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. In the parabrachial nucleus, Calca gene-expressing neurons, which react to the noxious US, also respond to a CS after its pairing with a US. This observation led us to hypothesize their involvement in SOC. To explore this possibility, we established an aversive SOC behavioral paradigm in mice and monitored Calca neuron activity via single-cell calcium imaging during SOC and subsequent recall phases. These neurons were activated not only by CS1 following its association with the US but also by CS2 after SOC. Chemogenetically inhibiting these neurons during second-order associations attenuated SOC. These findings suggest that activating the US pathway in response to 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.