and suggests that TLR9 and downstream RELA signaling could be promising targets for preventing neurocognitive disorders.A study reveals that the DNA-sensing TLR9 pathway plays a critical role in the formation of memory assemblies in hippocampal neurons. Following learning, some excitatory CA1 neurons exhibit DNA damage, nuclear envelope ruptures, and the release of histone and DNA fragments. These events trigger TLR9 signaling, leading to the accumulation of DNA repair complexes and the recruitment of neurons into memory circuits. TLR9 is essential for centrosome function, ciliogenesis, and the formation of perineuronal nets. Neuron-specific Tlr9 knockdown impairs memory formation and increases genomic instability, which is linked to cognitive impairments. The study also shows that TLR9 signaling is involved in the regulation of gene expression in memory-related neuronal clusters. TLR9-mediated DNA sensing is crucial for maintaining genomic integrity and preventing cognitive deficits. The findings suggest that TLR9 signaling is a key mechanism for integrating DNA damage repair with memory formation, and that maintaining TLR9 function is essential for preserving neurocognitive health. The study highlights the importance of TLR9 in the formation and maintenance of memory circuits, and suggests that TLR9 and downstream RELA signaling could be promising targets for preventing neurocognitive disorders.