Dynamic changes in amygdala and insula responses to the onset and offset of conditioned stimuli during threat learning.

The amygdala and insula are central to learning how to anticipate and respond to threats, yet their precise roles during different phases of fear conditioning remain incompletely understood. In this study, we used functional magnetic resonance imaging (fMRI) to examine amygdala and insula responses in a large sample (N = 286) of adult twins during fear conditioning. Participants were exposed to two virtual characters: one (CS +) was paired with a mild electric shock (US) on 50% of acquisition trials (partial reinforcement), while the other (CS -) was never paired with the US. During habituation, neither the amygdala nor the insula discriminated between the cues. During acquisition, amygdala responses were strongest and most consistent at CS + offset on non-reinforced CS + trials (i.e., when the US was expected but omitted), whereas insula responses differentiated CS + from CS - at both CS onset and CS offset. Whole-brain analyses showed that CS differentiation at CS onset expanded from early engagement of anterior insula and temporal regions to broader networks as learning progressed. CS differentiation at CS offset (non-reinforced CS +  > CS -) emerged in posterior insula and extended to broader predominantly contralateral networks in later acquisition blocks. Voxel-wise pattern similarity analyses indicated high within-event similarity across acquisition blocks (onset-with-onset; offset-with-offset), but lower similarity between event types (onset vs offset) and between either CS-related event and the US. Together, these findings are consistent with partially distinct large-scale activation patterns across cue onset, omission-related cue offset, and US delivery during threat learning.