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A scalable internal code for threat imminence in prefrontal ensembles

Reis, Fernando M. C. V.1; Maesta-Pereira, Sandra2; Miranda, Blake A.1; Blair, Garrett J.3; Adhikari, Avishek1

Published May 21, 2026 on Dryad. https://doi.org/10.5061/dryad.3tx95x6x9

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Abstract

Survival in dynamic environments requires the integration of multisensory cues and movement into a unified estimate of risk, a process governed by the internal variable of threat imminence. While the medial prefrontal cortex (mPFC) is known to regulate defensive behaviors, how it represents threat imminence remains unresolved. Here, we developed the FUGA task, in which mice are fear conditioning to a moving shock grid, eliciting the full topography of defensive actions, including avoidance, risk-assessment, freezing and flight upon grid approach (FUGA). Using longitudinal calcium imaging of mPFC excitatory ensembles, we show that following fear acquisition, mPFC activity patterns representing threat proximity became significantly correlated with the representation of the shock, revealing a learned neural association between shock and grid. Additionally, mPFC population trajectories tracked threat imminence, reflecting internal state rather than specific behaviors or kinematics. We also identified two non-overlapping ensembles that multiplex kinematic and contextual variables and that are respectively activated by high and low threat imminence states. Furthermore, ensemble synchrony and imminence encoding predicted the execution of successful escape trials prior to flight onset. Finally, optogenetic inhibition revealed that mPFC activity is essential for both the acquisition and expression of learned escape and avoidance. These discoveries identify the mPFC as a critical substrate for representing learned threat imminence to guide flexible behavior.