Dentate gyrus and CA3 activity mediates light-tone second-order conditioning expression in mice

Second-order conditioning (SOC) enables animals to form complex predictions about their environment, even in the absence of direct experience. While the neural mechanisms underlying first-order conditioning (FOC) are well characterized, the circuits supporting SOC expression remain poorly understood. To address this gap, we investigated the brain regions and cell types involved in SOC recall in mice and tackled the technical challenges of quantifying brain-wide neural activity. We employed a light–tone SOC paradigm in TRAP2:Ai14 mice, which allowed us to tag neurons active during SOC recall via tdTomato expression. Applying generalized linear models, we identified that the activity in the dentate gyrus (DG) and CA3 regions of the dorsal hippocampus significantly associated with SOC-related behavioral responses. To test their functional relevance, we used chemogenetic inhibition of CaMKII ⁺ neurons in these regions, which confirmed a causal role for DG/CA3 circuits in SOC recall. Together, our results highlight the dorsal hippocampus as a critical substrate for retrieving indirectly learned associations.