Second-order fear conditioning involves formation of competing stimulus-danger and stimulus-safety associations.

How do animals process experiences that provide contradictory information? The present study addressed this question using second-order fear conditioning in rats. In second-order conditioning, rats are conditioned to fear a stimulus, S1, through its pairings with foot-shock (stage 1); and some days later, a second stimulus, S2, through its pairings with the already-conditioned S1 (stage 2). However, as foot-shock is never presented during conditioning to S2, we hypothesized that S2 simultaneously encodes 2 contradictory associations: one that drives fear to S2 (S2-danger) and another that reflects the absence of the expected unconditioned stimulus and partially masks that fear (e.g. S2-safety). We tested this hypothesis by manipulating the substrates of danger and safety learning in the brain (using a chemogenetic approach) and assessing the consequences for second-order fear to S2. Critically, silencing activity in the basolateral amygdala (important for danger learning) reduced fear to S2, whereas silencing activity in the infralimbic cortex (important for safety learning) enhanced fear to S2. These bidirectional changes are consistent with our hypothesis that second-order fear conditioning involves the formation of competing S2-danger and S2-safety associations. More generally, they show that a single set of experiences can produce contradictory associations and that the brain resolves the contradiction by encoding these associations in distinct brain regions.