The mPFC-PAG in defensive coping: A correlational but not causal role in shifting strategy

Defensive behaviors under threat are fundamental for survival, and their dysregulation contributes to psychiatric disorders. The medial prefrontal cortex (mPFC) and periaqueductal gray (PAG) pathway are implicated in regulating defensive behaviors, but their specific role in choosing between coping strategies remains unclear. Here, we investigated these brain regions using the two-way active avoidance (TWAA) task in male rats. Anatomical tracing confirmed a robust unidirectional projection from layer V neurons of mPFC subregions (including ACC, PL, IL) to the PAG. Using c-Fos immunohistochemistry, we found significantly higher neuronal activation in the infralimbic cortex (IL), but not other mPFC subregions or the PAG, in rats exhibiting active avoidance (AA, active avoidance rate > 80 %) compared to those showing reactive escape (RE, active avoidance rate ＜50 %). However, combining retrograde tracing with c-Fos labeling showed no differences in activation of PAG projecting neurons between AA groups and RE groups across all mPFC subregions. Furthermore, optogenetic activation of IL neurons or PAG neurons did not alter established AA/RE coping preferences in the TWAA task, nor did it affect baseline locomotion in the open field test. These findings reveal a critical dissociation between regional neuronal activity and the causal control of defensive behaviors. While IL activity correlates with AA, acute global activation of the mPFC or PAG is insufficient to override established coping preferences. This suggests that adaptive strategy switching likely requires precisely timed, cell-type-specific activation or modulation during learning phase, rather than broad activation after strategies are consolidated. SIGNIFICANCE STATEMENT: This study demonstrates that while infralimbic cortex activity correlates with active avoidance behavior, acute optogenetic activation of the mPFC or PAG is insufficient to shift established defensive coping strategies. These findings reveal a critical dissociation between regional neural activity patterns and the causal control of defensive behaviors.