VIRAL VECTOR MANIPULATION OF THE FEAR MEMORY ENGRAMS IN THE CENTROMEDIAL AMYGDALA

Memory engrams represent the specific neurons whose activation encodes, stores, and later reactivates specific memories. Within the central amygdala, engrams have traditionally been localized to subnuclei such as the centrolateral amygdala and the capsular subdivision, while the centromedial amygdala has been regarded primarily as an output hub that drives conditioned fear responses. Here, we redefine this framework by identifying a fear memory engram within the centromedial amygdala itself. Using the [PM1] AAV9-pfos-hM4Di:mCherry:PEST construct, we transiently silenced this engram with Clozapine N Oxide shortly after fear acquisition. This manipulation induced a reduction in cFos expression[PM2], a selective deficit in fear memory expression test one day later along with a decrease in Delta FosB levels, indicating disruption of long‑term memory consolidation. In vivo calcium imaging in the centromedial amygdala in freely moving mice further revealed that this silencing induced a sustained reduction in neuronal activity for up to three hours, defining the temporal window of engram manipulation. During the first tone presentation of the fear memory expression test, animals displayed an increased proportion of inactive neurons, confirming functional suppression of the engram. Single-cell spatial proteomics uncovered a specific molecular identity for this centromedial engram, and whole-brain analyses of mCherry and cFos demonstrated that, when inhibited, this engram exhibits minimal projection activity or engagement with other brain regions. Together, these findings reveal that the centromedial amygdala not only executes fear responses but also contains a dedicated engram essential for their formation and expression, reshaping current models of amygdala-dependent memory.