Parabrachial Calca neurons mediate second-order conditioning

Learning to associate cues, both directly and indirectly, with biologically significant events is essential for survival. Second-order conditioning (SOC) involves forming an association between a previously reinforced conditioned stimulus (CS1) and a new conditioned stimulus (CS2) without the presence of an unconditioned stimulus (US). The neural substrates mediating SOC, however, remain unclear. Parabrachial Calca neurons, which react to the noxious US, also respond to a CS after pairing with a US, suggesting that Calca neurons mediate SOC. We established an aversive SOC behavioral paradigm in mice and monitored Calca neuron activity via single-cell calcium imaging during conditioning and subsequent recall phases. These neurons were activated by both CS1 and CS2 after SOC. Chemogenetically inhibiting Calca neurons during CS1-CS2 pairing attenuated SOC. Thus, reactivation of the US pathway by a learned CS plays an important role in forming the association between the old and a new CS, promoting the formation of second-order memories.